Recent Advances on Nutrition and the Prevention of Alzheimer’s Disease by Research Signpost

Recent Advances on Nutrition and the Prevention of Alzheimer’s Disease 2010 Editor

Charles Ramassamy INRS-Institut Armand Frappier, 531 Boulevard des Prairies, H7V1B7 Laval (Québec), Canada

Associate Editor

Stéphane Bastianetto Douglas Mental Health University Institute, 6875 LaSalle Boulevard Verdun, Québec, H4H 1R3, Canada

Transworld Research Network, T.C. 37/661 (2), Fort P.O., Trivandrum-695 023 Kerala, India

Published by Transworld Research Network 2010; Rights Reserved Transworld Research Network T.C. 37/661(2), Fort P.O., Trivandrum-695 023, Kerala, India Editor Charles Ramassamy Associate Editor StĂŠphane Bastianetto Managing Editor S.G. Pandalai Publication Manager A. Gayathri Transworld Research Network and the Editors assume no responsibility for the opinions and statements advanced by contributors ISBN: 978-81-7895-470-7

Preface This book touches upon the subject of nutrition and Alzheimerâ&#x20AC;&#x2122;s disease (AD). This subject is at the forefront of scientific research. The exponential increase in the number of scientific research is a strong indicator of the need for this book which provides an up-to-date guide to some mechanisms that underlie the crucial role of nutrition in the prevention of AD. However, this book does not intend to cover all nutritional approaches for the prevention of AD. The increase in life expectancy and the progressive aging of the total population represent a great challenge for the health care system. The quality of life in the elderly population is strongly linked to cognitive function whose impairment can be influenced by numerous factors, one of the most obvious, yet under-recognized, being the role of nutrition. AD is the most prevalent form of dementia, characterized by a progressive and irreversible cognitive deficit that affect memory and learning capacities, activities of daily living and quality of life in general. AD is a heterogeneous neurodegenerative disorder, with a complex combination of genetic and non-genetic components. The early-onset familial form represents only a small fraction of all AD cases ( 5%) and typically presents itself with the age of onset being younger than 65 years, while non-genetic or sporadic form represents the majority of AD cases. The development of late-onset AD is complex and supports the notion that multiple pathways can be involved. Indeed, neuronal loss, production of amyloid-Ă&#x; peptide, hyperphosphorylation of Tau protein, neuroinflammation and oxidative damages may act together to accelerate the downhill cascade of cognitive dysfunction as observed in AD. The exact mechanisms whereby these pathological features preferentially induce neuronal loss are not completely understood. Since the therapeutic options currently available have demonstrated limited efficacy, the search for preventive strategies for cognitive decline and dementia such as AD is mandatory. In the past few years, lifestylesrelated factors have been an area of intensive research as possible preventive options. Nutrition offers promising perspectives for the prevention of AD in particular for sporadic or late onset form of AD. Nutrition plays a role in the ageing process, and during the past decade, there has been an intensive research which has provided an evidence for a link between nutrient intake/status and cognitive decline. Nutrition has been suggested as one of the key factors likely to have a protective role in cognitive performance. For instance, subclinical deficiencies of essential

nutrients, such as vitamins B6, B12, C, E, and K, folate and Ă&#x;-carotene as well as nutrition-related disorders, such as hypercholesterolemia, diabetes, obesity have been suggested as nutrition-related risk factors for dementia. The potential of nutritional components for neuronal maintenance rather than as energy substrate is illustrated by the increasing evidence that nutrients not only stimulate neural plasticity but also attenuate the pathological burden in the brain. Although epidemiological studies report conflicting results, an association between nutritional elements and the risk for AD has been observed. These studies support the idea that AD patients as well as those with mild cognitive impairment may benefit from nutritional intervention. Additionally, the individual genetic pattern associated to some specific risk factors or co-morbidities such as cardiovascular disease, high blood pressure, diabetes, insulin resistance could further drive the need for higher intake of specific nutrients. For instance, high intake of omega-3 polyunsaturated fatty acids may be associated with a reduced risk of developing AD, whereas antioxidants or vitamins, folate represent potentially beneficial nutritional components in AD. Intake of fruits, vegetables has been linked to a lower risk of AD. In contrast, a decrease in homocysteine levels leads to an increased risk of AD. There is substantial evidence that a Mediterranean diet might be effective in the prevention of AD, which emphasizes the role of multiple ingredients rather than a single nutrient. These include the potential protective effects of polyunsaturated acids, vitamins, antioxidants and polyphenols. These macro and micronutrients may provide bioactive compounds that can be used for membrane and synapse formation and to attenuate oxidative damages. This books will summarize the current knowledge regarding the role of nutrition in AD with the description of several modes of action of polyphenolic compounds (chapter #1), new evidence from epidemiological studies with the convergence or inconsistent results (chapter #2), the role of the Mediterranean diet, the negative effects of saturated fatty acids and the protective effects of polyunsaturated fatty acids against cognitive decline (chapter #3), the association of alcohol consumption and cognitive impairment (chapter #4), the possible link between fish and seafood or DHA intakes and cognitive impairment (chapter #5), the manipulation of a high fat diet on the accumulation of the amyloid beta peptide and tau in the brain from transgenic mice model (chapter # 6), the role of vitamin A and retinoic acid signaling in AD (chapter #7), the role of homocysteine as a risk factor for neurodegenerative disorders and AD (chapter #8), the neuroprotective actions of catechins from green tea and resveratrol from red wine (chapter # 9), of oxyresveratrol, a natural hydroxystilbene from mulberry

fruit (chapter # 10), of wolfberry products composed of polysaccharides, carotenoids and trace elements (chapter # 11), and C.elegans as a model to study the aging process and age-related diseases and for screening nutraceuticals compounds. Limitations of some studies are discussed and future research directions are proposed. This book could never have been accomplished without the hard work of each author. I would like to take this opportunity to express my sincere thanks to all authors for their magnificent contributions who have participated with great enthusiasm in this project for providing these excellent chapters summarizing new developments in their specific research fields. I appreciate their professionalism and it was a great pleasure for me to interact with them. I am extremely grateful to each contributor in this volume who spent their time and fundamentally contributed to the success of this book. I hope that a number of readers will enjoy and find a lot of information to develop new ideas in this rapidly ongoing field of investigation. In this book, we have distinguished researchers and scholars from the Nutritional epidemiology Team and the Nutrition & Neurosciences Unit from the University of Victor Segolen at Bordeaux (France), the Center for Aging Brain from the University of Bari (Italy), the Research Center of Neurology from Moscow University (Russia), the Laboratory of Neurodegenerative Diseases from Hong Kong University (Hong Kong), the School of Pharmacy from the University of Maryland from Baltimore (USA) and several contributors from QuĂŠbec (Canada) as the Research Center on Aging from the University of Sherbrooke, the Faculty of Pharmacy & Centre Hospitalier de lâ&#x20AC;&#x2122;UniversitĂŠ Laval, the Douglas Mental Health University Institute from McGill University. Finally, I wish to thank the staff at Transworld Research Network for their assistance, in particular the managing editor, Dr. Pandalai for his patience and support. Charles Ramassamy

Contents

Chapter 1 Towards a nutritional approach for the prevention of Alzheimer’s disease: Promise of polyphenolic components Charles Ramassamy, Sihem Doggui, Madeleine Arseneault and Lé Dao Chapter 2 Diet and Alzheimer’s disease: New evidence from epidemiological studies C. Féart, C. Samieri and P. Barberger-Gateau Chapter 3 Dietary fatty acids, alcohol intake, and cognitive decline in the elderly Francesco Panza, Vincenza Frisardi, Cristiano Capurso Alessia D’Introno, Anna M Colacicco, Antonio Capurso and Vincenzo Solfrizzi Chapter 4 Alcohol consumption and cognitive impairment Luc Letenneur Chapter 5 Are fish and docosahexaenoic acid linked to lower risk of cognitive decline and Alzheimer’s disease? M.E. Bégin, M. Plourde, F. Pifferi and S.C. Cunnane

Chapter 6 Manipulation of dietary fatty acids: Impact on neuropathological markers of AD in transgenic models Frédéric Calon, Carl Julien, Meryem Lebbadi, Cyntia Tremblay and Vincent Émond

Chapter 7 Vitamin A and Alzheimer’s disease Valérie Enderlin and Véronique Pallet

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Chapter 8 Molecular mechanisms of homocysteine toxicity and possible protection against hyperhomocysteinemia Alexander Boldyrev Chapter 9 Catechins and resveratrol as protective polyphenols against beta-amyloid-induced toxicity: Possible significance to Alzheimer’s disease Stéphane Bastianetto, Yvan Dumont and Rémi Quirion

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Chapter 10 Neuroprotective effects of oxyresveratrol from fruit against neurodegeneration in Alzheimer’s disease Raymond Chuen-Chung Chang, Jianfei Chao, Man-Shan Yu and Mingfu Wang

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Chapter 11 Medicinal and nutraceutical uses of wolfberry in preventing neurodegeneration in Alzheimer's disease Raymond Chuen-Chung Chang, Yuen-Shan Ho, Man-Shan Yu and Kwok-Fai So

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Chapter 12 Nutraceutical modulation of the aging process and age-related diseases – What the worm has taught us Marishka K. Brown and Yuan Luo

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Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Recent Advances on Nutrition and the Prevention of Alzheimer’s Disease, 2010: 1-18 ISBN: 978-81-7895-470-7 Editors: Charles Ramassamy and Stéphane Bastianetto

1. Towards a nutritional approach for the prevention of Alzheimer’s disease: Promise of polyphenolic components Charles Ramassamy1,2, Sihem Doggui1, Madeleine Arseneault1 and Lé Dao3 1

INRS-Institut Armand Frappier, 531 Boulevard des Prairies, H7V1B7 Laval (Québec), Canada 2 INAF, Université Laval, Québec, Canada; 3INRS-EMT, 1650, boul. Lionel-Boulet Varennes (Québec), J3X 1S2, Canada

Abstract. Aging is the major risk factor for Alzheimer’s disease (AD). A large body of evidence indicates that oxidative stress is involved in the pathogenesis of AD. Some studies highlight a role for free radicalmediated injury to brain regions from early stages of AD. There is a growing interest on the beneficial role of nutrition in AD and particularly for sporadic or late-onset form of AD. An increasing number of in vitro and in vivo studies show that polyphenol components from nutrition can counteract some specific aspects of the neurodegenerative and neuropathological processes as observed in AD. The beneficial intake of nutritional flavonoids and polyphenols components is also supported by recent epidemiological studies. We will describe the molecular basis of the neuroprotective activities of dietary polyphenols, with emphasis on their ability to control intracellular signaling cascades considered as relevant targets in the approach of prevention of neurodegenerative diseases. The aim of this chapter is to provide the current knowledge regarding the role of nutritional polyphenolic components in the management of AD, the challenges and the future prospects in this promising area. Correspondence/Reprint request: Prof. Charles Ramassamy, INRS-Institut Armand Frappier, 531 Boulevard des Prairies, H7V1B7 Laval (Québec), Canada. E-mail: Charles.Ramassamy@iaf.inrs.ca

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Introduction The extent in life expectancy and progressive aging of the population represents the biggest challenge for the worldwide health care system for the next few decades. Quality of life at older age is strongly linked to the preservation of cognitive function that can be influenced by numerous factors and one of the most obvious but yet under-recognized, being the role of nutrition. Aging may be regarded as the main risk factor for all forms of dementia and particularly for AD affecting up to 15 million people worldwide and this situation will worsen due to the aging of the population and to the lack of efficient treatment. The prevalence of AD is only 1% above the age of 60 years old but it increases dramatically to 50% above 85 years old. AD is a heterogeneous neurodegenerative disorder, clinically characterized by progressive and irreversible cognitive deficits and behavioral alterations that affect memory and learning ability, activities of daily living and quality of life. AD is multifactorial with a complex combination of genetic and non genetic components. It is clinically characterized by memory dysfunctions, loss of lexical access, spatial and temporal disorientation and impairment of judgment. Early-onset familial form with an autosomal dominant transmission represents only a small fraction of all AD cases ( 5%) with the age of onset younger than 65 years, while non-genetic or sporadic form represents the majority of AD cases with the age of onset higher than 65 years-old. Therefore the latter form represents a significant and growing public health burden. Rare early-onset form of AD are caused by an autosomal dominant mutations in one of three different genes coding for the amyloid precursor protein (APP) on chromosome 21, presenilin 1 on chromosome 14, and presenilin 2 on chromosome 1 [1-3]. While these AD-causing mutations occur in 3 different genes located on 3 different chromosomes, they all share a common biochemical pathway, i.e., the altered production of the amyloid Ă&#x; peptide (AĂ&#x;) with an overabundance of the AĂ&#x;1-42 species, which leads to neuronal death and dementia. The cause(s) of late-onset are not yet clarified but several environmental and genetic risk factors have been identified with the most potent being the 4 allele of the apolipoprotein E gene located on chromosome 19q13 [4, 5]. The increased risk with the 4 allele of the apolipoprotein E has been consistently replicated in a large number of studies across many ethnic groups. Unlike the mutations in the known early-onset familial form genes, 4 allele of the apolipoprotein E is neither necessary nor sufficient to cause AD but instead operates as a genetic risk factor by decreasing the age of onset in a dose-dependent manner.

Polyphenols and Alzheimer's disease

1. Evidence for oxidative stress in AD In addition to the genetic components, there is considerable evidence that oxidative stress is an early and critical event in the pathogenesis of AD [6]. For instance, F-4 isoprostanes, derived from free radical oxidation of docosahexaenoic acid, are increased in brain cortex regions [7]. Moreover, the level of lipid peroxidation in hippocampus and frontal cortex from Alzheimer’s tissues is dependent on the apolipoprotein E genotype and level [8, 9]. Protein carbonyls are present in both tangles- and non-tangles-bearing neurons [10] and hippocampal neurons demonstrate intense cytoplasmic staining with 8-hydro-2-deoxyguanosine [11]. Interestingly, F-2 isoprostanes, prostaglandin-like compounds derived from free radical-catalysed peroxidation of arachidonic acid, are also elevated in plasma, urine and cerebrospinal fluid of patients suffering from AD [12]. Elevation of oxidative-induced damages was described in different transgenic mice with selected facets of AD pathology [13]. Further studies on the pathogenesis of AD highlight the involvement of free radical-mediate damages to brain regions from early stages of this illness. The early involvement of oxidative stress in AD is demonstrated by recent studies on cell culture models, on transgenic animal models of AD, on brain post-mortem studies and on biologic fluids from AD and mild cognitive impairment (MCI) subjects. Nunomura et al. [6] found that oxidative damage precedes Aß deposition in brain tissue from Down syndrome. Moreover, subjects with MCI or very mild AD display increaseing levels of lipid peroxidation and nucleic acid oxidation in post-mortem brain tissue [14, 15], in cerebrospinal fluid, plasma, urine and peripheral cells [16, 17]. Other marker of oxidative stress such as heme oxygenase-1 (HO-1) was also upregulated in glial cells from MCI post-mortem brain [18]. These results obtained in human studies are supported by experimental studies using cell culture and transgenic animal models with particular aspects of AD. Indeed, an elevation of lipid and protein oxidation, and a decrease in the activity of Cu/Zn superoxide dismutase activity precede Aß deposition in transgenic mice and C.elegans models of AD amyloidosis [19-22]. On the other hand, oxidative stress induces intracellular Aß accumulation and tau phosphorylation in cell cultures [23-25]. The elevation of Aß following oxidative stress has led to the postulation of an antioxidant role of Aß [26]. However, this possibility has not been rigorously investigated. An increase in the levels of Aß in brain, plaque burden and number was described in partial deficient MnSOD transgenic mice model carrying a double mutation on human APP construct [27]. Studies using transgenic mice model tell us that many different factors could affect the progress of AD, and as such, combination therapies targeting both proteins Aß and Tau, immunologic

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modulation, oxidative stress and lifestyle modifications may be effective treatments. Interestingly, several in vitro and in vivo studies have repeatedly reported that oxidative markers are attenuated by the administration of different polyphenolic compounds with antioxidant activities [28].

2. Classification, bioavailability and metabolism of polyphenols In addition to vitamins and minerals, fruits and vegetables provide a range of non-nutrient constituents that are considered as being at least partly responsible for their health benefits. Fruits, vegetables, beverages (tea, wine, juices), and some herbs are loaded with powerful antioxidants known as polyphenols. These compounds include different chemical families ranging from the very simple p-hydroxy-benzoic acid, to complex oligomers and polymers. Most of polyphenolic compounds are combined with different sugars and aliphatic, aromatic acids, as well as glucuronides and sulphates. These compounds are responsible for colour, flavor, aroma and appearance of fruits and vegetables. Polyphenols constitute a large and ubiquitous group of phytochemicals. They are characterized by having at least one aromatic hydrocarbon ring attached with one or more hydroxyl groups. The simplest one is gallic acid. Polyphenols are mostly found as sugar conjugated and can be broadly divided into two categories i.e. flavonoids and non-flavonoid polyphenols. Flavonoids, the target class of polyphenols may be divided into different subclasses according to the degree of oxidation of the heterocyclic ring: flavonols, flavones, flavan-3-ols, flavanones, anthocyanins and isoflavones. In general they are hydroxylated, methoxylated, and/or glycosylated derivatives. The linked sugar is often glucose or rhamnose. The number of sugar-moieties is commonly one, but could be two or three and there are several positions of substitution on the polyphenol. Flavonols are the most widespread in food kingdom and the main flavonols are quercetin, kaempferol, isorhamnetin, and myricetin. Quercetin, present in many fruits, vegetables and beverages, is the main flavonol in our diet and its mean intake was estimated around 16 mg/day [29]. Flavones are structurally similar to flavonols and differ only in the absence of hydroxylation at the position 3 on the C-ring. The main flavones present in the diet are apigenin and luteolin. They are not widely distributed and are reported to be present in celery, parsley and artichoke. Thus their dietary intake is very low. Flavan-3-ols are widely distributed in the plant kingdom and are found in apricots, cherries, grapes, berries and so on. They are the most complex subclass of flavonoids ranging from the simple monomers such as catechin to

Polyphenols and Alzheimer's disease

the oligomeric and polymeric proanthocyanidins which are also known as tannins. Flavan-3-ols can undergo esterification with gallic acid to form catechin gallates and hydroxylation reactions to form gallocatechins found in green tea. Proanthocyanidins are polymeric flavanols and are usually present in plants. They are responsible for the astringency of food and common sources are apples, pears, grapes, red wine, tea and chocolate. Flavanones are represented mainly by naringenin, eriodictyol and hesperitin. They are highly reactive and are reported to undergo hydroxylation, glycosylation and O-methylation. Flavanones are mainly found in citrus fruit, in the citrus peel, grapefruit, orange and banana and contribute to their flavour. Anthocyanins are pigments responsible for the red, blue and purple colours of many fruits such as berries, grapes, strawberries, plums and cherries. Their contents could vary from 0.15 to 4.5mg/g in fresh fruit. The common anthocyanidins are pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin. Anthocyanidins are abundantly found in berries, cherries and plums. Isoflavones are mainly found in soy, which contains around 1 mg of genistein and daidzein/g dry bean[30]. Both isoflavones have received considerable attention due to their estrogenic-like properties. Phenolic acid, hydroxycinammates and stilbenes are non-flavonoids polyphenolic compounds. The main phenolic acid is gallic acid or hydroxybenzoates. This compound is the base unit of gallotannins. The most common hydroxycinnamates are p-coumaric, caffeic and ferulic acids. Stilbenes are phytoalexins produced by plants in response to attack by fungal, bacterial and viral pathogens. They are not widely distributed and refer exclusively to resveratrol as the content of other stilbenes such as piceatannol, astringin, pterostilbene or viniferins is very low. Dietary sources of resveratrol are scarce. It is present in red wine and peanuts and is present at low levels in some berries and spinach. Red wine content of resveratrol can vary greatly on the wine making process, grape variety, and infections during growing, geographical area and harvesting year. Resveratrol has recently received great attention for its anticarcinogenic properties and for its neuroprotective effect.

3. Several modes of action of polyphenols A large range of polyphenols were thought to protect cells against oxidative damage through their antioxidant property. This was observed on in vitro experiments with cell cultures or cell free systems using various

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methods. While this mode of action remains the focus for many polyphenols, recent data indicate that the protective effect of polyphenols may extend beyond their antioxidant activity. For instance, green tea is rich in flavonoids (30% of dry weight of a leaf) [31] and the main compounds are epigallocatechingallate (EGCG), (-)epigallocatechin (EGC), (-)-epicatechin (EC) and (-)-epicatechin-3-gallate (ECG) [32]. These flavonoids display antioxidants properties in this order EGCG>ECG>EGC>EC [33]. Green tea polyphenols have shown beneficial effects in animal models of stroke/cerebral ischemia, AD and Parkinson’s disease. Neuroprotection in ischemia by EGCG may be mediated through reducing iNOS expression, peroxynitrite formation and preservation of mitochondrial complex activity and integrity [34, 35]. There are substantial in vitro studies that describe the protective effect of different catechins against the Aß induced- damages [36-38]. Different mechanisms could be involved in this neuroprotective effect since in neuronal cell culture, EGCG could promote the non amyloidogenic α-secretase pathway [38] while EC could reduce the formation of amyloid β-fibrils [39]. In primary neuronal cells derived from transgenic mice model overexpressing APP with the mutation Sweden, EGCG significantly reduced Aβ peptide generation (Aβ1-40 and Aβ1-42) by 38% [40] with purified EGCG being more potent than green tea. Green tea catechins, especially ECGC, also modulate a number of signaling pathways such as MAPK [41], protein kinase C [38] and phosphatidylinositol-3-kinase (PI-3 kinase)-Akt [42] and these modulations may mediate some of the neuroprotective mechanisms of EGCG. In neuronal cell line and primary cell culture models, EGCG prevented the decline in ERK1/2 induced by 6-hydroxydopamine or oxidized low-density-lipoproteins [43, 44]. MAPK are also involved in the regulation of the expression of proapoptotic and anti-apoptotic genes. EGCG treated SH-SY5Y neuroblastoma cells have decreased expression of pro-apoptotic genes Bax, Bad, cell cycle inhibitor Gadd45, Fas ligand, and tumour necrosis factor mediated apoptosis ligand TRAIL [43]. EGCG, at doses of 1-10 µM, protected against Aβ peptide and 6-hydroxydopamine-induced cell death by activation of protein kinase C [38] that plays a central role in neuronal cell survival and loss of its activity is frequently observed in neuronal insults such as in the presence of Aβ peptide accumulation and other neurotoxins. EGCG could also prevent LPS-induced neuronal cell death as well as the expression of inflammatory proteins, inducible nitric oxide synthetase and cyclooxygenase-2 [45]. Recently, Wang et al., reported that the consumption of grape seed extract containing catechins prevents Aß deposition and attenuates inflammation in brain from APP(Swe)/PS1 transgenic mice [46]. Finally, the administration

Polyphenols and Alzheimer's disease

of EGCG could prolong the inhibition of the acetylcholinesterase activity induced by huperzine A [47]. In summary, green tea and its active component EGCG exert several intracellular mechanisms relating to neuroprotection. Curcumin is a low molecular weight molecule with potent antioxidant and anti-inflammatory activities. The yellow curry spice is part of Indian vegetables. When fed to aged APP(Swe)/PS1 transgenic mice with advanced amyloid accumulation, curcumin reduced Aß levels and plaques. Interestingly, curcumin also blocked Aβ aggregation and fibril formation in vitro with an IC50 =0.8µM [48] and this property could be implicated in the reduction of amyloid plaques burden observed in vivo after curcumin treatment in APP(Swe)/PS1 transgenic mice. Curcumin is also a good inhibitor of expression of inflammatory cytokines, Cox-2 and iNOS likely by inhibition of JNK/AP-1 and NF-κB mediated gene transcription [49]. All of these factors (IL-1, TNFα, Cox-2, iNOS, JNK, NF-κB) are also implicated in Aβ toxicity [50, 51]. Moreover, curcumin could chelate the redox active metals such as iron and copper [52]. Begum et al., reported that when curcumin was fed to APP(Swe)/PS1 transgenic mice, indices of oxidative stress i.e. oxidized proteins were significantly reduced [53]. Remarkably, recent evidence has demonstrated that curcumin is a potent inducer of HO-1 in vascular endothelial cells [54]. HO-1 induction occurs through the antioxidant response element (ARE) [55]. Recently, Ma et al., demonstrated that mice fed with the combination of fish oil and curcumin for 1 month had significant effects on Y-maze, a cognitive performance test, and the combination induced higher inhibition of JNK and tau phosphorylation [56]. Curcumin was also investigated as an inhibitor of glycogen synthase kinase-3 beta (GSK-3 beta). GSK-3 is a serine/threonine kinase and its name derived from its substrate glycogen synthase (GS) as GSK-3 is a key enzyme involved in a variety of cellular processes ranging from glycogen metabolism, insulin signaling, cell proliferation and neuronal function. Its high expression in brain is associated with a variety of neurological disorders such as AD. There are several GSK-3 inhibitors being developed for AD (see review by [57]). Curcumin was found to fit within the binding pocket of GSK-3 beta via several attractive interactions with key amino acids. Hence, curcumin was found to potently inhibit GSK-3 beta with an IC50 = 66.3 nM [58]. Altogether, curcumin, a highly lipophilic compound, can protect cells against Aß toxicity by preventing Aβ peptide aggregation and by reducing plaques burden, through its antioxidant and anti-inflammatory activities. Moreover, curcuminoids and all individual components also possess a pronounced acetylcholinesterase inhibitory activity [59]. These various pharmacological activities of curcumin indicate that

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curcumin is a promising compound in the development of disease-modifying drugs to prevent and/or cure AD. Resveratrol (trans-3, 4, 5-trihydroxystilbene) is the most relevant and the main biologically active non flavonoid found in grapes and red wine. A number of studies have demonstrated the antioxidant, anti-inflammatory, antimutagenic and anticarcinogenic effects of this compound [60, 61]. Several epidemiological studies indicate an inverse correlation with wine consumption and incidence of AD [62-64]. At cellular levels, resveratrol could protect PC12 cells against Aß-induced toxicity and accumulation of intracellular reactive oxygen species (ROS) [65]. The inhibition of Aß secretion by resveratrol could be implicated in this neuroprotective effect since the secretion of Aß is reduced in two cell lines, HEK 293 and N2a, transfected with APP695 and treated with resveratrol [66]. This effect was not mediated by β and γ-secretases activities but may be through the elevation of degradation of Aβ peptide. Other neuroprotective mechanisms involve the modulation of NF κB/Sirt1 pathways since in vitro and in vivo studies have shown that resveratrol is a specific activator of Sirt1 [67, 68]. This property could be implicated in the protective effect against Aß involving the inhibition of the NF-κB activity [69]. In cultured PC12 cells, resveratrol could also upregulate HO-1 gene expression via the activation of NF-E2-related factors 2 (NRf2) and protected against H2O2 induced cell death [70]. APP(Swe)/PS1dE9 transgenic mice fed with 2% of grape seed extract containing 592.5 mg/g of total phenolic, at 3 months of age for 9 months, display a significant reduction of brain Aß burden and microglia activation [71]. In summary, in addition to its antioxidants effects, the efficacy of resveratrol and grape seed extract against Aß toxicity involves several pathways. Thus resveratrol and phenolic compounds from grape seeds are promising compounds in delaying the development of AD. Their bioavailability needs to be addressed due to its rapid metabolization in liver and intestinal epithelials cells. Moreover, the efficacy of these compounds in the treatment of AD will also depend on the extent to which resveratrol metabolites become bioavailable. Several dietary supplements with either spinach, strawberries or blueberries extracts have been reported to reduce some neurological deficits in aged animal models [72-74]. In blueberries (Vaccinium ashei reade), catechin was the major flavonoid found with 387 mg/100 g fresh weight, epicatechin ranged from 34 to 129 mg/100 g fresh weight and total anthocyanins ranged from 84 to 113 mg/100g fresh weight [75]. It has estimated that 0.543-1.69 mg/L of total anthocyanins was present in human serum after a consumption of 100g of blueberries containing 1.20g of total anthocyanins and maximal level was reached 4 hours after the

Polyphenols and Alzheimer's disease

consumption. Interestingly, a significant positive correlation between serum anthocyanin content and postprandial antioxidant status has been observed [76]. This absorption could have some positive effects in the brain through different processes as it has been demonstrated in different animal studies. Thus, dietary supplementation for 8 weeks with blueberries extracts reversed cognitive deficits in Morris water maze performance test in 19-month-old rats [77]. These results could be correlated to the presence of different classes of polyphenols in brain areas associated with cognitive performance following blueberry supplementation. Thus, several anthocyanins (cyanidin-3-Obeta-galactoside, cyanidin-3-O-beta-glucoside, cyanidin-3-O-beta-arabinose, malvidin-3-O-beta-galactoside, malvidin-3-O-beta-glucoside, malvidin-3-O-betaarabinose, peonidin-3-O-beta-arabinose and delphinidin-3-O-beta-galactoside) were found in the cerebellum, cortex, hippocampus or striatum from the 19 month-old rats supplemented with 2% of blueberry extract for 8-10 weeks [78]. These findings indicate that some polyphenolic compounds are able to cross the blood brain barrier and localize in various brain regions important for learning and memory. It is now well established that the effect of blueberries extracts on cognitive functions might involve more than their antioxidants actions. Thus, aged rats with blueberries extracts diet had significantly lower levels of NF-κB than aged control diet [79]. These results are in accordance with the known effect of flavonoids on cell signaling such as on the activity of NF-κB [80, 81]. Additional evidence was seen in a recent study with the double APP(Swe)/PS1 transgenic mice model, in which genetic mutations promote the production of the Aß peptide and hallmark of AD-like senile plaques in several regions. When these mice were supplemented with blueberries extracts (2% of diet) from 4 months and continued until 12 months of age, their performance in a Y-maze test is similar to that of non transgenic mice and significantly better than that of non-supplemented transgenic mice [82]. However the examination of the brain of these mice revealed that blueberries extracts supplementation did not affect the Aß peptide production or deposition or the number of plaques. These data suggest that the impairment of cognitive functions observed in these transgenic mice may not necessarily be the result of deposition of the Aß peptide. In these mice supplemented with blueberries extracts, the activities of hippocampal ERK as well as striatal and hippocampal protein kinase C are higher than in transgenic mice supplemented with control diet. Both protein kinase C and ERK have been shown to be involved in early and late stages of memory formation [83, 84]. These results indicate that blueberries extracts supplementation might prevent cognitive deficits through neuronal signaling pathways. Diet supplemented with blueberries extracts could also protect the brain against apoptosis as rats

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receiving blueberries extracts had significantly lower caspase-3 activity in the ischemic hemisphere [74]. Taken together, these studies demonstrate that blueberries extracts-supplemented diets could protect neuronal loss and prevent the decrease of cognitive functions against different insults through the antioxidant, anti-apoptotic activity and regulation of some cell signaling pathways. Pomegranates (Punica granatum L.) contain very high level of polyphenols as compared to other fruits and vegetables [85]. The most important polyphenols are ellagic acid, punicalagin and hydrolysable tannins such as ellagitannins and gallotannins. Recently, the administration of pomegranate juice (PJ) to APP(Swe)/PS1 transgenic mice from 6 to 12.5 months of age exhibited improvements in cued and spatial learning tasks as compared to sugar water control [86]. Additionally, PJ-treated mice had significantly reduced burden of plaque load and soluble AÎ˛ 1-42 in hippocampus. Grape juice is also a rich source of flavonoids that include catechins, epicathechins, quercetins, anthocyanins, and proanthocyanidins [87]. When aged Fisher 344 rats were given 10% or 50% of grape juice from 19 to 21 months of age, their performance motor functions in rod walk and cognitive performance on the Morris water maze were improved [88].

4. Bioavailability and metabolism of polyphenols Polyphenols are common in our diet and are good for health. This has powered the interest in determining their biological activity. Their bioaccessibility, bioavailability and metabolism are not well established and should be taken into consideration in all studies regarding the determination of the biological activity of polyphenols. Bioaccessibility refers to the amount of potentially absorbable form of ingredients into the body whereas the bioavailability refers to the amount of active ingredients actually absorbed into the body. The bioavailability is tremendously dependent on the bioaccessibility while the bioaccessibility is influenced by the chemical structure of the active ingredients. Most of them have low intrinsic activity or are poorly absorbed from the intestine, highly metabolized or rapidly eliminated. Furthermore, the metabolites that are found in blood and organs may differ from the native substances. Extensive research regarding the bioavailability of polyphenols and their metabolites is required if their health effects are to be understood. Glycosylation influences the chemical, physical and biological properties of the polyphenols and their absorption by small intestine [89, 90]. If the phenolic compounds contain a sugar molecule such as glucose and galactose, they are absorbed through the small intestine by the

Polyphenols and Alzheimer's disease

hydrolytic activity of cytosolic Î˛-glucosidase/lactase phorizin hydoxylase. Those linked to rhamnose moiety are degraded by the action of rhamnosidases by colonic microflora. The same applies to the polyphenolics linked to arabinose or xylose, although this has not been specially studied. As a general rule, glycosides with rhamnose are absorbed less rapidly and less efficiently than aglycones and glucosides. In the case of quercetin glucosides, absorption is higher than that of aglycone itself [89, 90]. Acylated flavonoids such as epicatechin and epigallocatechin are absorbed without deconjugation and hydrolysis [90]. Proanthocyanidins are very poorly absorbed and may exert local activity in the gastrointestinal tract or activity mediated by phenolic acids produced through the microbial degradation. Hydroxycinnamic acids, when ingested in the free form, are rapidly absorbed from small intestine and are conjugated or glucuronidated in the same way as flavonoids are [91]. Once absorbed, polyphenols are subjected to 3 main types of conjugation: methylation, sulfation and glucuronidation. Catechol-O-methyl transferase (COMT) catalyses the transfer of methyl group to polyphenols having a catechol moiety. Quercetin, catechin, caffeic acid, and luteolin undergo methylation. Sulfotranferases and UDP-glucurnosyltransferase carry out sulfation and glucuronidation, respectively. Sulfation is generally a higheraffinity, lower capacity pathway than is glucuronidation, so when the ingested dose increases, a shift from sulfation towards glucuronidation occurs [92]. Regardless of the respective contributions of methylation, sulfation, and glucuronidation, in general, the capacity for conjugation is quite high. The concentration of polyphenols is usually very low in plasma after the intake of nutritional dose, except for tea catechins (up to 77% for EGCG) [93]. Plasma concentration and half life reached after polyphenol consumption vary highly according to the nature of polyphenols and the food source. For instance, resveratrol has a short initial half-life (8-14 minutes for the primary molecule) [94, 95] and is metabolized extensively in the body. Wall et al., [96] showed that the bulk of an intravenous dose of resveratrol is converted to sulphate conjugates within 30 minutes in human. Total sulphate conjugates accounted for 37% of the metabolites in the urine and total glucuronide conjugates for 19%, with the remainder being made up of unknown metabolites. Half-life of the total resveratrol metabolites was 9.2 hours. Several metabolites retain the ability to activate Sirt1 and inhibit cyclooxygenase in vitro (A.Mesecae, personal communication). About 40-85 % of ingested curcumin is absorbed in the gastrointestinal tract, most being metabolized in the intestinal mucosa and liver [97] but it has a short half life of only 40 minutes [98]. Curcumin undergoes O-glucuronidation and sulfation and is also reduced to tetrahydrocurcumin, hexahydrocurcumin and hexahydrocurcuminol in rats and mice [99]. Certain curcumin metabolites, such as tetrahydrocurcumin, possess anti-inflammatory

Charles Ramassamy et al.

and antioxidant activities similar to those of their metabolic progenitor. Green tea polyphenols especially EGCG, are well absorbed in unconjugated form and plasma half life of EGCG is 173 minutes after intravenous injection [100]. Thus, bioavailability and pharmacokinetics of polyphenolics is governed by plethora of factors i.e. its native form (glycosylated/aglycone) and the type of sugar moiety present and its physiochemical properties. Moreover some of the metabolites still possess inherent biological activities of their progenitor molecule.

5. Some challenges for research on polyphenols The state of knowledge in this field has progressed the last few years with recent progress in analytical techniques such as mass spectrometry. In addition, one of the major difficulties of elucidating the beneficial effects of polyphenols is the large number of polyphenolic compounds found in fruits, vegetables and beverages and even larger numbers of their metabolites. Moreover, their bioavailability differs from one consumer to another in addition to intraindividual response occurring with physiopathological conditions. One of the major challenges for the next coming years is to understand the conjugation of polyphenols with the phase II enzymes, to identify the concentration of polyphenols and their metabolites reached in plasma and in target cells. Apart from appearance, flavor and texture, another important indication of quality in fresh-cut fruits and vegetables is the nutritive value. These quality parameters depend upon the cultivar, preharvest cultural practices, climatic conditions, maturity of harvest and harvesting method. Time between harvest and preparations have a major impact on the quality of foods. Postharvest treatment, handling and storage can have a large influence on the phenolic content in fruits and vegetables because the biosynthesis of anthocyanins in some fruits (i.e. berries) tends to continue after harvest and during storage. The effect of temperature on the rate of anthocyanins formation during storage may also depend on the maturity of the fruits at harvest. Different postharvest approaches have been considered to increase resveratrol level in grapes. For instance, an increasing of the biosynthesis of stilbenoid during storage at 22oC and 95% room humidity could be obtained with exposure to ozone or UV-C [101]. Storage of berries at high temperature (15-25oC) increased the levels of anthocyanins. For instance, raspberry pulp stored at 4oC for 90 days retained much higher levels of total anthocyanins than product stored at 20oC or 37oC. Leak of water-soluble polyphenols occurred during storage of fruits packed in liquid canning. For instance, in canned peaches stored for 3 months procyanidin

Polyphenols and Alzheimer's disease

monomers, dimers, trimers and tetramers decreased by 10, 16, 45 and 80%, respectively [103]. Also consumer preferences influence the health promoting effect of the intake of polyphenols. For instance, peeling apples and peaches before consumption will reduce the intake of polyphenols. In some cases, peeling will eliminate most of flavonols that are exclusively located in the peel. Discarding the seeds of grapes will decrease the intake of antioxidants. The way in which oranges fruits are peeled can also affect the intake of flavonones as these compounds are mainly present in the albedo (the white tissue between the flavedo and the fruit). Another consumer preference that influences phytochemical intake is steam cooking vegetables which always preserves higher content of phytochemicals as compared to boiling and discarding the water. Carrot purees thermally processed with periderm tissue contained higher levels of phenolic acids, total carotenoids, and sugars than samples processed without periderm tissue [102]. Thus, the polyphenols intake can vary 10-fold depending on consumer choices and culinary habits. Nutritional recommendations in the majority of developed countries encourages increasing the consumption of fruit and vegetables (five per day). However, the consumption of fruits and vegetables remains below the recommended levels in many countries because the notion of portion needs to be clearly indicated. Although considerable progress has been made in understanding the beneficial effects of polyphenols components from fruits and vegetables in AD, there are still important gaps in our knowledge concerning the biology and chemistry of these compounds. Future studies should aim to enhance our knowledge of the roles and functions that the compounds display at the cellular and molecular levels and on the bioactivities of different metabolites including glucuronidated, sulfated and methylated derivatives. Moreover, the product formed from the action of colonic microflora on polyphenolic components may also contribute health benefits by their consumptions. The consequence of the interactions of the compounds with the food matrix should be investigated. Moreover, the synergistic action between different polyphenols should be investigated. Finally, future research in this area should analyze the gene-nutrient interactions.

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Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Recent Advances on Nutrition and the Prevention of Alzheimer’s Disease, 2010: 19-40 ISBN: 978-81-7895-470-7 Editors: Charles Ramassamy and Stéphane Bastianetto

2. Diet and Alzheimer’s disease: New evidence from epidemiological studies C. Féart, C. Samieri and P. Barberger-Gateau «Nutritional epidemiology » team, INSERM, U897, Bordeaux, F-33076 France; Univ Victor Segalen Bordeaux 2, Bordeaux, F-33076, France

Abstract. Nutrition offers promising perspectives for the prevention of Alzheimer’s disease (AD) and, more generally, cognitive decline. Several cohort studies have recorded dietary behaviour and then documented cognitive decline and incidence of dementia through repeated neuropsychological testing over many years of follow-up. They have yielded increasing evidence for a protective role of antioxidants, homocysteine-related vitamins (vitamin B12 and folate) and n-3 poly-unsaturated fatty acids (PUFA) against AD. Fruits and vegetables are major providers of anti-oxidant compounds and folate. Several observational epidemiological studies have evidenced an inverse association between higher consumption of vegetables and lower risk of cognitive decline or dementia, whereas the association with fruit was less consistent. However, intervention studies of the prevention of cognitive decline with anti-oxidant supplements, most of which were at very high dosages, have shown disappointing results. A regular consumption of fish also seems to exert protective effects which may be attributed to eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), two long-chain n-3 PUFA. Conversely, an imbalanced diet with excessive intake of n-6 PUFA is associated with a higher risk of dementia. Despite very convincing results from several observational epidemiological studies, the single intervention Correspondence/Reprint request: Dr. Catherine Féart, INSERM U897, University of Bordeaux 2, 146, rue LeoSaignat, F-33076 Bordeaux, France. E-mail: Catherine.Feart@isped.u-bordeaux2.fr

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trial for the primary prevention of dementia by n-3 PUFA has yielded negative results. The analysis of dietary patterns is in favor of a synergy between dietary sources of n-3 PUFA and anti-oxidants. Further research is needed to better identify the joint mechanisms of action of fatty acids and anti-oxidants, the optimal dosages, and to understand the interaction with genetics before implementing nutritional interventions in targeted individuals.

Introduction With the aging of the population, pathological brain aging has become a major public health concern [1]. More than 5 % of the population aged 65 and over suffer from dementia and its prevalence steadily increases with aging. Alzheimer’s disease (AD) is the most frequent cause of dementia, accounting for 50–60% of all cases [2]. Unfortunately, there is presently no causal treatment of AD and its main risk factor, beside age, is genetic since possession of at least one ε4 allele of the apolipoprotein E (ApoE4) gene increases by about 4-fold the risk of late-onset AD. These factors offer of course no possibility of prevention. There is therefore a need to identify environmental risk factors on which we could act to decrease the risk of AD. As neuro-degeneration in AD is estimated to start 20–30 years before clinical onset [2], there is a large window for prevention in order to slow down the pathological process or reinforce non specific neuro-protection. The dementia stage is preceded by a long phase of progressive cognitive decline with minimal impact on daily living named “Mild Cognitive Impairment” (MCI). At this stage, the cognitive deficits are still potentially reversible [3], and therefore susceptible to secondary prevention. Nutrition offers promising perspectives for the prevention of AD and, more generally, cognitive decline [4]. Indeed, the role of nutrition in cardiovascular disease is well documented [5] regarding in particular the protective effect of poly-unsaturated fatty acids of the omega 3 series (n-3 PUFA) [6]. We could therefore expect a protective effect of the same nutrients against vascular dementia but also against the vascular component of AD. A healthy diet could then contribute to simultaneously decrease the risk of several conditions whose incidence sharply increases with aging. The brain is also particularly susceptible to oxidative stress because of, on one hand, its high content in easily peroxidizable long-chain PUFA, in particular DHA, and on the other hand, the high level of in-site production of free radicals. In AD patients, the accumulation of the β-amyloid protein is associated with increased free radical production and increased lipid peroxidation [7]. Important oxidative damage has also been observed in subjects with MCI, suggesting an early role of oxidative stress [8].

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As described in other chapters of this book, in vitro and animal studies have yielded increasing arguments for a protective role of anti-oxidants, homocysteine-related vitamins (vitamin B12 and folate) and n-3 PUFA against AD. However, epidemiological studies are necessary to assess the impact of these nutrients in humans. A task force of the International Academy on Nutrition and Aging recently published a comprehensive review of epidemiological studies on nutrition and cognitive decline [9]. In this chapter we will update this review with recent epidemiological findings, show the convergence or inconsistence of results, and suggest new directions for future research. In order to better understand some potential discrepancies, this chapter will begin with a brief reminder of the contribution and pitfalls of nutritional epidemiology applied to the relationships between nutrition and AD.

1. What contribution of epidemiological studies? 1.1. Study design: Observation vs. intervention Epidemiological studies yield an increasing degree of evidence depending on their design. In observational studies, the spontaneous dietary behaviour of the participants is recorded and linked to their health status. Cross-sectional studies are of little interest since they cannot ascertain causality, i.e. is the observed dietary behaviour the cause or the consequence of cognitive impairment. In casecontrol studies, past dietary behaviour of AD patients is compared to that of cognitively normal controls. The bias is obvious in a disease whose main symptom is memory impairment. Longitudinal studies are still scarce. Indeed, few cohort studies in the world have recorded dietary behaviour at baseline and then documented cognitive decline and incidence of dementia through repeated neuropsychological testing over many years of follow-up. Most of these studies were conducted in Europe (mainly France, the Netherlands, Scandinavia and Italy) and North-America. Observational studies are subject to measurement errors in reported dietary intake and residual confounding bias by lifestyle. In intervention studies, the best scheme is the randomized controlled trial (RCT) where participants are randomized to one or several intervention arms or to a control arm. Participants in each arm are therefore similarly distributed for each known or unknown characteristic except the intervention to be tested. The intervention may try to modify the spontaneous dietary behaviour of the participants in a pragmatic way. Alternatively, the intervention arm may consist in nutritional supplements, either in combination or alone, at nutritional dosages. In that case, the aim of the intervention will be to identify which specific nutrients have protective

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effects in order to better target further dietary recommendations. The choice of the kind of intervention also depends on the target: primary prevention, secondary prevention (at the MCI stage) or treatment of dementia, in adjunction to classical anti-dementia drugs. In the latter case, supplements are given at very high pharmacological dosages. This chapter will therefore focus on dietary intake, not supplements, except for nutritional intervention studies which are still very scarce in the field of dementia.

1.2. Variables: From dietary patterns to the bioavailability of nutrients Several sets of variables are necessary to assess the impact of nutrition on brain aging in epidemiological studies (figure 1). The main explanatory variable is dietary behaviour in terms of frequency of food consumption as recorded by Food Frequency Questionnaires (FFQ). However, foods are not consumed in isolation but combined in various dietary patterns which reflect lifestyle. Similarly, nutrients are associated in foods and even more in dietary patterns where they can exert cumulative, synergistic or opposite effects which makes the study of their individual contribution quite problematic. Dietary patterns combine multiple dietary components as a single exposure [10]. Techniques for identifying dietary patterns include a priori methods, based on previous knowledge and recommendations on “healthy foods” (e.g. the Mediterranean diet), or a posteriori methods where combinations of foods Confounding factors: Age, gender, education, income, lifestyle, vascular risk factors, genetics…

Exposure:

Outcome:

Diet

Cognitive health status

- Foods

- Incidence dementia, Alzheimer

- Dietary patterns

- cognitive decline

- Dietary nutrients

(neuropsychological tests)

- Bioavailable nutrients (plasma, membranes, tissues…)

- neuro-imaging: MRI, SPECT… - neuro-pathology

Mechanisms? - energy: obesity, metabolic syndrome.. - vascular: cholesterol, triglycerides… - inflammation: cytokines - oxidative stress: isoprostanes… - nuclear receptors: PPARs… - neuroprotection

Figure 1. Variables to be recorded in epidemiological studies of dietary factors in dementia and cognitive decline.

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associated with positive or negative outcomes are identified by statistical techniques. Given the lack of previous knowledge on the specific protective effect of food on cognitive decline or dementia, the latter method is the most suitable to study the role of diet in brain aging. However, the specific role of nutrients must also be individualized in order to verify the underlying physio-pathological assumptions, to establish biological plausibility and causality, and to contribute to the conception of functional foods or supplements whose impact could be definitely ascertained in RCT. Quantitative data derived from FFQ or food records or 24 h-dietary recalls can be converted into nutrients by the use of adequate food composition tables. Several causes of measurement error can therefore add up: error on portion size, nutrient content of the food, day-to-day intra-individual variability. If quantitative methods can provide a fairly good estimation of mean nutrient intakes, the estimation of their variance is much more prone to bias. The combination of quantitative and qualitative (food frequency) data is therefore necessary. Macronutrients also provide energy, in particular lipids with 9 kcal/g. The higher the total lipid intake of an individual, the higher will also be his or her energy intake. An excessive amount of energy intake leads to obesity which was shown to be associated with increased dementia risk [11]. The problem also arises for micronutrients which are consumed in combination with lipids such as fat-soluble vitamins A, D, E and K for instance. The adverse effects of high energy intake can then counterbalance the expected protective effects of some nutrients. Adjustment for total energy intake is therefore necessary [12]. Measuring dietary intake does not provide information about the real bioavailability of nutrients in the body. Many factors can interact to compromise or enhance the absorption of nutrients in the digestive tract. Biological data are also necessary in various compartments of blood and tissues in order to assess the bioavailability or incorporation of nutrients and their mechanisms of action. Finally, in observational studies the many potential confounding factors linked to both the outcome of interest and dietary behaviour should also be recorded in order to be accounted for by statistical adjustment (figure 1). For the study of dementia they include gender, education, income, genetics (apoE4), vascular risk factors, and lifestyle [13].

2. Dietary anti-oxidants and risk of dementia or Alzheimerâ&#x20AC;&#x2122;s disease 2.1. Dietary anti-oxidants The main dietary antioxidant is vitamin E, a fat-soluble vitamin mostly found under the forms of alpha- and gamma-tocopherol. Dietary vitamin E is

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mainly brought by vegetable oils, in particular walnut and sunflower oils, seeds, and wheat-germ. The Dietary Reference Intake (DRI) is 15 mg (equivalent of 22.5 IU of natural vitamin E)/day of α-tocopherol for adults (Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids, Institute of Medicine, 2000). Vitamin C is a hydrophilic vitamin abundant in many fruits (e.g. citrus, kiwi…) and vegetables (e.g. pepper, broccoli, cabbage…). The DRI are 90 mg/day for adult men and 75 mg/day for adult women. Carotenoids are a vast family of anti-oxidant molecules including pro-vitamin A carotenoids (alpha- and beta-carotene, betacryptoxanthin), but also lycopene and xanthophylls (e.g. lutein, zeaxanthin…) which have no vitamin A activity. There are no DRI for carotenoids since total intake must also consider dietary retinol. Carotenes are found in coloured fruits and vegetables while xanthophylls are mainly provided by green vegetables, corn, egg yolk and lycopene by tomato. The contribution of polyphenols to the anti-oxidant capacity of the human diet is much larger than that of vitamins [14]. Polyphenols occur in all plant foods and may contribute to the beneficial health effects of fruit and vegetables. They can be divided in several sub-classes including nonflavonoid compounds, such as lignans, and flavonoids. Important dietary sources of flavonoids in Western societies are onions (flavonols); cocoa (proanthocyanidins); tea, apples, and red wine (flavonols and catechins); citrus fruit (flavanones); berries and cherries (anthocyanidins); and soy (isoflavones) [14]. Vitamin C, some carotenoids and phenolic antioxidants also contribute to the regeneration of vitamin E. Finally, several elements which act as cofactors of anti-oxidant enzymatic defence systems glutathione peroxidases, Cu/Zn superoxide dismutase and catalase are also found in the diet such as selenium, zinc, and manganese.

2.2. Epidemiological data Fruits and vegetables are therefore major providers of anti-oxidant compounds. Several recent observational epidemiological studies have evidenced an inverse association between higher consumption of fruits or vegetables and lower risk of cognitive decline or dementia (table 1). Most studies found a protective association against AD or cognitive decline with vegetable consumption, whereas the association with fruit consumption was less consistent. Some studies have also linked dietary intake of specific anti-oxidant nutrients and risk of AD or cognitive decline (table 2). However, their results are hampered by the inclusion of supplement users who have considerably higher total intakes of anti-oxidants. As discussed above, supplement users

Table 1. Main prospective observational epidemiological studies relating fruit or vegetable consumption and risk of cognitive decline or AD.

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Table 2. Main prospective epidemiological studies relating dietary intake of antioxidant nutrients and risk of dementia or cognitive decline.

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may be particularly concerned by their health status and prone to adopt a general healthy lifestyle. On the other hand, high-dose supplements may be more harmful than beneficial [15] and therefore mask the protective effect of dietary antioxidants. Another interesting point is the inverse association between dietary flavonoid intake and risk of dementia or cognitive decline observed in the Personnes AgĂŠes QUID (PAQUID) and Rotterdam studies. Tea is an important source of polyphenols in some populations. Recently, a cohort study of older Chinese adults, the Singapore Longitudinal Ageing Studies, reported an inverse association between risk of cognitive decline and the quantity of tea drank [16]. Nutritional biomarkers of anti-oxidant intake are also correlated with dementia or cognitive decline. Low plasma vitamin E was linked to increased risk of incident dementia or cognitive decline in several studies [17, 18] while a U-shaped relationship was observed in another study [19]. However, plasma concentrations of fat-soluble compounds such as vitamin E or carotenoids depend on genetic polymorphisms in genes involved in lipid metabolism, in particular lipoproteins [20]. More specifically, an interaction with the ApoE genotype suggests a functional vitamin E deficiency in ApoE4 patients with AD [21]. Several cross-sectional studies found lower plasma carotenoids in patients with MCI or AD [22-24]. In the Rotterdam study, less peri-ventricular white matter lesions were observed in participants with higher serum carotenoids, in particular zeaxanthine [25]. Two different longitudinal studies found less cognitive decline over 7 years in participants with higher dietary consumption of carotene [26] or higher serum betacarotene [27] but this relationship was observed only if they were ApoE4 positive in the latter study. Despite their limitations, these observational studies suggest a protective effect of vitamins E, C, flavonoids and carotenoids from food sources against AD and cognitive decline. However, the interactions with smoking or apoE genotype deserve further research. Several RCT have been conducted for the primary prevention of cognitive decline with anti-oxidants, most of which at very high dosages far above the DRI. They have produced disappointing results. The first doubleblind placebo controlled trial was conducted in 185 healthy volunteers followed-up for 1 year [28]. This study did not show any effect of a combination of several anti-oxidants. A sub-study of the Age-Related Eye Disease Study showed no protective effect of an association of vitamins C, E, beta-carotene, zinc and copper against cognitive decline in 2166 participants followed-up for 6 years [29]. More recently, an ancillary study of the Womenâ&#x20AC;&#x2122;s Health Study showed no global effect of the supplementation with vitamin E at high dosages (600 IU on alternate days) on cognitive decline

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over 4 years, except in a subgroup of women with dietary intake below the median of the group, which was very low in this study [30]. In men, the Physician’s Health study combined two trials: the first one with 18 years of follow-up (N=4052) and a new one with only one year of follow-up (N=1904) [31]. This trial showed a protective effect of beta-carotene (50 mg on alternate days) only in the group with the longest follow-up. However, many participants were lost to follow-up or deceased in that group, which might be attributed to higher mortality rates associated with beta-carotene supplementation [15]. Many anti-oxidants (polyphenols, some carotenoids) are found in vegetables which are also rich in folate such as green leafy vegetables. Low folate intake is associated with raised plasma homocysteine, a risk factor for dementia and AD [32]. However, observational studies have yielded very conflicting results regarding the potential protective effect of folate against cognitive decline [33]. Despite these inconsistencies, a recent review identified 14 intervention trials with vitamins B6 or B12 or folic acid either alone or in combination [34]. Some of these studies evidenced a homocysteine lowering effect, but they failed to show any effect on cognitive functioning. Nevertheless, some hope comes from the most recent study in which 800 µg folic acid were given daily to subjects aged 50 to 70 with elevated homocysteinemia but normal vitamin B12 [35]. In these subjects, supplementation was associated with lower rate of decline on tests of memory and cognitive speed compared to placebo.

3. Dietary fats and risk of dementia or Alzheimer’s disease 3.1. Mechanisms of action The putative mechanisms of action of fatty acids in brain aging are examined in depth in other chapters of this book and will be only briefly reminded here. Most dietary fat is under the form of triglycerides which also account for about 95% of fat in the body. Triacylglycerols are formed from a molecule of glycerol combined with three fatty acids. Fatty acids are classified according to their number of double bonds as saturated (no double bond), mono-unsaturated (a single double bond) or poly-unsaturated (several double bonds) fatty acids. There are two families of PUFA: the omega 3 (or n-3) and the omega 6 (or n-6) named on the basis of the position of their last double bond on the carbon chain. In each family, there is a precursor or essential fatty acid with 18 carbon atoms which cannot be synthesized by human beings and must therefore be found in the diet, mainly in vegetable oils and seeds. From these precursors, animals and human beings can

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synthesize long-chain derivatives. The same elongase and desaturase enzymes are involved in the n-3 and n-6 series and there is therefore a competition between the two families of PUFA for the biosynthesis of longchain derivatives. In the n-6 family, the precursor is linoleic acid (LA) mainly found in sunflower and grape seed oils, from which arachidonic acid (AA, 20 carbons with 4 double bonds) is obtained. Dietary sources of AA include meat and eggs. In the n-3 series, the precursor is alpha-linolenic acid (ALA) found in canola, walnut and soya oils, and in flaxseed. The main long-chain n-3 derivatives include eicosapentaenoic acid (EPA, 20 carbons and 5 double bonds) and docosahexaenoic acid (DHA, 22 carbons and 6 double bonds). However, the rate of conversion of ALA to EPA and then to DHA is very low [36]. A dietary supply of EPA and DHA by fatty fish (e.g. salmon, tuna, herring, mackerel…) is therefore necessary. The n-3 and n-6 families have opposite biological effects. In addition to their role in the composition and fluidity of neuron membranes [37] and their vascular properties [38, 39], several mechanisms can be proposed to explain the effects of PUFA on cognition. Firstly, the role of PUFA in inflammation could explain their effect in brain aging. Most mediators formed from EPA and DHA are anti-inflammatory, whereas those formed from AA are proinflammatory [40]. Many of the effects of n−3 and n−6 fatty acids are also exerted through altered gene expression via their binding to the nuclear peroxisome proliferator-activated receptors (PPAR). Nuclear receptors are a family of ligand-activated transcription factors that directly and indirectly control several genes of lipid metabolism and inflammatory signalling [40]. DHA is also the precursor of neuroprotectin D1 which is a potent regulator of an intrinsic neuroprotective, anti-inflammatory, and anti-apoptotic geneexpression program that promotes survival in stressed human brain cells [41]. A balance between dietary intake of n-3 and n-6 PUFA is therefore necessary.

3.2. Dietary intake Several epidemiological studies have evidenced an inverse association between fish consumption and risk of incident dementia or cognitive decline (table 3). The Rotterdam Study was the first to show such a protective relationship [42]. However, their follow-up was limited to 2 years on average and no relationship with any type of dietary fat was found with a longer follow-up of the same cohort. The lower risk of dementia or cognitive decline in fish consumers was nevertheless confirmed in several independent cohort studies (table 3). Interestingly, consumption of fatty fish, but not fried fish, was also associated with lower prevalence of subclinical infarcts and white

Table 3. Main prospective observational epidemiological studies relating dietary fat consumption and risk of dementia or cognitive decline.

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Table 3. Continued

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matter abnormalities on MRI examinations in older participants in the Cardiovascular Health Study [43]. Some of these studies also evidenced an inverse association between consumption of EPA or DHA and risk of AD or cognitive decline (table 3). Conversely, an increased risk of dementia or cognitive decline was associated with higher consumption of saturated or trans fat. Dietary fat intake at midlife also affects cognitive performance and occurrence of MCI later in life. In the Cardiovascular risk factors, Aging and Dementia (CAIDE) study, higher saturated fat intake from milk products and spreads at midlife was associated with poorer global cognitive function and prospective memory and with an increased risk of MCI at older ages [44]. Conversely, frequent fish consumption was associated with better global cognitive function and semantic memory whereas higher dietary PUFA-toSFA ratio was associated with better psychomotor speed and executive function 20 years later in the same study. Two studies found an interaction with the apoE genotype, the protective effect of fish consumption being observed only in those who did not have the ApoE4 allele [45] [46]. Conversely, in the Washington Heights-Inwood Columbia Aging Project (WHICAP) a deleterious effect of total energy and total fat intake on risk of AD was observed only in those who were ApoE4 positive [47].

3.3. Biological data Few epidemiological studies have investigated the relationship between fatty acid profiles in plasma or erythrocytes membranes and risk of dementia or cognitive decline. Cross-sectional studies provide limited evidence since they cannot ascertain causality [48] [49]. Longitudinal studies are still scarce and they yielded divergent results. In the Framingham Heart Study, the top quartile of plasma Phosphatidyl Choline DHA level was associated with a significant 47% reduction in the risk of developing all-cause dementia [50]. This quartile corresponded to a dietary intake of 180 mg DHA per day or about 3 servings of fish per week. In the Three-City (3C) study, higher plasma EPA but not DHA was associated with lower incidence of dementia independently of depressive status [51]. Conversely, higher AA-to-DHA and n-6-to-n-3 ratios were related to increased risk of dementia particularly marked in depressive subjects [51]. In the Atherosclerosis Risk in Communities Study, higher total EPA plus DHA reduced the risk of decline in verbal fluency over 10 years whereas the risk of global cognitive decline increased with elevated palmitic acid in plasma in cholesteryl esters and phospholipids, and with high AA and low LA in cholesteryl esters [52].

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In the placebo arm of the FACIT trial, higher plasma n-3 PUFA proportions were associated with lower decline in the speed-related cognitive domains over 3 years [53]. The EVA study found an inverse association between the DHA proportion and total n-3-to-n-6 ratio in red blood cell RBC membranes and cognitive decline [54]. A single study found an interaction with the ApoE genotype, where lower cognitive decline was associated with higher total erythrocyte n–3 PUFA content only in the absence of the ApoE4 allele [55]. The Canadian Study of Health and Aging produced unexpected results, since subjects who developed cognitive impairment had higher mean relative concentration of EPA than normal controls and those who developed dementia had higher mean relative concentrations of DHA [49]. Such discrepancies can be explained by several methodological differences between studies. The protective effect of plasma EPA or DHA observed in most studies may be related to higher fish and n-3 rich oils consumption. However, only 25% of the variation in plasma n-3 PUFA was explained by fish and fish-oil consumption in the European Prospective Investigation into Cancer-Norfolk United Kingdom cohort [56]. Plasma PUFA levels reflect a shorter-term lipid intake than do PUFA from RBC membranes [57]. Since plasma PUFA turnover is more rapid than of the RBC, direct measures of PUFA in plasma may better reflect their bio-availability as precursors of other active molecules such as eicosanoids, in particular for EPA. The main effect of n−3 PUFA on plasma lipids is a reduction of the concentration of plasma triacylglycerols [58]. Analyses must therefore be adjusted for plasma triacylglycerol concentration, which was not always the case in published studies and may also explain some discrepancies. Plasma antioxidants could also confound the relationship between plasma PUFA and dementia, since they may exert a protective effect on dementia by decreasing lipid peroxidation [7]. Adjustment for plasma vitamin E as a marker of overall antioxidant status [21] or for intake of fruits and vegetables which are good sources of antioxidants is therefore necessary in observational studies.

3.4. Intervention studies Despite very convincing results from several observational epidemiological studies showing a protective effect of dietary consumption of fish or n-3 PUFA, a single intervention trial for the primary prevention of dementia by an association of EPA and DHA was recently published [59]. In that study, cognitively healthy older participants were randomly assigned to receive 1,800 mg/d EPA+DHA, 400 mg/d EPA+DHA, or placebo capsules for

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26 weeks. There were no significant differential changes in any cognitive domain among the three groups at either 13 or 26 weeks except slightly more decline in memory at 13 weeks in the 400 mg group than the placebo, which might be attributed to chance. An interaction with ApoE genotype was observed, i.e. ApoE4 carriers in both fish oil groups showed an improvement on the cognitive domain of attention after 26 weeks of intervention compared with placebo. However, such post-hoc analyses in small subgroups must be taken with caution all the more since the interaction is in the reverse direction comparatively to observational studies [45, 46] [55]. Previous trials focused on patients who already suffered from various stages of dementia. They produced mixed results. A small single-blind RCT showed positive effects on memory, mood and appetite of 60 AD patients receiving 0.5 ml/day of a mixture of LA (0.92g/ml) and ALA (0.90 g/ml). However, this trial suffered from major methodological limitations, in particular because outcomes were reported by caregivers. In Japan, a very small RCT on 20 patients suffering from dementia from thrombotic cerebrovascular disease showed an improvement on the Mini-Mental Status score with 720 mg DHA per day for 6 months [60]. A small pilot study on the efficacy of ethyl-EPA in the treatment of Alzheimerâ&#x20AC;&#x2122;s disease did not evidence any effect on cognition in a 12-week period [61]. The OmegAD RCT was the first large-scale trial that examined the effect of 0.6 g EPA + 1.7 g DHA supplementation per day on cognitive functioning in patients with mild to moderate AD [62]. Although no overall effect was observed at 6 months, positive effects were found in a small sub-group of patients with very mild AD. Such disappointing results of RCT may be explained by the relatively good level of functioning of the participants at baseline in the single primary prevention study, and by the short duration of the supplementation in all studies. More RCT are therefore necessary to assess the potential impact of n-3 PUFA supplementation over the long term. The discrepancy between negative RCT and observational studies showing a protective effect of fish consumption might also be explained by a wrong targeting of the protective nutrients brought by fish. For instance, fish is also a good dietary source of selenium which acts as an antioxidant. In the EVA study, plasma selenium was negatively correlated with RBC n-6 PUFA and positively with n-3 PUFA [63]. Plasma selenium also increased with fish consumption in the same study as well as in a sample of institutionalized elderly [64]. Selenium could therefore enhance the effect of EPA and DHA by protecting them against peroxidation.

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4. Dietary patterns The synergistic effect of dietary sources of n-3 PUFA and antioxidants on cognitive functioning can be examined by determining dietary patterns. Dietary patterns have been seldom investigated in elderly populations [65]. Non-specific a priori “healthy” dietary patterns were derived based on previous knowledge. A healthy diet indicator (HDI) based on the World Health Organisation's guidelines for the prevention of chronic diseases was proposed by Huijbregts et al [66]. There was a tendency towards a lower prevalence of cognitive impairment associated with increased HDI score in four out of five European cohorts of older persons [67]. Similarly, a better HDI score was associated with a lower prevalence of cognitive deficit in another sample of older Italians [68]. A posteriori dietary patterns were also derived using multidimensional methods which do not imply previous hypotheses. A hybrid cluster analysis lead to the identification of a genderspecific “healthy” cluster characterized by higher consumption of fish in men and fruits and vegetables in women. This cluster was associated with lower cognitive symptoms and better perceived health in both genders [69]. However, a major limitation of these studies is their cross-sectional design. A Mediterranean diet (MeDi) score was proposed to measure adherence to the so-called “Mediterranean diet” characterized by higher consumption of vegetables, legumes, fruits and nuts, cereal, and fish, lower consumption of meat, poultry, and dairy products, moderate alcohol intake, and higher monounsaturated to saturated lipids ratio [70]. The MeDi score was reproduced in an American population and linked to decreased risk of incident Alzheimer’s disease in the WHICAP study [71]. The association was independent of vascular risk factors suggesting that other protective mechanisms might be implicated [72]. These dietary patterns were not specifically derived to evidence cognitive benefits. In the 3C study, we defined “good” or “poor” dietary patterns according to the regular consumption of foods whose components were expected to be protective (sources of n-3 PUFA such as fish or oils; sources of antioxidants such as fruits and vegetables) or conversely deleterious (intake of n-6 PUFA not balanced by intake of n-3 PUFA) [46]. A very poor diet characterized by infrequent consumption of fish, fruits and vegetables, and no regular use of n-3 rich oils was associated with a significantly increased risk for all cause dementia and AD. Regular consumers of n-6 rich oils but not n-3 rich oils nor fish also had a considerably increased risk of dementia but only if they were not ApoE4 carriers. Conversely, individuals consuming fish at least weekly or n-3 rich

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oils and fruits and vegetables daily had a significantly decreased risk of dementia. Finally, the dietary pattern “having a single good habit” (whatever its kind, anti-oxidants or n-3 PUFA) was not associated with decreased risk of dementia [46]. A combination of dietary sources of n-3 PUFA and anti-oxidants seems therefore necessary for a protective effect against dementia.

5. Conclusion These data considered altogether suggest that a lower risk of dementia is associated with regular consumption of fruits and vegetables, fish and n-3 rich oils. The protective effect of fruits and vegetables may be explained by their content in anti-oxidants such as vitamins C and E, carotenoids, or flavonoids, but also to folate. A regular consumption of fish also seems to exert protective effects which may be attributed to EPA or DHA. There seems to be a synergy between dietary sources of n-3 PUFA and antioxidants. Indeed, anti-oxidants protect long-chain n-3 PUFA against peroxidation to which they are very sensitive due to their multiple doublebounds. Conversely, an imbalanced diet with excessive intake of n-6 PUFA will contribute to exacerbate oxidative stress. However, primary prevention trials with anti-oxidants or long-chain n-3 PUFA are all negative. We cannot therefore exclude that unmeasured lifestyle characteristics associated with a « healthy » diet, such as physical and cognitive activities, may decrease dementia risk and thus act as residual confounders in the apparently protective relationship between anti-oxidants n-3 PUFA and dementia. Further research is needed to better identify the mechanisms of action of fatty acids and anti-oxidants, the optimal dosages, and to understand the interaction with genetics. Based on this previous knowledge, more efficient intervention studies could be implemented associating n-3 PUFA and antioxidants in high risk older persons such as those with nutritional deficiencies or mild cognitive impairment. Presently, there is no evidence of any effect of supplements for the prevention of cognitive decline, except maybe in some specific groups. There are nevertheless realistic possibilities of prevention of dementia and cognitive decline through diet. The French National Program for Nutrition and Health (http://www.mangerbouger.fr/menu-secondaire/pnns/) recommends to eat at least 5 fruits and vegetables a day, fish twice a week, and to prefer canola, soya or walnut oils which are n-3 rich oils. These dietary recommendations could contribute to decrease the incidence of dementia, in addition to cardiovascular disease and cancer.

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Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Recent Advances on Nutrition and the Prevention of Alzheimer’s Disease, 2010: 41-62 ISBN: 978-81-7895-470-7 Editors: Charles Ramassamy and Stéphane Bastianetto

3. Dietary fatty acids, alcohol intake, and cognitive decline in the elderly Francesco Panza1, Vincenza Frisardi1, Cristiano Capurso2, Alessia D’Introno1 Anna M Colacicco1, Antonio Capurso1 and Vincenzo Solfrizzi1 1

Department of Geriatrics, Center for Aging Brain, Memory Unit, University of Bari, Bari, Italy 2 Department of Geriatrics, University of Foggia, Foggia, Italy

Abstract. Since the therapeutic options currently available have demonstrated limited efficacy, the search for preventive strategies for cognitive decline and dementia is mandatory. A possible role of vascular and lifestyle-related factors was recently proposed for age-related changes of cognitive function, predementia syndromes, and cognitive decline of degenerative (Alzheimer’s disease, AD) or vascular origin. At present, cumulative evidence suggested that vascular risk factors may be important in the development of mild cognitive impairment (MCI), dementia, and AD. Among vascularrelated factors, metabolic syndrome has been associated with the risk of cognitive decline and overall dementia. Currently available epidemiological evidence suggested that an increase of saturated fatty acids (SFA) could have negative effects on cognitive functions, while increased polyunsaturated fatty acids (PUFA) and monounsaturated fatty acids (MUFA) may be protective against cognitive decline. In a Southern Italian elderly population from the Italian Longitudinal Study on Aging (ILSA), a clear reduction of risk of age-related cognitive decline (ARCD) has been found with elevated intake of PUFA and MUFA. Furthermore, in the ILSA, Correspondence/Reprint request: Dr. Francesco Panza, Department of Geriatrics, Center for Aging Brain Memory Unit, University of Bari, Policlinico, Piazza Giulio Cesare, 11, 70124 Bari, Italy E-mail: geriat.dot@geriatria.uniba.it

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while dietary fatty acids intake were not associated with incident MCI, high PUFA intake appeared to have borderline non-significant trend for a protective effect against the development of MCI. Moderate alcohol drinking has been proposed as a protective factor against MCI and dementia in several longitudinal studies, but contrasting findings also exist. These epidemiological findings on predementia syndromes, i.e. MCI or ARCD, together with a recent randomised controlled trial on a possible effect on cognitive and depressive symptoms of ω-3 PUFA supplementation in patients with very mild AD, suggested a possible role of fatty acids intake in maintaining adequate cognitive functioning and possibly in preventing or delaying the onset of dementia. However, in most cases, these were only observational studies, and results are awaited from large multicenter randomized clinical trials in older persons. At present, vascular risk factor management, diet rich in PUFA and MUFA, moderate alcohol intake, lifestyle changes, and drugs could be employed together to delay the onset of dementia syndromes.

Introduction Dementia is a syndrome definite by impairments in memory and other cognitive functions that are severe enough to cause significant decline from a previous level of social and occupational functioning [1]. Dementia is estimating as affecting approximately 6% of the population aged 65 and older, with the prevalence increasing exponentially with age, being 40 to 70% at the age of 95 years and above [2]. In occidental countries, the most common forms of dementia are Alzheimer’s disease (AD) and vascular dementia (VaD), with respective frequencies of 70% and 15% of all dementias [3]. Therefore, AD is the most common dementia and primary neurodegenerative disorder in the elderly, that gradually leads to a complete psychological and physical dependency and finally to death within one to two decades. The transitional phase between mild nondisabling cognitive decline and disabling dementia is an ambiguous diagnostic period during which it is unclear whether mild cognitive deficits predict incipient dementia or not. The term “predementia syndrome” identify all conditions with age-related deficits in cognitive function reported in the literature, including a mild stage of cognitive impairment based on a normality model and pathological conditions considered predictive or early stages of dementia [4, 5]. Such predementia syndromes have been defined for AD and VaD, but have not yet been operationalized for other specific forms of dementia. Therefore, the term “predementia syndromes” included different conditions and among these predementia syndromes, mild cognitive impairment (MCI) is, at present, the most widely used term to indicate nondemented aged persons with no significant disability and a mild memory or cognitive impairment that cannot be accounted for any recognized medical or psychiatric condition [6, 7].

Fatty acids, alcohol, and cognitive decline

There is now ample evidence that MCI is often a pathology-based condition with a high rate of progression to AD [6]. Therefore, MCI was also identified as the predementia syndrome for AD [4, 5]. The more recently proposed multiple subtypes of MCI were intended to reflect the heterogeneity of different types of dementia. Actually, the recent subclassification of MCI according to its cognitive features (dysexecutive MCI and amnestic-MCI (aMCI) or aMCI and non-amnestic MCI (naMCI): single domain aMCI and multiple domain aMCI or single domain naMCI, and multiple domain naMCI) [7], clinical presentation (MCI with parkinsonism or cerebrovascular disease) [8], or likely etiology (MCI-AD, vascular MCI, or MCI-Lewy Body Disease) [9] represents an attempt to control this heterogeneity [10]. The causes of cognitive decline and dementia are unknown and, at present, there is no curative treatment for dementia and AD, or therapeutical approach to prevent the conversion of MCI to dementia. However, some studies have suggested that these conditions may be prevented [11, 12]. Epidemiological evidence supported the hypothesis that modifiable vascular and lifestyle-related factors are associated to the development of dementia and predementia syndromes in late life, opening new avenues for the prevention of these diseases [11-14]. Given the lack of effective pharmacological therapies, lifestyle changes may be possible treatment options for predementia syndromes, i.e. MCI or age-related cognitive decline (ARCD). In older subjects, particularly over age 85 years, the prevalence of vascular factors and other medical conditions, that impair cognition, increases substantially including chronic cardiovascular, respiratory metabolic disease, and severe sensory deficits [5]. Recent evidence from population-based longitudinal epidemiologic studies suggesting that cerebrovascular disease (CVD) and vascular risk factors may contribute to the heterogeneity of MCI [5,15-18]. Because such vascular risk factors may be modifiable, identification and subsequent management of these possible risk factors may help to prevent, and reduce conversion rates of MCI to dementia [5]. Special attention was paid to the possible role of metabolic syndrome (MetS) in predementia and dementia syndrome.

Metabolic syndrome, dementia, and cognitive decline MetS is defined as a cluster of abdominal obesity, impaired fasting glucose, hypertension, low high-density lipoprotein (HDL) and/or high triglycerides. Most of the MetS components have been shown to be independent risk factors for coronary artery disease (CAD) and stroke, and MetS itself was already evidenced to be an independent risk factor for CAD, CAD mortality, and fatal and non-fatal stroke [19-21]. As been above,

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several individual components of MetS have been linked to risk of developing dementia and MCI [5, 15, 22]. MetS has also been previously shown to increase the risk of cognitive decline in different ethnic groups [23-26], overall dementia [27], AD in both population-based and case control studies [28, 29], and frontal-subcortical syndrome [30]. However, the association between MetS and accelerated cognitive decline seems to disappear at the age 85 and older [31]. Among lifestyle- and vascular-related factors, the impact of diet, particularly, Mediterranean diet, has been the subject of recent interest [32].

Mediterranean diet, dietary fatty acids, and cognitive decline The typical dietary pattern of Mediterranean diet is characterized by high intakes of vegetables, fruits and nuts, legumes, cereals, fish, and monounsaturated fatty acids (MUFA); relatively low intakes of meat, and dairy products, and moderate consumption of alcohol. MUFA consequently to the high consumption of extra-virgin olive oil, represent the most important fat in Mediterranean diet. Cumulative evidence suggests that extra-virgin olive oil may have a role in the protection against cognitive decline, other than against coronary disease and several types of cancer because of its high levels MUFA and polyphenolic compounds. The cross-sectional association between dietary macronutrients and cognitive impairment was examined in 278 nondemented elderly subjects aged 65-84 years from the Italian Longitudinal Study on Aging (ILSA), a large population-based prospective study with a sample of 5,632 subjects 65-84 years old. After adjustment for educational level, the odds ratios (ORs) of cognitive decline (MMSE score < 24) decreased exponentially with the increase of MUFA energy intakes. Despite the lower education (â&#x2030;¤ 3 years), MUFA energy intake over 2400 kJ/day was associated with a reduction in OR of cognitive impairment. The age as a confounder of the interaction term â&#x20AC;&#x153;education by MUFAâ&#x20AC;? was associated with a further increase in OR of cognitive impairment. Furthermore, selective attention performances were independently associated with MUFA intake [33]. Very recently, in the Doetinchem Cohort Study, after adjusting for age, gender, education, alcohol consumption, smoking, and energy intake, higher dietary cholesterol was associated with an increased risk of impaired memory function and cognitive flexibility cognitive function, whereas higher SFA intake was associated with an increased risk of impairment in memory function, psychomotor speed, and cognitive flexibility by 15% to 19%, although not significantly. Fatty fish and marine n-3 polyunsaturated fatty (PUFA) consumption were significantly associated with a decreased risk of

Fatty acids, alcohol, and cognitive decline

global cognitive function impairment and psychomotor speed by 19% to 28%. These associations appeared to be independent of differences in cardiovascular risk factors [34]. High fish consumption, an important source of long-chain n-3 PUFA, tended to be inversely associated with cognitive impairment and cognitive decline at 3-year follow-up, but not significantly [35]. Finally, recent findings from the Chicago Health and Aging Project (CHAP) showed that in a large populationbased sample of 2,560 persons, aged 65 years and older, a high intake of saturated and trans-unsaturated fat were associated with a greater cognitive decline over a 6-year follow-up. Intake of MUFA was inversely associated with cognitive change among persons with good cognitive function at baseline and among those with stable long-term consumption of margarine, a major foodsource. Slower decline in cognitive function was associated with higher intake of PUFA, but the association appeared to be due largely to its high content of vitamin E, which shares vegetable oil as a primary food source and which is inversely related to cognitive decline. Finally, cognitive change was not associated with intakes of total fat, animal fat, vegetable fat, or cholesterol [36]. Moreover, in a total of 732 men and women, 60 years or older, participating in the EPIC-Greece cohort (European Prospective Investigation into Cancer and Nutrition) and residing in the Attica region, six to 13 years later, seed oil consumption may adversely affect cognition, whereas adherence to the Mediterranean diet, as well as intake of olive oil, MUFA and SFA exhibited weakly positive but not significant associations [37]. Therefore, on the basis of the previous significant suggestions [38], we tested further the hypothesis that high MUFA and PUFA intakes may protect against the development of cognitive impairment over time in a median follow-up of 8.5 years of the ILSA. The major finding of this study was that high MUFA, PUFA, and total energy intake were significantly associated with a better cognitive performance in time. Total energy intake should be considered an important confounder of diet-ARCD relationships and, as we proposed in our methodological approach, suggesting that association between macronutrient intake and cognitive decline should be adjusted by total energy intake. The association between high MUFA, PUFA intakes and cognitive performance remained robust even after adjustment for potential confounding variables such as age, sex, educational level, Charlson comorbidity index, body mass index, and total energy intakes [39]. Finally, recent findings from the ILSA demonstrated that while dietary fatty acids intakes were not associated with incident MCI, high PUFA intake appeared to have borderline non-significant trend for a protective effect against the development of MCI [40]. In the Figure, we summarized the evidence on the possible effects of dietary fatty acids on predementia and dementia syndromes.

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Suggested protective factors

Higher n-6 polyunsaturated fatty acids intakes

Suggested risk factors

Higher total fat intakes

Predementia and dementia syndromes Higher monounsaturated fatty acids intakes Higher saturated fatty acids intakes Elevated fish consumption

Figure. Synopsis of the evidence on the possible effects of dietary fatty acids on predementia and dementia syndrome.

Very recently, Freund-Levi and colleagues examined the effects of dietary ω-3 PUFA supplementation randomizing 204 patients with moderate AD to receive docosohexaenoic acid (DHA) and eicosapentaenoic acid (EPA) (for a total dose of 1,720 mg DHA/600 mg EPA) or placebo for 6 months (OmegAD Study). After the treatment period, all the subjects received open label ω-3 PUFA for another 6 months. The supplementation did not delay the rate of cognitive decline but, in the group of 32 patients with the most mild AD (MMSE >27, Clinical Dementia Rating Score 0.5-1), ω-3 PUFA supplementation slowed the decline in MMSE scores [41]. In addition, the subjects in the placebo group of these very mild AD patients also showed a statistically significant slowing of decline when they were switched to treatment between 6 and 12 months, suggesting that ω-3 PUFA might be of benefit to slow the progression of the disease in MCI or very mild AD [41]. Furthermore, in the OmegAD Study, supplementation with

Fatty acids, alcohol, and cognitive decline

ω-3 PUFA in patients with mild to moderate AD did not result in marked effects on neuropsychiatric symptoms except for possible positive effects on depressive symptoms in non-apolipoprotein E (APOE) ε4 carriers and agitation symptoms in APOEε4 carriers [42]. Some experimental evidence suggested that essential ω-3 PUFA protect against neuronal deficits, decrease β-amyloid (Aβ levels, and decrease the number of activated microglia in the brain using transgenic mouse models of AD [43, 44]. At present, the effect of arachidonic acid and DHA (240 mg/day) after a 90-day supplementation upon MCI, organic brain lesions, or AD showed a significant improvement of the immediate memory and attention score for MCI patients, and a significant improvement of immediate and delayed memories for patients with organic brain damages [45]. The AD group showed no improvement after the supplementation of arachidonic acid and DHA, and the placebo group showed no significant improvement of cognitive functions by the supplementation of 240 mg/day of olive oil (high MUFA content) [45]. The lack of cognitive effects of the olive oil supplementation may be probably explain from the very small amount of olive oil administered in comparison with ILSA sample in which the mean consumption of olive oil was particularly high: 46 g/day (12.6 to 113.1 g/day) [32]. A recent study in people with mild age-related memory complaints, demonstrated the positive effects of a 14-day healthy longevity lifestyle program on word fluency, and activity in the left dorsolateral prefrontal cortex at [fluorine-18]fluorodeoxyglucose (FDG) positron emission tomography (PET) scans in comparison of the control. Cardiovascular conditioning and brief relaxation exercises designed to lower stress are recommended each day. Suggested shopping lists and menus guide subjects to follow a healthy diet plan, including five daily meals emphasizing antioxidant fruits and vegetables, ω-3 PUFA, and low glycemic index carbohydrates [46]. The conceptual basis for the healthy diet plan was that diets high in ω-3 PUFA from olive oil or fish, as well as those rich in antioxidant fruits and vegetables, are associated with less ARCD [47]. On the basis of these evidences, predementia syndromes may be a high-risk condition for progression to dementia of vascular and degenerative origin, intervention trials using measures of dietary supplementation similar to the OmegAD Study to determine if such supplements will slow cognitive decline [48].

Dietary unsaturated fatty acids and cognitive decline: Possible mechanisms The mechanisms by which high UFA intake could be protective against cognitive decline and dementia in healthy older people are, at present,

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unknown. In the older subjects of the ILSA, which fulfilled a Mediterranean dietary pattern, total fat is 29% of energy, with a high consumption of olive oil (46 g/d), a MUFA energy intake of 17.6% of total energy, 85% of which derived from olive oil, and a SFA intake of only 6% [33]. In our population, the prolonged protection of MUFA intake against age-related changes in cognitive functions, may be linked to the relevant quota of antioxidant compounds in olive oil, including low molecular weight phenols [49]. In fact, animal studies suggested that diets high in antioxidant-rich foods, such as spinach, strawberries, and blueberries, rich in anthocyanins and other flavonoids may be beneficial in slowing age-related cognitive decline [50]. The possible role of antioxidant compounds from olive oil do not diminish or otherwise alter the argument concerning the fatty acids, because this is only a possible explanation of the role of MUFA on age-related cognitive changes in our population, in which MUFA intake derived for a large part from olive oil. The protective effect of dietary UFA could be related to the role of fatty acids in maintaining the structural integrity of neuronal membranes, determining the fluidity of synaptosomal membranes and thereby regulating neuronal transmission. Furthermore, essential fatty acids can modify the activity of certain membrane-bound enzymes (phospholipase A2, protein kinese C, and acetyltranferase), and the function of the neurotransmitters’ receptors. Finally, free fatty acids, lipid metabolites, and phospholipids modify the function of membrane proteins including ion channels [51]. Moreover, fatty acid composition of neuronal membranes in advancing age demonstrated an increase in MUFA content and a decrease in PUFA content [52]. There is also evidence associating a dietary deficiency of n-3 PUFA with changes in cortical dopoaminergic function [53]. The ω-3 PUFA from fish may be inversely associated with dementia because it lowers the risk of thrombosis [54], stroke [55], cardiovascular disease [56], and cardiac arrhythmia, reducing the risk of thromboembolism in the brain and consequently of lacunar and large infarcts that can lead to VaD and AD. Furthermore, the ω-3 PUFA may be important as lipids in the brain, particularly for the possible influence of DHA on the physical properties of the brain that are essential for its function [57]. Furthermore, fish oil was a better source than α-linolenic acid for the incorporation of ω-3 PUFA into rat brain phospholipid subclasses [4]. On the contrary, high linoleic acid intake (ω-6 PUFA) may increase the susceptibility of LDL cholesterol to oxidation, which makes it more atherogenic [58], even if the association between linoleic acid and atherosclerosis is controversial [59]. Therefore the ratio of dietary ω-3/ω-6 PUFA intake may influence the potential role of PUFA on cognitive decline and dementia, the optimal ratio of ω-6:ω-3 should be <5:1

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[60]. Finally, a high dietary intake of SFA and cholesterol increases the risk for cardiovascular disease, and therefore for cognitive decline, VaD, and AD [11]. On the contrary, treatment for four weeks with a Mediterranean-inspired diet rich in Ď&#x2030;-3 PUFA decreased blood lipids in healthy individuals with a low-risk profile for cardiovascular disease, with a beneficial effect also on vascular function and oxidative stress [61].

Alcohol consumption, dementia, and cognitive decline Among lifestyle factors related to diet, several studies have assessed alcohol consumption and cognitive function among older adults, but whit inconsistent results [4]. There is also much emerging and a lot of older data, mainly from population-based longitudinal cohort studies but also including some case-control studies, which suggest that alcohol consumption, particularly red wine, within limits and/or of certain types, is associated with a decreased risk of cognitive impairment or decline, predementia, and dementia syndromes [62,63], despite chronic alcohol abuse causing progressive neurodegenerative disease [64]. However, a possible source of variability in the findings of these studies were cross-sectional design, restriction by age or sex, or incomplete ascertainment [65]. It is especially important to examine data for men and women separately when alcohol consumption is a predictor variable, because their consumption levels are very different. In virtually every study which included both sexes, women consumed alcohol less frequently and in smaller amounts than men. Moreover, education, smoking or APOE Îľ4 allele often modified the association between alcohol drinking and cognitive impairment or decline. In fact. the association between low education and predementia and dementia syndromes is supported by the majority of studies, but very few studies have investigated whether this association may be attributed with lifestyle factors that covary with education [11]. Socio-economic and educational factors, determining drinking patterns in different populations and countries, might influence the strength of association of alcohol and cognitive impairment or decline. Furthermore, the APOE Îľ4 allele is the most important currently known genetic risk factor for AD [66], and it could be a possible effect modifier for the associations between alcohol/vascular risk factors and dementia syndromes [11] Launer and colleagues showed in the Zutphen Elderly Study that men with CAD or diabetes and low-to-moderate alcohol intake had a significantly lower risk of poor cognitive function compared to abstainers [67]. In the Framingham Heart Study the association between alcohol consumption and cognitive performance was analyzed separately for

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men and women, since the researchers expected a different alcohol-cognition relationship for male and female drinkers [68]. Some have claimed there is a J or U shaped relation between alcohol drinking and cognitive impairment [67, 69,70]; that is, light to moderate alcohol drinking might have a protective effect compared with total abstention and heavy drinking. Moreover, epidemiological studies have recently reported an association between wine consumption and the incidence of AD. Particularly, red wine was investigated in the PAQUID study, in which the relative risk for dementia and AD among 318 subjects who drank three or four glasses of wine each day in comparison with 971 total abstainers were 0.21 and 0.25, respectively. Among the 922 older subjects who drank no more than one or two glasses of wine each day respect to the abstainers the relative risk for AD was reduced significantly (0.55) [71]. More recently, the relation between alcohol consumption and risk of dementia (AD, VaD, or other dementia) was examined in the Rotterdam Study. The findings of this study, with an average follow-up of 6 years, suggested that light-to-moderate alcohol consumption is associated with a reduced risk of dementia in individuals aged 55 years or older; this effect seems to be unchanged by the source of alcohol [72]. In the Rotterdam study, the protective effect of alcohol drinking was found mainly for VaD, and the authors suggested that moderate alcohol intake might protect against dementia via a reduction in vascular risk factors [72]. In a nested case-control study on 373 cases with incident dementia and 373 controls who were among 5,888 adults aged 65 years and older, and participated in the Cardiovascular Health Study (CHS), the adjusted odds ratio for dementia among whose weekly alcohol consumption was less than 1 drink were 0.65, compared with abstention; 1 to 6 drinks, 0.46; 7 to 13 drinks, 0.69; and 14 or more drinks [73]. In the Copenhagen City Heart Study, the risk of developing dementia was significantly lower among occasional wine drinkers, in weekly drinkers and, but not significantly, in daily drinkers. An increased risk for beer and for spirits was found in occasional, weekly, and daily drinkers [74]. A trend toward greater odds for dementia associated with heavier alcohol consumption was most apparent among men and participants bearing an APOE Îľ4 allele, with similar relationships of alcohol use with AD and VaD [73]. The findings from the CHS were consistent with the PAQUID Study [71] and the Rotterdam Study [72], but suggested a higher risk of dementia with consumption greater than 2 drinks per day. The results of the CHS were also consistent with those of the Epidemiology of Vascular Aging Study which found that alcohol intake was associated with a lower risk of cognitive deterioration among subjects without an APOE Îľ4 allele, but a higher risk in

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APOE ε4 carriers [75]. Surprisingly, the Rotterdam Study found that the lower risk of dementia associated with alcohol use was more consistent among individuals with an APOE ε4 allele [72], but no significant interaction was detected. In the Washington Heights Inwood-Columbia Aging Project, with 908 subjects aged 65 years and older, consumption of up to three servings of wine daily is associated with a lower risk of AD in elderly individuals without the APOE ε4 allele [76]. Finally, in the Prospective Population Study of Women in Goteborg, Sweden, in a 34-year follow-up, wine was protective for dementia, and the association was strongest among women who consumed wine only. In contrast, consumption of spirits at baseline was associated with slightly increased risk of dementia [77]. Very recently, we evaluated the impact of alcohol consumption on the incidence of MCI in 1,445 non–cognitively impaired individuals and on its progression to dementia in 121 patients with MCI, aged 65 to 84 years, participating in the ILSA, with a 3.5-year follow-up. Patients with MCI who consumed up to 1 drink/day had a reduction in the rate of progression to dementia in comparison with patients with MCI who never consumed alcohol. Overall, vs non-drinkers, patients with MCI who consumed 1.0 to 14.9 g of alcohol/day, derived mostly from wine, had a decrease in the rate of progression to dementia of about 85 percent. Moderate intake of alcohol deriving from wine, in drinks controlled for the intake of alcohol deriving from other sources within each level of total intake, was also associated with a significantly lower rate of progression to dementia. No significant associations were found between any levels of drinking and the incidence of MCI in non–cognitively impaired individuals vs abstainers [78]. To the best of our knowledge, only two other studies have examined the effect of alcohol consumption on the risk for the incidence of MCI [79,80]. After an average follow-up of 23 years, non-drinkers and frequent drinkers were both more than twice as likely to have MCI in old age than occasional drinkers [79]. However, the APOE genotype seemed to modify the relationship, such that the risk of old age dementia increased with increasing midlife alcohol consumption only among carriers of the APOE ε4 allele [79]. In our report on ILSA sample, we failed to confirm these findings, but we note that the alcohol consumption reported was a midlife determination [77]. Probably, a follow-up period longer than 3.5 years would have revealed that a moderate alcohol consumption might influence the incidence of MCI. On the other hand, our findings are consistent with those obtained in the Women’s Health Initiative Memory Study with a 4.2 year-follow-up, which found that moderate alcohol intake was associated with an approximately 50 percent

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reduced risk of combined probable dementia and MCI [80]. However, after adjusting for demographic and socioeconomic factors, and baseline Modified Mini Mental State Examination (3MSE), the significance disappeared [80]. Currently, ours is the first study in which alcohol consumption was associated with the rate of progression of MCI to dementia, in fact, up to 1 drink/day of alcohol or wine may decrease the rate of progression to dementia in patients with MCI [76]. It is also possible that moderate lifestyles in general, which obviously vary according to different cultural environments, protect from cognitive impairment. Thus, it may not be the direct effect of alcohol or specific substances in alcoholic drinks that provide the protection, but moderate alcohol drinking may be an indicator of a complex set of favorable social and lifestyle factors. A protective effect of alcohol on cognitive function in moderate drinkers may be due to a relatively poor health status among abstainers or because cognitive status influences alcohol consumption and overall health status.

Alcohol intake and protection against cognitive decline: Possible mechanisms The mechanism by which low alcohol intake could be protective against cognitive decline impairment or decline in older age or against predementia and dementia syndromes is, at present, unclear. In particular, the association between alcohol drinking and cognitive functions could have different explanations in relation to cognitive domains explored. White matter lesions (WMLs) would play a neuropathological role [81], given that alcohol drinking influenced in particular measures of psychomotor speed, episodic memory, and executive function [82]. In fact, alcohol drinking has been associated with fewer brain infarcts and was shown to have a U shape relationship with WMLs [83]. WMLs and infarcts, in turn, may reflect a vascular mechanism responsible for the observed association between alcohol and cognitive functions [84]. Furthermore, different mechanisms may underly the adverse effects of heavy drinking and the potential beneficial effects of low to moderate drinking, and may also partly explain why deficits are reported seen in certain functions (e.g., delayed recall) [65], whereas benefits are seen in others (e.g., learning) [85]. In fact, higher doses of alcohol may affect cognitive functioning through increased release of acetylcholine from the hippocampus [65], which was linked to problems with memory and attention [81]. On the other hand, animal evidence indicated that low doses of alcohol may stimulate the release of hippocampal acetylcholine [86, 87], that regulates learning and memory. Finally, moderate alcohol

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consumption was associated with reduced cardiovascular risk factors [88, 89] and lower rates of cardiovascular disease [89, 90], which might protect brain vasculature and prevent subclinical strokes, resulting in better cognitive performance [83, 91]. Alcohol consumption might protect from unspecified dementia by effects on the cerebral vasculature, supporting the observation that light to moderate alcohol intake might be protective against ischemic stroke [92]. Moreover, a light to moderate alcohol use is associated with a lower prevalence of MRI-defined WMLs and sub-clinical infarcts [93], although MRI abnormalities, HDL cholesterol levels, and fibrinogen levels only marginally influenced the association of alcohol consumption and dementia in the CHS [94]. The suggested protection of low to moderate alcohol use against CVD may explain the possible protection also against VaD. In the Rotterdam study, the protective effect of alcohol consumption was found mainly for VaD, and the authors suggested that moderate alcohol intake might protect against dementia via a reduction in vascular risk factors [74]. In fact, moderate doses of alcohol may increase prostacyclin concentrations, reduce the generation of thromboxane A2, and inhibit platelet function [73, 95]. They may increase plasma levels of endogenous tissue-type plasminogen activator, a serine protease that regulates intravascular fibrinolysis [96], and fibrinolytic activity while decreasing plasma fibrinogen levels [97]. It is also known that alcohol is associated with increased levels of HDL cholesterol, its subfractions HDL2 and HDL3, and its associated apolipoproteins A-I and A-II [98,99]. The association with HDL cholesterol is deemed to account for up to a half of the reduction in coronary events associated with moderate alcohol consumption [100]. While the above-cited factors affected the risk of unspecified dementia and, probably, of Vad, other experimental and clinical findings may partly explain the suggested protection of low to moderate alcohol consumption against AD. In fact, small amounts of alcohol have been reported to be associated with a lower prevalence of vascular brain findings and, in APOE Îľ4 carriers, with hippocampal and amygdalar atrophy as assessed by MRI [101]. Experimental studies found than ethanol initially increases hippocampal acetylcholine release, which could conceivably improve memory performance [102]. Processes that originate, modulate, or precipitate the deposition of amyloid beta (Î˛A) in the brain, such as oxidative stress, rather than vascular processes, may better explain the development of AD, and the vascular effects of the alcohol component of alcoholic beverages may not be enough to explain the protective effects of the moderate intake of alcohol from dementia. Wine consumption may exert a protective effect, either through alcohol intake itself, through the antioxidant effects of polyphenols richly

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represented in red wine [102, 103], or through both. The latter effects, of course, are independent of alcohol and, in fact, have been also associated with alcohol-free red wine [104]. The constituents of red wine have also potentially beneficial vascular effects [105, 106], enhance endothelial nitric oxide release [107], and reduce atherosclerosis in APOEâ&#x20AC;&#x201C;deficient mice [108, 109]. Red-wine polyphenols are a complex mixture of flavonoids (mainly anthocyanins and flavan-3-ols) and non-flavonoids (such as resveratrol and gallic acid). Flavan-3-ols are the most abundant, with oligomeric and polymeric procyanidins (condensed tannins) often representing 25â&#x20AC;&#x201C;50% of the total phenolic constituents [110]. A recent study identified procyanidins as the principal vasoactive polyphenols in red wine and showed that they are present at higher concentrations in wines from areas of south-western France and Sardinia, where traditional production methods ensure that these compounds are efficiently extracted during vinification [111]. Given the link between VaD, vascular risk, and the increasing body of evidence suggesting that AD may be influenced by vascular factors [112, 113], it may be concluded that the vascular protection associated to wine consumption decreases the risk of incident dementia/AD. In fact, in the 5-year follow-up PAQUID cohort, a significant inverse association between flavonoid intake and the risk of dementia was found [114]. It has been also suggested that the antioxidant properties of the flavonoids in wine may help prevent the oxidative damage implicated in dementia. In fact, oxidative stress may also develop in the brain, leading to neuronal death by various mechanisms such as formation of Î˛A4 protein, DNA damage, and abnormal tau protein [115, 116]. The presence in wine of nonalcoholic components, such as particular antioxidants, could explain a differential effect of wine on dementia. In fact, liquor has been shown to have less antioxidant activity than wine [117]. Nonetheless, in some studies on the neuroprotective role of moderate alcohol consumption, the most typically consumed alcohol types were beer and spirits [82,118]. It is also possible that moderate lifestyles in general, which obviously vary according to different cultural environments, protect from cognitive impairment. Thus, it may not be the direct effect of alcohol or specific substances in alcoholic drinks that provide the protection, but moderate alcohol drinking may be an indicator of a complex set of favourable social and lifestyle factors. A protective effect of alcohol on cognitive function in moderate drinkers may be due to a relatively poor health status among abstainers or because cognitive status influences alcohol consumption and overall health status [80]. Moreover, given the suggested role of different components of Mediterranean diet, it could be possible that alcohol influences the metabolism of PUFA and that this interaction could explain the

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role of both alcohol and PUFA in dementia and AD. In fact, alcohol is a potent modulator of fatty acid metabolism and is known to influence the fatty acid profiles of different organs [119]. In animal studies, chronic alcohol exposure has been shown to decrease long-chain PUFA concentrations, especially docosahexaenoic acid and arachidonic acid in liver and brain tissues, depending on dosage and length of alcohol exposure [120-122]. In this picture, the suggested neuroprotective role of low to moderate alcohol consumption may be a marker for a more complex dietary pattern. Further studies with dietary patterns more similar to the original Mediterranean model are needed to confirm the protective role of this diet against cognitive decline and dementia.

Conclusions Drugs currently used in the treatment of cognitive impairment and dementia have a very limited therapeutic value, overall on the management of psychiatric and behavioural symptoms, rather than cognitive symptoms. It results evident the necessity to potentially individualize new strategies able to prevent and to slow down the progression of predementia and dementia syndromes. In the past few years, vascular and lifestyle-related factors for predementia and dementia syndromes have been an area of intensive research. At present, cumulative evidence suggested the vascular risk factors may be important in the development of MCI, dementia, and AD. Also, MetS, defined as a cluster of vascular risk factors, appeared to be a possible, independent risk factor for cognitive decline and dementia. Moderate alcohol drinking has been proposed as a protective factor against MCI and dementia in several longitudinal studies, but contrasting findings also exist. In fact, many of these studies were limited by cross-sectional design, restriction by age or sex, or incomplete ascertainment. Whether, the divergent findings can be explained by the drinking patterns has not been extensively investigated. At present, there is no evidence indicating that starting to drink at a later age would be beneficial. It thus seems that from an epidemiologic point of view, the way to curb the dementia epidemic is through strict attention to vascular risk factors. In fact, several studies have confirmed that good control of hypertension can prevent dementia (both AD and VaD) and treatment with statins also has the same effect [123]. Obviously, even rigid attention to these risk factors will not be able to prevent dementia altogether. For one, the most important vascular risk factor is probably age, which still cannot be manipulated. Secondly, the data concerning the reduction of the incidence of dementia should better be interpreted as delay in the onset rather than prevention. However, because the prevalence of dementia doubles every

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5 years, delay in the onset of dementia by five years is equivalent to a reduction of the prevalence by half in any given age group [124]. Probably, at present, since several important factors are already identified, vascular risk factor management, lifestyle changes, and drugs should be employed together to delay the onset of dementia syndromes.

Acknowledgements Funding for this study was provided by the Italian Longitudinal Study on Aging (ILSA) (Italian National Research Council - CNR-Targeted Project on Ageing - Grants 9400419PF40 and 95973PF40).

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Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Recent Advances on Nutrition and the Prevention of Alzheimerâ&#x20AC;&#x2122;s Disease, 2010: 63-69 ISBN: 978-81-7895-470-7 Editors: Charles Ramassamy and StĂŠphane Bastianetto

4. Alcohol consumption and cognitive impairment Luc Letenneur INSERM, U897, Bordeaux, France; Universite Victor Segalen, Bordeaux, France

Abstract. Moderate alcohol consumption has been found to be associated with a lower risk of developing dementia in several prospective epidemiological studies. When analysing the type of alcoholic beverage consumed, moderate wine intake has been systematically associated with lower risk. Similar results were found when analysing cognitive impairment such as the incidence of Mild Cognitive impairment. However, moderate consumption has very different definitions across studies, ranging from monthly or weekly drinking to 3-4 drinks per day. In addition, different results have been observed according to gender: some studies found the same effect in men and women, while others found either no association or a stronger association in women. All of these results lead to the conclusion that the observed association is fragile and needs further confirmation.

Dementia is the most common disorder affecting the brain in older people. Epidemiological studies have reported several risk factors and a consensus has emerged that gender, education level, dietary, and vascular factors are likely to be important. Among dietary factors, alcohol consumption was found to be associated with a lower risk for dementia in some studies. We shall review several prospective studies on this topic. Correspondence/Reprint request: Dr. Luc Letenneur, INSERM, U897, ISPED, Case 11, Universite Victor Segalen, 146 rue LĂŠo Saignat, 33076 Bordeaux Cedex, France. E-mail: luc.letenneur@inserm.fr

Luc Letenneur

The first prospective study to explore alcohol intake was the PAQUID program [1]. A sample of 3,777 subjects aged 65 years or older was followed for 3 years and 99 incident cases of dementia were diagnosed. Alcohol consumption data were collected at baseline, with wine the main type of alcohol consumed, usually on a daily basis. Four categories of individuals were defined: non-drinkers, mild drinkers (consuming 1 or 2 drinks per day), moderate drinkers (consuming 3 or 4 drinks per day) and heavy drinkers (consuming more than 4 drinks per day). Lower risks of developing dementia were found among drinkers compared with non-drinkers, but the relationship was significant only for moderate drinkers (mild drinkers: risk ratio [RR]=0.81; moderate drinkers: RR=0.19; heavy drinkers: RR=0.31). No modification effect was found according to gender and the association did not change after adjusting for age, gender, education, occupation, and baseline cognition. The Rotterdam Study followed 5,395 subjects aged 55 years or older over a period of 6 years; 197 incident cases of dementia were diagnosed [2]. The number of drinks of alcohol (beer, wine, fortified wine, or spirits) was collected at baseline and five categories of intake were studied: no drink consumed; less than 1 drink per week; more than 1 drink per week but less than 1 per day; 1 to 3 drinks per day; more than 3 drinks per day. The risk of developing dementia was lower among drinkers compared with non-drinkers (Table 1) and was significant in the 1-3 drinks per day category. The pattern was different in men and women. No association was found in women, whereas a lower risk was found for men drinking 1-3 drinks per day. A modification effect was found when the Apolipoprotein E4 allele (Apo E4) was taken into consideration: the risk was lower among drinkers with an ApoE4 allele, whereas it was less clear for drinkers without the ApoE4 allele (Table 1). No difference was found according to beverage type, although beer tended to give marginally lower risk than wine. In a prospective study of elderly people living in North Manhattan [3], 2,126 subjects were followed for 4 years and 260 incident cases of dementia were diagnosed. The number of drinks per week was collected at baseline and subjects were classified as non-drinkers, light drinkers (less than 1 drink per month to 6 drinks a week), moderate drinkers (1-3 drinks a day), and heavy drinkers (more than 3 drinks a day). Light and moderate categories were aggregated because of a low number of moderate drinkers. Indeed, in this sample, 70% of the subjects were non drinkers. When analysing the association between each alcoholic beverage type and dementia, wine was significantly associated with a lower risk among light to moderate drinkers (hazard ratio [HR]=0.64, p=0.018). When analysing the risk of Alzheimerâ&#x20AC;&#x2122;s disease adjusted for age and gender, a decreased risk was observed in wine

Alcohol consumption and cognition

Table 1. Hazard ratios of dementia according to alcohol consumption in the Rotterdam Study.

Total

No alcohol 1.00

< 1 drink per week 0.82 (0.56-1.22)

≥ 1 per week but <1 per day 0.75 (0.51-1.11)

1-3 drinks per day 0.58 (0.38-0.90)

≥ 4 drinks per day 1.0 (0.39-2.59)

Men

1.00

0.60 (0.27-1.34)

0.53 (0.28-1.0)

0.40 (0.21-0.74)

0.88 (0.322.44)

Women

1.00

0.91 (0.58-1.44)

0.91 (0.55-1.49)

0.85 (0.47-1.57)

ApoE4 absent

1.00

1.26 (0.67-2.37)

1.39 (0.73-2.64)

0.67 (0.31-1.46)

ApoE4 present

1.00

0.69 (0.35-1.34)

0.46 (0.23-0.94)

0.60 (0.30-1.21)

drinkers (HR=0.59, p=0.018) but the association became insignificant when education and the ApoE4 genotype (HR=0.69, p=0.11) were included. The risk ratios were greater than 1 for light to moderate beer or spirits drinkers (beer: HR=1.39, p=0.094, spirits: HR=1.34, p=0.152). When wine, beer and spirits were analysed simultaneously with full adjustment, the risk for Alzheimer’s disease was lower in wine drinkers (HR=0.55, p=0.015), but higher for beer (HR=1.47, p=0.065) or spirits (HR=1.51, p=0.062) drinkers. A modification effect was found with the ApoE4 genotype. A significantly lower risk of dementia was found in light to moderate wine drinkers without an ApoE4 allele (HR=0.44, p=0.004) compared with non-drinkers, whereas the association disappeared for ApoE4 allele bearers (HR=1.10, p=0.093). No modification effect by gender was found. The association between alcohol intake and risk for dementia was also examined in studies originally designed to explore cardiovascular events. During follow up, cognitive functioning was explored and nested casecontrol studies were performed. In the Copenhagen City Heart study [4], a nested case-control included 83 cases of dementia and 1,626 controls. Alcohol intake was collected in two ways: the number of drinks per week (less than 1, 1-7, 8-14, 15-21, 22 or more) and the frequency of intake (never / hardly ever, monthly, weekly, daily). No association was found between the number of drinks of alcohol consumed per week and the risk of dementia. When beer, wine and spirits intake were analysed simultaneously, a reduced risk was observed only for wine drinkers (monthly: HR=0.43 [0.23-0.82];

Luc Letenneur

weekly: HR=0.33 [0.13-0.86]; daily: HR=0.57 [0.15-2.11]). Beer drinkers tended to have a higher risk (monthly: HR=2.28 [1.13-4.60]; weekly: HR=2.15 [0.98-4.78]; daily: HR=1.73 [0.75-3.99]) and no clear association was found in spirits drinkers (monthly: HR=0.81 [0.42-1.57]; weekly: HR=1.65 [0.74-3.69]; daily: HR=1.12 [0.43-2.92]). No difference was found between men and women. Another nested case-control study was performed within the Cardiovascular Health Study, which included 373 cases of dementia and 373 controls [5]. Levels of alcohol intake were defined as: 0 drinks per week; less than 1 drink per week; 1-6 drinks per week; 7-13 drinks per week; 14 or more drinks per week. The association between alcohol intake and the risk of dementia followed a J shaped curve, with a nadir for the category of 1-6 drinks per week (Table 2). The pattern was different for men and women: all drinker categories were associated with a lower risk in women, whereas a J-shaped curve was found for men (Table 2). A modification effect was observed by Apo E4: when the ApoE4 allele was absent, the risk was significantly lower among subjects who consumed 1-6 drinks per week. When the ApoE4 allele was present, the HR was below 1.00 only for light drinkers, and above 1.00 for heavier drinkers (Table 2). The odds of dementia were lower (although not significantly) for wine drinkers (less than 1 drink per week: HR=0.72 [0.46-1.11]; 1-6 drinks per week: HR=0.72 [0.39-1.33]; more than 6 drinks per week: HR=0.62 [0.25-1.50]). However, the trend was not the same for beer (less than 1 drink per week: HR=0.84 [0.48-1.47]; 1-6 drinks per week: HR=0.74 [0.36-1.54]; more than 6 drinks per week: HR=1.96 [0.71-5.47]) or spirits drinkers (less than 1 drink per week: HR=0.84 [0.48-1.45]; 1-6 drinks per week: HR=1.17 [0.59-2.30]; more than 6 drinks per week: HR=1.08 [0.55-2.13]). Several other prospective studies have reported an association between alcohol consumption and dementia. A Canadian study [6] reported that at least weekly consumption of alcohol was associated with a decreased risk of Alzheimerâ&#x20AC;&#x2122;s disease (OR=0.68, [0.47-1.00]. In Sweden [7], the risk of dementia was estimated to be 0.5 [0.3-0.7] among light to moderate drinkers (1 to 21 drinks per week in men, 1 to 14 drinks per week in women). In China [8], light to moderate drinkers (1 to 21 drinks per week in men, 1 to 14 drinks per week in women) had a lower risk (RR=0.52 [0.32-0.85]) than nondrinkers, but a non-significant increased risk was observed in heavy drinkers (RR=1.45 [0.43-4.89]). A greater reduction of risk was observed for men (RR=0.37) than for women (RR=0.76). The same pattern was observed when analysing cognitive performance or cognitive decline. Ngandu et al [9] in the CAIDE study showed that the participants who did not drink alcohol at midlife had a poorer performance in

Alcohol consumption and cognition

Table 2. Odds of incident dementia according to alcohol consumption (drinks per week) in the Cardiovascular Health Study. None

1-6

7-13

≥ 14

Total

1.00

0.65 (0.41-1.02)

0.46 (0.27-0.77)

0.69 (0.37-1.31)

1.22 (0.60-2.49)

Men

1.00

0.82 (0.38-1.78)

0.36 (0.17-0.77)

1.42 (0.58-3.48)

2.40 (0.86-6.64)

Women

1.00

0.52 (0.30-0.90)

0.57 (0.28-1.17)

0.23 (0.09-0.61)

0.39 (0.14-1.10)

ApoE4 absent

1.00

0.56 (0.33-0.97)

0.37 (0.20-0.67)

0.64 (0.30-1.38)

0.60 (0.24-1.51)

ApoE4 present

1.00

0.60 (0.24-1.52)

0.62 (0.21-1.81)

1.49 (0.33-6.65)

3.37 (0.67-17.1)

episodic memory, psychomotor speed, and executive function in late life as compared with infrequent and frequent drinkers. In the Women’s Health Initiative Memory Study, Espeland et al [10] showed that compared with no intake, intake of ≥1 drink per day was associated with higher baseline Modified Mini-Mental State Examination scores (p < 0.001) and a covariateadjusted odds ratio of 0.40 (95% CI [0.28 - 0.99]) for significant declines in cognitive function. Associations with incident probable dementia and mild cognitive impairment were of similar magnitude but were not statistically significant after covariate adjustment. Stampfer et al [11] in the Nurses’ Health Study showed that moderate drinkers had better mean cognitive scores than nondrinkers and the relative risk of a substantial decline in performance over a two-year period was 0.85 (95% CI [0.74 - 0.98]) among moderate drinkers, as compared with nondrinkers. Ganguli et al [12] in the Monongahela Valley Independent Elders Survey (MoVIES) showed that after seven years of follow-up, compared to no drinking, both minimal and moderate drinking were associated with lesser decline on the MMSE and Trailmaking tests. Minimal drinking was also associated with lesser decline on tests of learning and naming. Finally, when analysing the risk of developing a Mild Cognitive impairment (MCI), Antilla et al [13] showed that participants who drank no alcohol at midlife and those who drank alcohol frequently were both twice as likely to have mild cognitive impairment in old age as those participants who drank alcohol infrequently. Solfrizzi [14] estimated the impact of alcohol consumption on the incidence of MCI and its progression to dementia. No significant associations were found between any levels of drinking and the

Luc Letenneur

incidence of MCI in non窶田ognitively impaired individuals vs abstainers. However, subjects with MCI who were moderate drinkers (less than 1 drink/ day of wine), had a lower rate of progression to dementia than abstainers (HR = 0.15; 95% CI [0.03 - 0.78]). Finally, there was no significant association between higher levels of drinking (竕･ 1 drink/day) and rate of progression to dementia in patients with MCI vs abstainers.

Discussion All these studies tend to show the same result: light to moderate alcohol consumption is associated with a lower risk of developing dementia or cognitive impairment. Which mechanisms may be involved in the risk reduction of cognitive impairment? One possibility is that alcohol might act by reducing cardiovascular risk factors, either through an inhibitory effect of ethanol on platelet aggregation, or through the alteration of the serum lipid profile. A second possibility is that alcohol might have a direct effect on cognition through the release of acetylcholine in the hippocampus. Finally, another possible mechanism is through antioxidant activity of alcohol, particularly wine, which has been found to have important antioxidant effects. However, the definition of light to moderate alcohol intake varies considerably across the studies reviewed. The classification of drinking as moderate ranges from monthly or weekly drinking to 3-4 drinks per day, and many studies reported an association for an intake of less than 1 drink per day. As alcohol intake is self-reported, it is also expected to be underreported. However, neither a linear dose-response nor a J-shaped curve were systematically found over all studies, and the association sometimes differed according to gender. The type of alcohol does not appear to be consistent across studies, yet wine intake is systematically associated with lower risk. If alcohol per se were associated with a decreased risk of developing dementia, the same pattern would be expected for beer and wine drinkers, yet beer has been found to be associated with higher risk in several studies. When analysing these results and discrepancies, one can wonder about the nature of the association between alcohol consumption and the risk of dementia. It can be hypothesised that alcohol intake (especially light to moderate intake) is only a marker of a broader psychosocial behavior that is associated with a decreased risk of developing dementia. However, the analyses were controlled for many other risk factors and the association with alcohol was still significant. It is possible that important confounders (not yet identified) were not considered, which might explain some of the discrepancies between optimal intake, gender, or type of alcohol. Light to moderate wine drinkers may prove to be moderate with regard to other risk factors of dementia,

Alcohol consumption and cognition

and alcohol intake would only be an indicator of such behavior. In summary, is it the drink or the drinker? Until such factors have been identified, we must be careful in how we interpret results relating to alcohol consumption. People should not be encouraged to drink more in the belief that this will protect them against dementia.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Orgogozo, J.M., Dartigues, J.F., Lafont, S., Letenneur, L., Commenges, D., Salamon, R., Renaud, S., and Breteler, M. 1997, Rev Neurol, 153: 185-192. Ruitenberg, A., van Swieten, J.C., Witteman, J.C., Mehta, K.M., van Duijn, C.M., Hofman, A., and Breteler, M. 2002, Lancet, 359: 281-286. Luchsinger, J., Tang, M.X., Siddiqui, M., Shea, S., and Mayeux, R. 2004, J Am Geriatr Soc, 52: 540-546. Truelsen, T., Thudium, D., and Gronbaek, M. 2002, Neurology, 59: 1313-1319. Mukamal, K., Kuller, L., Fitzpatrick, A., Longstreth, W., Mittleman, M., and Siscovick, D. 2003 JAMA, 289(11): 1405-1413. Lindsay, J., Laurin, D., Verreault, R., HĂŠbert, R., Helliwel, l.B., Hill, G., and Mc Dowell, I. 2002. Am J Epidemiol, 156: 445-453. Huang, W., Qiu, C., Winblad, B., and Fratiglioni, L. 2002, J Clin Epidemiol, 55: 959-964. Deng, J., Zhou, D., Li, J., Wang, Y., Gao, C., and Chen, M. 2006, Clin Neurol Neurosurg, 108(4): 378-383. Ngandu, T., Helkala, E.L., Soininen, H., Winblad, B., Tuomilehto, J., Nissinen, A., and Kivipelto, M. 2007, Dement Geriatr Disord, 23: 140-149. Espeland, M., Gu, L., Masaki, K., Langer, R., Coker, L., Stefanick, M., Ockene, J., and Rapp, S. 2005, Am J Epidemiol, 161: 228-238. Stampfer, M., Kang, J.H., Chen, J., Cherry, R., and Grodstein, F. 2005, N Engl J Med, 352: 245-253. Ganguli, M., Vander Bilt, J., Saxton, J., Shen, C., and Dodge, H. 2005, Neurology, 65: 1210-1217. Antila, T., Helkala, E.L., Viitanen, M., Kareholt, I., Fratiglioni, L., Winblad, B., Soininen, H., Tuomilehto, J., Nissinen, A., and Kivipelto, M. 2004, Brit Med J, 329(7465): 539. doi: 10.1136/bmj.38181.418958.BE. Solfrizzi, V., D'Introno, A., Colacicco, A.M., Capurso, C., Del Parigi, A., Baldassarre, G., Scapiccho, P., Scafato, E., Amodio, M., Capurso, A., and Panza, F. 2007, Neurology, 68: 1790-1799.

Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Recent Advances on Nutrition and the Prevention of Alzheimer’s Disease, 2010: 71-95 ISBN: 978-81-7895-470-7 Editors: Charles Ramassamy and Stéphane Bastianetto

5. Are fish and docosahexaenoic acid linked to lower risk of cognitive decline and Alzheimer’s disease? M.E. Bégin, M. Plourde, F. Pifferi and S.C. Cunnane Research Center on Aging, Health and Social Services Centre-Sherbrooke Geriatrics University Institute and Department of Medicine, Université de Sherbrooke, Sherbrooke, Québec J1H 4C4, Canada

Abstract. We review here the published studies examining the possible links between fish and seafood consumption or docosahexaenoic acid (DHA) intake and cognitive impairment, cognitive decline and dementia including Alzheimer’s disease (AD). We focused on three types of human studies: 1) epidemiological studies of fish and seafood intake, 2) analysis of DHA intake, and DHA content of blood and brain tissues, and 3) clinical trials using DHA-enriched oils as treatment for cognitively impaired nondemented elderly and for patients with dementia including AD. Our assessment is that prospective epidemiological studies are most supportive for a protective role of fish and seafood intake against the risk of cognitive decline, all-cause dementia and AD. However, the commonly discussed link between lower dietary DHA, lower blood DHA or lower brain DHA and mild cognitive impairment, cognitive Correspondence/Reprint request: Dr. S.C. Cunnane, Research Center on Aging, Health and Social Services Centre-Sherbrooke Geriatrics University Institute and Department of Medicine, Université de Sherbrooke Sherbrooke, Québec, J1H 4C4, Canada. E-mail: stephen.cunnane@usherbrooke.ca

M.E. Bégin et al.

decline or AD does not appear to be consistently supported by a full overview of published reports. Moreover, in intervention studies, DHA-enriched oils induced only modest clinical benefits against mild cognitive impairment and may have a role in primary prevention of AD but not in treatment of manifest disease. At present, there is no clear explanation for the apparent lack for a consistent association between dietary DHA, blood DHA or brain DHA and the presence or risk of mild cognitive impairment, cognitive decline or AD, but it may be more methodological than biological. Directions for future research are proposed.

List of abbreviations AD: ApoE ε4: ARA: CIND: DHA: DPA: EPA: MMSE: PC: PE: PUFA: ω3: ω6:

Alzheimer’s disease apolipoprotein E epsilon 4 arachidonic acid cognitively impaired non-demented docosahexaenoic acid docosapentaenoic acid eicosapentaenoic acid Mini-Mental State Examination phosphatidylcholine phosphatidylethanolamine polyunsaturated fatty acids omega-3 omega-6

Introduction Cognitive decline is an increasing burden since its prevalence, leading to dementia and AD, is expected to increase as populations age. The extent and rate of cognitive decline can vary considerably between individuals. Small cognitive deficits may represent an early manifestation of the pathology of Alzheimer’s disease (AD). These small cognitive deficits may progressively increase in severity to the levels reached in clinically diagnosed AD, the most frequent form of dementia and the primary neurodegenerative disorder in the elderly [1]. Most of the subjects with mild cognitive deficits will progress to AD at a rate of 10 -15% per year compared with healthy control subjects who convert at a rate of 1 - 2% per year [1, 2]. The cause of the progression of mild cognitive deficits to dementia and AD is as yet unknown, but once it is clinically diagnosed, there is little prospect of improving the prognosis of AD. Cognitive decline in the elderly, particularly in the form of AD, constitutes a major challenge to the quality of life for the elderly and their caregivers, and to healthcare resources.

Fish and DHA in cognitive decline and Alzheimer’s disease

Since aging is unavoidable and since AD is as yet incurable, strategies to identify environmental factors for lowering the risk of AD, such as diet, are essential. One such strategy may be the consumption of fish and seafood (shellfish and crustacean) naturally containing ω3 polyunsaturated fatty acids (PUFA). Indeed, ω3 PUFA are closely associated with better functioning of the brain throughout life, especially docosahexaenoic acid (DHA), which is a major component of brain membrane phospholipids. Fish and seafood consumption is the main dietary source of preformed DHA. Whether ω3 PUFA from fish and seafood, especially DHA, might be the principal nutrients in preventing cognitive impairment, cognitive decline, dementia and AD in the elderly is presently debated. Therefore, we have reviewed here the evidence for a possible link between fish and seafood or DHA intakes and cognitive impairment, cognitive decline and dementia including AD with emphasis on three types of human studies – epidemiological studies on fish and seafood intake or DHA intake, analysis of DHA levels in blood or brain tissues, and clinical trials of supplementation with DHA-enriched oils in cognitively impaired non-demented (CIND) elderly and in demented patients. We examined the following questions: 1) Does the intake of fish and seafood protect against cognitive impairment, cognitive decline and dementia including AD? 2) What is the biological evidence from blood and brain fatty acid analyses that DHA plays a significant role in the protective effect of fish and seafood consumption? 3) What is the clinical benefit of DHA treatment against cognitive impairment and AD?

Definitions and classification In this review, we will use the terms cognition, cognitive impairment, cognitive decline, cognitive dysfunction, dementia and AD. Cognition. Mental activities involved in the acquisition, storage, retrieval and use of information are referred to here by the term “cognition” [3]. Manifestations of cognitive behaviour are achieved through the integration of a variety of processes and activities such as perception, imagery, memory, speed of information processing, reasoning, problem solving, decision making and language. Cognitive impairment. Cognitive impairment refers to a condition between normal aging and AD in which persons experience objective cognitive deficits to a greater extent than one would expect for age, yet they do not meet currently accepted criteria for clinically probable dementia [1, 4]. The expression mild cognitive impairment represents preclinical AD or very

M.E. Bégin et al.

mild AD and clinical subtypes may be differentially related to risk of AD [1, 2, 5, 6]. Cognitively impaired non-demented (CIND) persons. Throughout this review, non-demented persons showing cognitive impairment or mild cognitive impairment will be referred to as cognitively impaired non-demented (CIND) persons. CIND persons have a higher risk of progression to dementia, but the evolution is heterogeneous, as some persons can improve over time or remain stable [1, 2]. Cognitive decline. Cognitive decline will specify the progression of cognitive impairment over time. Cognitive dysfunction. Cognitive dysfunction is a generic term that includes conditions ranging in severity from the relatively benign mild cognitive impairment to dementia. All-cause dementia. Dementia is a syndrome caused by damage or disease that affects the brain. It is characterized by a progressive decline in cognitive function beyond what might be expected from normal healthy aging. It constitutes a large category that includes AD, stroke or vascular dementia, and fronto-temporal lobe dementia (Pick’s disease). In most of the studies reported here, dementia was diagnosed according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders including an initial screening of the participants with the Mini-Mental State Examination (MMSE) or by neuropsychological tests and a clinical examination. Alzheimer’s disease. The most common form of dementia is AD. The neurodegenerative disorder is characterized by progressive memory loss, decline in abstract reasoning, visuospatial perceptual changes, reduced time of reaction and language difficulties. In thirteen of fourteen of the studies examined here, the diagnosis of AD was based on the criteria defined by the National Institute of Neurological and Communicative Disorders and StrokeAlzheimer’s Disease and Related Disorders Association.

Fish consumption, DHA intakes, cognitive decline and dementia Three cross-sectional studies investigated the impact of fish and seafood consumption or ω3 PUFA on the risk of cognitive impairment in nondemented subjects (Table 1). Only one of them reported that both fish and long-chain ω3 PUFA intakes lowered the risk of cognitive impairment significantly in nondemented middle-aged adults [7]. In the other two studies involving older

Fish and DHA in cognitive decline and Alzheimer’s disease

Table 1. Cross-sectional studies showing fish or ω3 PUFA intake lowers (A) or does not lower (B) the risk of cognitive impairment in non-demented middle-aged and old adults. Reference

Participants N Age (y)

Exposurea EPA + DHA Fish g/d g/d

Risk value

A. LOWER RISK Kalmijn et al. [7]

1613

45-70

0.17

0.8b

B. NO RISK REDUCTION Kalmijn et al. [8]

476

69-89

>20

0 to 2

van Gelder et al. [9]

210

70-89

0 to >20

0.02 to 0.4

The fish and ω3 PUFA intakes were derived from food frequency questionnaires or a dietary histories b odds ratio with p ≤ 0.05 DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; PUFA: polyunsaturated fatty acids; NS: not significant.

participants, neither fish consumption nor ω3 PUFA intakes were associated significantly with lower risk of cognitive impairment [8, 9]. Therefore, there is not much support for protection against cognitive impairment by fish and long chain ω3 PUFA intakes when evaluated by a cross-sectional design. In prospective studies following CIND elderly during 3 to 5-years (Table 2), an intake of more than 105 g of fish per week lowered the rate of cognitive decline by 10 to 75% [8-10]. The estimated long-chain ω3 PUFA or eicosapentaenoic acid (EPA) + DHA intakes corresponding to the amount of fish consumed gave inconsistent results. Two studies [9, 11], but not three others [2, 8, 10], reported that they were associated significantly with lower risk of cognitive decline. Hence, the effects of EPA+DHA or long-chain ω3 PUFA (EPA + docosapentaenoic acid (DPA) + DHA) intakes do not appear to always reflect that of fish intake. The possible difference in ω3 PUFA composition among different species of fish consumed may account in part for the variation in the amounts of the estimated ω3 PUFA intakes between the studies. Most support for a protective role of fish and seafood consumption comes from prospective studies investigating their effects against the risk of dementia or AD (Table 3). Four studies revealed that 1 to 2 servings of fish per week lowered the risk of all-cause dementia significantly [12-15]. Similar findings were reported for AD [12-15]. In contrast, estimated ω3 PUFA or ω6 + ω3 PUFA intakes were associated with lower risk of dementia in two of three studies [13, 17], and with no risk reduction of AD in three of four studies [13, 16-18]. Thus, the effects of estimated EPA+DHA or long-chain ω3 PUFA intakes did not

M.E. Bégin et al.

Table 2. Prospective studies showing fish or ω3 PUFA intake lowers (A) or does not lower (B) the risk of cognitive decline in cognitively impaired non-demented adults. Reference

Participants N Age (y)

Followup (y)

Exposurea

% rate reduction or risk value

1. FISH INTAKE A. LOWER RISK Morris et al., [10]

3718

≥65

≥1 fish serving/wk

10*

van Gelder et al. [9]

210

70-89

>140 g fish/wk

75*

B. NO RISK REDUCTION Kalmijn et al. [8]

342

69-89

105 g fish/wk

2. ω3 PUFA INTAKE A. LOWER RISK Beydoun et al. [11]

2251

50-65

increase of 1 SD in EPA+DPA+DHA (% energy intake)

0.79b,* (verbal fluency only)

van Gelder et al. [9]

210

70-89

0.38 g EPA+DHA/d

73*

B. NO RISK REDUCTION Kalmijn et al. [8]

342

69-89

Morris et al. [10]

3718

≥65

0.0 – 2 g EPA+DHA/d 1.6 g ALA+EPA+DPA +DHA/d

Solfrizzi et al. [2]

278

65-84

2.6

≥9g PUFAc/d

The fish and ω3 PUFA intakes were derived from food frequency questionnaires or a dietary histories b odds ratio c amounts and types of ω6 or ω3 PUFA not reported * p < 0.05 ALA: alpha-linolenic acid; DHA: docosahexaenoic acid; DPA: docosapentaenoic acid; EPA: eicosapentaenoic acid; NS: not significant; PUFA: polyunsaturated fatty acids; SD: standard deviation

Fish and DHA in cognitive decline and Alzheimer’s disease

Table 3. Prospective studies showing fish or ω3 PUFA intake lowers (A) or does not lower (B) the risk of all-cause dementia (D) or Alzheimer’s disease (AD). Reference

1. FISH INTAKE A. LOWER RISK Kalmijn et al. 1997b

Participants N Age (y)

Followup (y)

Amount fish/wk a PUFA g/d a (mean or range)

Risk value D

5386

2.1

>130 g serving

0.4b

0.3b

5944

≥65

1 serving

0.6c,†

0.7c

Barberger-Gateau et al. 2007 Barberger-Gateau et al. 2002 Huang et al. 2005

1416

≥68

≥1 serving

0.7c

1570

≥65

5.4

>2 servings

0.7c,†

0.6c,†

B. NO RISK REDUCTION Morris et al., 2003

815

65-94

3.9

≥1 serving

Huang et al. 2005

474

≥65

5.4

>2 servings

NS‡

Barberger-Gateau et al. 2007

1479

≥65

1 serving

NS‡

815

65-94

3.9

1.8 g ALA+EPA+DHA 0.06 g DHA 0.5 - 0.8 g ω6+ω3 0.9 - 2.9 g ω6+ω3

0.4 0.5d,‡

0.4b NS 0.4d,‡

2. ω3 PUFA INTAKE A. LOWER RISK Morris et al. 2003

NS NS‡

0.3b d,‡

Laitinen et al. 2006

1449

65-80

Barberger-Gateau et al. 2007

7783

≥65

ω3e

0.4c

5395

16.6 g total ω6 1.3 g total ω3

B. NO RISK REDUCTION Engelhart et al. 2002

derived from food frequency questionnaires or dietary histories relative risk c hazard ratio d odds ratio e from ω3 rich oils, amount and type of ω3 PUFA not reported † for apolipoprotein E ε4 non-carriers only ‡ for apolipoprotein E ε4 carriers only Results are significant at p ≤ 0.05 ALA: alpha-linolenic acid; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; NS: not significant; PUFA: polyunsaturated fatty acids. b

M.E. Bégin et al.

consistently match that of fish intake. The lack of a consistent association may well be related to methodological differences such as varying follow-up periods, variability of ω3 PUFA composition among different species of fish consumed, and to other nutrients in fish and seafood such as proteins, iodine and/or selenium, which may interact with ω3 PUFA and assist in lowering the risk for dementia and AD [13, 19]. Another possibility could be that fish and seafood consumption or ω3 PUFA intakes are associated with lower risk of cognitive decline but only in specific cognitive domains rather than with a single global measure of cognitive function. Indeed, five studies have addressed the association of fish and seafood consumption or ω3 PUFA intakes with performance in memory, language, speed and visuospatial specific domains. It was reported that moderate intakes of fish and seafood products or ω3 PUFA could lower the risk of decline in verbal fluency [20] and in speed-related domains [7, 21]. However, others have found no improvement in language [21] or speedrelated domains [20, 22]. High fish and seafood consumers (70-74 y) whose daily intake of marine products was about 85 g/d had significantly better performance in episodic memory, perceptual speed, visuospatial skills and verbal fluency tests than those whose intake was <10 g/d [23]. Conversely, Whalley et al. [22] reported that fish-oil users improved only in visuospatial skills but not in the other specific cognitive domains. Overall, in the few studies looking at specific cognitive domains, most supported a protective effect of moderate consumption of fish and seafood against cognitive decline in language, visuospatial and speed-related domains. The inconsistencies of the findings in some specific cognitive domains might be related to the use of different neuropsychological tests by the authors. All together, we suggest that the available epidemiological literature provides good arguments for the potential of fish and seafood consumption to protect against cognitive impairment, cognitive decline and dementia including AD. However, the effects of the estimated EPA+DHA or longchain ω3 PUFA intakes do not consistently reproduce those obtained by fish and seafood consumption.

Blood DHA, cognitive decline and Alzheimer’s disease Measurements of ω3 PUFA in plasma or in erythrocytes are generally considered as better estimates of ω3 PUFA status than those obtained from dietary assessments since they can be regarded as an integrated measure of short to medium-term dietary fatty acids intake, absorption, and individual metabolism [24-31]. Because of the importance of DHA in brain function [32] and since DHA is brought to the brain via the blood, several investigators

Fish and DHA in cognitive decline and Alzheimer’s disease

Table 4. Blood docosahexaenoic acid (DHA) in cognitively impaired non-demented (CIND) elderly. Reference

Cognitive status

Age (y) Mean

Followup (y)

DHA % of total fatty acids

Control

4.6 ± 0.4

CIND

3.7 ± 0.2

Control

2.1 ± 0.8

CIND

2.1 ± 0.8

Control

≥65

3.3 ± 1.1

CIND

≥65

3.1 ± 1.1

Control

2.0 ± 0.7

CIND

2.1 ± 0.8

Control

2111

2.9 ± 0.9

CIND

140

3.0 ± 0.9

Control

2111

0.5 ± 0.2

CIND

140

0.5 ± 0.2

Control

725

2.3 ± 0.1

CIND

153

2.2 ± 0.1

Control

219

DHA in CIND (% control)

PLASMA TPL Conquer et al. [33]

Laurin et al. [34]a

Manzato et al. [35]

Laurin et al. [34]b

Beydoun et al. [20]

80*

100

105

103

PLASMA CE Beydoun et al. [20]

100

PLASMA TL Cherubini et al. [36]

ERYTHROCY TE TPL 4 Heude et al. [37]

6.3 ± 1.1 4

CIND a

cross-section analysis

prospective analysis

*p < 0.05 CE: cholesteryl esters; TL: total lipids; TPL: total phospholipids.

5.9 ± 1.0

94*

M.E. Bégin et al.

have analyzed the link between blood DHA status and the risk of cognitive decline and dementia. Plasma DHA can be determined in four major lipid classes, namely phospholipids, triglycerides, cholesteryl esters and free fatty acids. Phospholipids can be separated into different classes among which phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are two major components. Erythrocyte DHA has been determined from the total phospholipid or total lipid fractions of the erythrocyte membrane. As shown in Table 4, a limited number of studies have documented DHA proportions in plasma total phospholipids, cholesteryl esters and total lipids, or in erythrocytes in middle-aged and older subjects with CIND. Among these studies, only two reported significantly lower DHA proportions in plasma total phospholipids [33] or in erythrocytes [37] in elderly with cognitive impairment or cognitive decline, respectively, compared to healthy controls. Table 5. Blood docosahexaenoic acid (DHA) in all-cause dementia (D) or Alzheimer’s disease (AD). Reference

Cognitive status

Age (y) Mean

DHA % of total fatty acids

DHA in D, AD (% control) or risk value

Control

5.6 ± 0.6

MID

5.5 ± 0.2

4.4 ± 0.9

79*

Control

4.6 ± 0.4

3.1 ± 0.2

Control

2.1 ± 0.8

2.3 ± 0.8

Control

2.0 ± 0.7

2.7 ± 1.2

135

>4.2%

0.5c,*

>4.2%

PLASMA TPL Corrigan et al. [38]

Conquer et al. [33]

Laurin et al. [34]a

Laurin et al. [34]b

67*

110

PLASMA PC Schaefer et al. [39]

Fish and DHA in cognitive decline and Alzheimer’s disease

Table 5. Continued PLASMA CE Corrigan et al. [38]

Tully et al. [40]

Control

0.4 ± 0.4

MID

2.0 ± 1.0

500*

0.6 ± 0.5

150*

Control

1.2 ± 0.9

AD1

0.6 ± 0.5

Control

725

2.3 ± 0.1

2.0 ± 0.2

Control

N/A

41.2 ± 20.92

35.3 ± 21.52

AD3

5.4 ± 1.64

AD5

4.2 ± 1.24,6

Control

3.2 ± 2.5

4.4 ± 2.2

50*

PLASMA TL Cherubini et al. [36]

ERYTHROCYTE TL Boston et al. [41]

Wang et al. [42]

ERYTHROCYTE TPL Corrigan et al. [38]

cross-sectional study

prospective study

relative risk

1 2

138*

quartile with lowest MMSE (score = 12.9 ± 3.9) µg/g

mild AD (MMSE score = 24-27) mmol/L 5 moderately severe AD (MMSE score = 16-19) 4

values between moderately severe AD and mild AD are not significantly different * p < 0.05, CE: cholesteryl esters; MID: multi-infarct dementia; MMSE: Mini-Mental State Examination; N/A: not available; PC: phosphatidylcholine; NS: not significant; TL: total lipids; TPL: total phospholipids.

M.E. BĂŠgin et al.

The link between the % composition of DHA in plasma total phospholipids, PC, cholesteryl esters or total lipids, or in erythrocytes, and the presence or risk of all-cause dementia or AD is reported in Table 5. One prospective study with a follow-up of a mean of 9.1 years reported that 76 y subjects with plasma PC DHA levels greater than 4.2% of total fatty acids experienced a significantly lower risk (relative risk = 0.5) of developing allcause dementia compared with participants with levels lower than 4.2% of total fatty acids [39]. A similar trend was observed for the development of AD but the differences in relative risks did not reach statistical significance. If AD is associated with lower fish intakes, elderly with AD should have lower DHA in plasma or erythrocytes. However, among the published studies, three reported significant lower plasma DHA in AD compared to healthy controls, two reporting for plasma total phospholipids [33, 38], and the other for plasma cholesteryl esters [40]. Surprisingly, Corrigan et al. [38] obtained significantly higher DHA levels in plasma cholesteryl esters and in erythrocyte total phospholipids in AD compared to healthy controls. Hence, inconsistent data between studies were reported within each of the plasma total phospholipids or cholesteryl ester fractions as well as in the erythrocytes. These inconsistencies in blood DHA data between studies may be due to the variability in the DHA levels found in CIND or demented participants and/or in age-matched healthy controls. We have shown previously that upon fish oil supplementation, healthy elderly subjects 70-79 y had higher DHA into plasma lipids than 18-30 y old adults [43]. Since DHA levels in plasma of CIND or AD elderly were compared to those of age-matched healthy control subjects, age as a possible confounder may not explain the incongruities. Rather, these inconsistencies might reflect differences in food intake patterns of different populations from different geographical regions if we assume that DHA into blood lipids is similar among elderly of 60 to 80 years old [44, 45]. The differences in the number of demented participants across the studies do not appear to be a plausible explanation since the total number of demented subjects (N=190) with lower blood DHA levels is close to the number of demented subjects who do not have lower plasma DHA (N=194). As a whole, the current literature does not adequately support the commonly discussed link between lower blood DHA and cognitive impairment, cognitive decline, dementia or AD. To date, no lipid fraction in particular stands out as ideal for the analysis of the relation between blood DHA, cognitive impairment, cognitive decline, dementia and AD. Since there is substantial variability observed in the blood DHA proportions in AD subjects, it may represent large variations in individual biological responses, but whether both are linked to development of dementia is unknown at present.

Fish and DHA in cognitive decline and Alzheimer’s disease

Brain DHA and Alzheimer’s disease Cognitive decline being the central clinical feature of AD and since DHA is a major constituent of brain membrane phospholipids, comparison of DHA content in brain phospholipids in AD subjects relative to age-matched healthy controls could reveal more directly the nature and the importance of the link between DHA status and cognitive decline in AD than the analyses described above. During normal aging, the brain total phospholipid content of healthy participants reportedly decreases by about 43% from 30 - 100 y old [46]. In AD, brain PE and PC concentrations were 20 - 30% lower in the frontal cortex and about 14% lower in the hippocampus compared to those of age-matched healthy elderly respectively ([47, 48], data not shown). Thus, the loss of brain PE and PC concentrations is more extensive in AD than in healthy elderly. Although there is a loss of brain PE and PC content in AD subjects compared to healthy elderly, the proportions of DHA (% DHA) relative to other fatty acids in phospholipids in three brain regions normally affected by AD showed inconsistent results (Table 6). The few studies published reveal that - 1) only DHA in PE of the hippocampus was significantly lower in AD than in age-matched controls [48, 50], and 2) except for the study of Soderberg et al. [48], no significant change in DHA proportions in PE or PC between AD and age-matched healthy controls was observed in the frontal cortex or the parahippocampus. In AD, a decrease in brain DHA proportions (absolute data not shown) appears not to be specific for DHA. Indeed, either arachidonic acid (ARA) and/or adrenic acid were also decreased [48, 51, 52], or the lower DHA was a function of a decrease mostly in plasmalogen PE [47]. Indeed, increases (up to 50%) in DHA [47, 49, 50] and 4 fold in adrenic acid [49] have also been reported for some regions of AD brain. In summary, besides lower DHA in the hippocampus, the available literature suggests that elderly with AD have similar DHA proportions to age-matched healthy elderly. The changes in brain phospholipid concentrations during AD may be equally or more relevant than the possible changes in fatty acid composition of these lipid classes [44, 53].

APOE ε4, fish and ω3 PUFA intakes A combination of genetic and environmental factors most likely contributes to the etiology of AD. Among the genetic factors, the apolipoprotein E (ApoE) ε4 genotype is the genetic risk factor most associated with AD [54] and has been associated with greater risks of cognitive decline [55, 56] and progression from the preclinical state to AD [57, 58]. There are three isoforms (ApoE ε2, ApoE ε3 and ApoE ε4) that differ in their abilities to accomplish the critical

M.E. BĂŠgin et al.

Table 6. Docosahexaenoic acid (DHA) in brain phospholipids in healthy elderly (controls) compared to elderly with Alzheimer's disease (AD)a. Reference

Cognitive

DHAb

Age (y)

DHA in AD

status

(% control) TL

54*

57*

121

47*

55*

FRONTAL CORTEX

Soderberg et al. [48]

Control

8-10

n/a

23.5

1.4

8-10

n/a

12.6

0.8

Control

2.1c

1.1

2.5

0.8

Control

18.6

17.3

Control

3.6

4.6

Control

8-10

n/a

16.9

0.9

8-10

n/a

7.9

0.6

Control

1.0c

121

0.6

Control

20.7

1.6

20.5

1.4

Guan et al. [47] Skinner et al. [49]1

Skinner et al. [49]2

130

HIPPOCAMPUS

Soderberg et al. [48]

Prasad et al. [50]

PARAHIPP OCAMPUS

Corrigan et al. [51]

Fish and DHA in cognitive decline and Alzheimer’s disease

Table 6. Continued Skinner et al. [49]1

Control

16.9

16.6

Control

5.1

5.0

Skinner et al. [49]2

Modified from Bégin et al. 2009 Unless otherwise noted, the DHA values are % of total fatty acids c µmol/g wet tissue 1 Total lipids of gray matter 2 Total lipids of white matter *p < 0.05 n/a: not available; PC: phosphatidylcholine; PE: phosphatidylethanolamine; TL: total lipids b

functions of redistributing lipids among brain cells for normal lipid homeostasis, repairing injured neurons and maintaining synapto-dendritic connections [58]. ApoE ε4 is associated with neurodegenerative pathology due to its unique structural features that distinguish it from the other ApoE [58]. It is thus plausible that genes involved in lipid metabolism and transport interact with environmental factors for lowering or increasing the risk of AD. ApoE ε4 genotype may be an important confounder in lowering the risk of dementia and AD by fish and seafood consumption. Indeed, two prospective studies reported in Table 3 show that ApoE ε4 carriers were not protected against all-cause dementia when consuming fish and seafood [13, 15]. However, when looking at the risk for AD, mixed results were obtained: one study showed that there was no interaction between fish consumption and ApoE ε4 genotype on the risk of AD [13] while another study reported that frequent fish consumption reduced the risk of AD but only in ApoE ε4 noncarriers. Conversely, Laitinen et al. [17] reported that moderate PUFA intake at midlife was protective against both dementia and AD, especially among ApoE ε4 carriers. However, they limited their estimated PUFA intakes to fat coming from spreads and they did not distinguish between ω3 and ω6 PUFA. Recently, we have shown that ApoE ε4 carriers may also have altered ω3 PUFA metabolism since they had 59% higher plasma ω3 PUFA levels and had a lower rise in plasma ω3 PUFA incorporation after ω3 PUFA supplementation compared to the non-carriers [59]. Given that ApoE ε4 is the most important genetic risk factor for AD and that mixed findings are reported in the literature, the interaction between fish and seafood consumption and ApoE ε4 genotype deserves further research.

M.E. Bégin et al.

DHA treatment of dementia Whether higher DHA intake can be clinically beneficial in preventing or reducing cognitive dysfunction in dementia, especially AD, is the central practical issue in linking low DHA to the risk of dementia. Table 7. Effect of docosahexaenoic acid (DHA) treatment of cognitively impaired non-demented (CIND) elderly and patients with Alzheimer’s disease (AD) or vascular dementia (VD). Reference

Cognitive

status

Age

Supplement

(y)

Dose

Duration

(g/d)

(mo)

Outcome

Kotani et al.

CIND

0.2

No change

[60]

CIND

DHA +

0.2

8 - 15%

ARA

higher scoresa

DHA +

0.2

No changeb

ARA Chiu et al.

CIND

55-90

1.8

-0.4 ± 1.4c

CIND

55-90

DHA+ EPA

0.7 +

-3.2 ± 3.8 c,d

[61] 1.1 AD

55-90

1.8

-3.7 ± 4.3 c

55-90

DHA+ EPA

0.7 +

-2.5 ± 2.6 c

75% of

1.1 Suzuki et al.

Control

[62]

DHA +

0.6 +

EPA

0.5

subjects improvede

Freund-Levi

et al. [63] AD

DHA +

0.6 +

55% of

EPA

0.5

subjects

P/DHA +

4.0/1.7

6/6

improvede No changef

EPA

+ 0.6

DHA +

1.7 +

No changef

EPA

0.6

Fish and DHA in cognitive decline and Alzheimerâ&#x20AC;&#x2122;s disease

Table 7. Continued Terano et al.

None

DHA

No change

17% higher

[64] 4.3

scoresg a

attention (p<0.05) and memory (p<0.01) tests

relative to placebo

change in scores of the cognitive portion of the Alzheimerâ&#x20AC;&#x2122;s Disease Assessment Scale

significantly different relative to placebo with p = 0.03

% of subjects with improved intelligence score

except in subjects with scores >27 on the Mini-Mental State Examination

p < 0.05 at 3 and 6 months, but not at 12 months after supplementation

ARA: arachidonic acid; EPA: eicosapentaenoic acid; P: placebo.

The published results of DHA treatment on cognitive dysfunction in nondemented and demented elderly are shown in Table 7. Only two placebocontrolled studies examined the impact of DHA supplementation in the elderly with diagnosed mild cognitive impairment (CIND; [60, 61]). Both studies suggested that a combination of DHA+ARA [60] or of DHA+EPA [61] improved cognitive performance modestly in CIND patients. The efficacy of DHA intervention in AD and vascular dementia cases was assessed in five intervention studies. Only two of these studies reported that DHA treatment improved cognitive performance and in both, the improvements were modest. Suzuki et al. [62] reported that 75% of healthy controls compared to 55% of elderly with AD had improved cognition after DHA treatment. Unfortunately, the improvements could not be matched to the changes in blood DHA after treatment. Similarly, in the pilot study by Terano et al. [64], subjects with mild to moderate vascular dementia supplemented with DHA monotherapy improved their dementia rating scores by 17% in parallel to an increase in serum DHA levels by 179%. In contrast, three studies reported that a combination of DHA and EPA or ARA (at combined doses ranging from 0.2 to 2.3 g/d) did not improve cognitive performance of AD patients [60, 61, 63]. In the largest study, patients with AD were randomized into a double-blind placebo-controlled clinical trial and received a supplement of DHA+EPA or placebo during the first 6 months of the study, after which all patients received the DHA+EPA supplement for an additional 6 months [63]. After the first 6 months, cognitive decline in patients with mild to moderate AD was not delayed, but was significantly slower in a small group of very mild AD patients with MMSE scores >27 [63].

M.E. BĂŠgin et al.

Thus, epidemiological studies suggest that DHA in combination with EPA or ARA may have a role in primary prevention of AD but these clinical trials do not yet support the utility of DHA in the treatment of manifest cognitive decline in the elderly. However, interpretation of the available clinical trials is limited by the fact that they have all been small and of short duration. Furthermore, since they used DHA in the presence of either EPA [61-63] or ARA [60], it remains to be established whether DHA has a beneficial clinical effect on its own. One interpretation is that PUFA other than DHA may possibly be required to induce a beneficial clinical effect on deteriorating cognition in the elderly. Finally, the doses of DHA used varied from 120 mg/d to 4.3 g/d, which is a 36-fold range. There is no clear explanation as to why the necessary dose exceeds many fold the apparent brain turnover of DHA of 4 mg/d [65].

Interpretating blood and brain DHA data in relation to fish intake Prospective studies on fish and seafood consumption are most supportive of a protective role of fish and seafood products against cognitive dysfunction in elderly with CIND or with dementia including AD. Contrary to what is commonly reported, DHA levels in plasma, erythrocytes or brain do not consistently explain the protective effect of fish and seafood consumption against cognitive impairment and dementia, due mainly to the inconsistencies of the findings and to the large dispersion of the data. At present, there is no clear explanation for this apparent lack of a consistent association between low DHA and risk of cognitive decline and dementia including AD. However, we suggest the two following possibilities: If low DHA intake is not linked to the risk of AD but low fish and seafood intake is, it is possible that nutrients other than DHA or EPA in fish and seafood products may actually be the nutrients protective against AD, such as proteins, minerals (ex: selenium, iodine, zinc, manganese) or other bioactive substances like niacin, antioxidants, etc [13, 19, 66, 67]. Thus DHA intake may simply be the marker of dietary intake of a different nutrient to which it is closely linked in fish and seafood products or in other foods that are part of the diet of regular fish and seafood consumers. It is also plausible that the protective effect of fish and seafood may be the result of interactions between Ď&#x2030;3 PUFA, especially DHA and EPA, and such other nutrients found in fish and seafood products. The other possibility is that there is really a link between DHA and cognitive health in the elderly but that, owing methodological differences between studies or biological changes in DHA metabolism consequent to AD

Fish and DHA in cognitive decline and Alzheimer’s disease

itself, it is not revealed in the literature as it stands. If that is the case, further research needs to start by elucidating why the blood and brain DHA data in AD do not agree with the fish and seafood intake data. Because DHA synthesis from alpha-linolenic acid and its β-oxidation are minimal in humans [68], preformed DHA intake is required to maintain whole body DHA homeostasis in humans. For instance, net brain DHA levels may depend on the balance between processes linked to DHA degradation such as peroxidation or its loss due to cell death in the brain and variables linked to DHA utilisation and/or to DHA replenishment such as dietary DHA intake and its transport into the brain. At the present stage of investigation, the link between DHA intake and brain DHA may be more complex than anticipated: indeed, there is no a priori reason to believe that there should be a simple link between DHA intake, blood DHA and brain DHA in the healthy elderly, let alone in AD. Moreover, isolated biological DHA measurements may have little meaning given the opposite effects of ω3 and ω6 PUFA on cognitive impairment [12], cognitive decline [37], dementia [13] and neuroinflammation involved in neurodegenerative pathology [19, 69]. Indeed, DHA content in relation to other ω3 or ω6 PUFA, overall fatty acids or cholesterol might be more informative than DHA alone. Furthermore, a higher percentage of a specific fatty acid or group of fatty acids will interdependently bring a lower percentage of another. Hence, this problem of interdependence makes it potentially difficult to interpret the effect of a single constituent or group of constituents.

Limitations of studies Almost every aspect of the studies cited here showed inconsistent findings. Several factors may account for these inconsistencies: differences in sample size, heterogeneity of study populations, types of population analyses (cross-sectional vs prospective), adjustments for different confounders and the wide variety of the applied cognitive tests, different methods of estimating ω3 PUFA intakes, widely differing doses and PUFA composition of supplements used, and choice of tissues and lipid fractions analyzed. Comparison between different studies is difficult due to lack of uniform quantification of fish and seafood or ω3 PUFA and DHA intakes. DHA composition and DHA intake estimates may vary with species of fish and cooking methods [15, 70, 71]. Differences of food intake patterns in different populations and cultural backgrounds may complicate the comparison of findings from populations of different geographical areas. Most studies reviewed here, with few exceptions, adjusted the association between fish and seafood consumption or ω3 PUFA intake and cognitive

M.E. BĂŠgin et al.

impairment, cognitive decline, dementia including AD for a list of confounders that are linked with regular fish consumers such as dietary habits (intake of fruits, vegetables and alcohol) and lifestyles, socioeconomic characteristics (education, income), and medical conditions (hypertension, past stroke, and depressive symptoms), but the particular confounders examined varied between studies. Cross-sectional studies are of limited value because they cannot ascertain whether the diet was the cause or the consequence of the cognitive impairment. In particular, CIND elderly may have altered their diet as a consequence of the disease, or the diet of demented patients may have been modified by their caregivers and therefore not reflect their past dietary habits. Prospective studies with at least 6 y-follow-ups are recommended because this follow-up period was shown to be less prone to bias due to subclinical dementia [18] and was the time needed for significant cognitive decline in specific cognitive domains to become more apparent in susceptible individuals compared to a 3y follow-up period [72]. Part of the inconsistencies across studies between the fish and seafood intake data and blood or brain DHA levels may be due to artifacts caused by differences in methodology including different definitions of cognitive dysfunction (ex: various cut-off points indicative of cognitive impairment or cognitive decline in MMSE, different number and types of neuropsychological tests), diverse analytical methods, possible different criteria of â&#x20AC;&#x2DC;healthyâ&#x20AC;&#x2122; age-matched controls, and differences between normal age-related cognitive decline and pathological cognitive dysfunction. Therefore, standardization of definitions, clinical classifications of severity of pathological cognitive dysfunction and of analytical procedures appears required in order to draw valid general conclusions made from comparable evaluations.

Future research directions Three years after the extensive review by Maclean et al. [73] have not clarified whether and, if so, how DHA is linked to AD. Whether DHA status plays an etiologic role in the development of dementia, especially AD or may constitute simply a secondary marker of the disease process remains to be established. Thus, it is crucial to investigate the true nature and importance of the link between DHA and AD. We propose that new directions of research be taken. For example, the study of DHA transporters (lipoproteins and other circulating DHA binding proteins) or free circulating DHA may be undertaken to help elucidate the link between plasma and brain DHA status and progression to, or severity of, dementia and AD. Aside from this new direction of biological analysis, well designed clinical studies could be directed at resolving the question of whether the discrepancies between the epidemiological and tissue DHA analysis in AD is methodological or biological. Specifically, they should answer the following

Fish and DHA in cognitive decline and Alzheimer’s disease

two questions: 1) do changes in DHA metabolism in AD raise plasma DHA, thereby counterbalancing the normal relation between low DHA intake and low plasma DHA?, and 2) does the amount of fish or DHA needed to protect against AD reproducibly change plasma DHA? Importantly, because this is pressing public health problem, development of effective strategies to reduce the risk of AD and eventually improve its treatment, clinical studies reporting whole marine food and/or DHA intakes, blood DHA levels of lipid fractions, and DHA transporting proteins while also undertaking a whole marine food and/or a DHA intervention in AD should also be carried out. Accordingly, well conceived strategies need to be developed both to maximize the protective effect of fish and seafood products and determine what ingredients in fish and seafood products are protective against cognitive dysfunction in AD and other types of dementia.

Acknowledgements Financial support was provided by the Canada Research Chairs program (SCC), Canadian Foundation for Innovation, Canadian Institutes for Health Research, Natural Sciences and Engineering Research Council, AFMNet, Fonds de la recherche en Santé du Québec (Fellowship to MP), Health and Social Sciences Center-University Institute of Geriatrics of Sherbrooke, Research Center on Aging, the Department of Medicine, Université de Sherbrooke (fellowship to MP and FP), and the Réseau Québecois de recherche sur le vieillissement.

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Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Recent Advances on Nutrition and the Prevention of Alzheimer’s Disease, 2010: 97-108 ISBN: 978-81-7895-470-7 Editors: Charles Ramassamy and Stéphane Bastianetto

6. Manipulation of dietary fatty acids: Impact on neuropathological markers of AD in transgenic models Frédéric Calon1,2, Carl Julien1,2, Meryem Lebbadi1,2, Cyntia Tremblay1,2 and Vincent Émond1,2

Faculty of Pharmacy, Laval University, Quebec (QC), Canada; 2Molecular Endocrinology and Oncology Research Center, Centre Hospitalier de l'Université Laval (CHUL) Research Center Quebec (QC), Canada

Abstract. Animal models provide a useful tool to determine how environmental factors interfere with the pathogenesis of Alzheimer’s disease (AD) in a controlled setting. In recent years, several research reports have shown that physiological processes important for the development of AD neuropathology in transgenic mice are affected by the brain fatty acid profile, which in turn is a mirror image of dietary intake. Consequently, it can be speculated that nutraceutical interventions could provide low-cost health strategies to alter the normal progression of neurodegenerative diseases.

Introduction It could be anticipated that brain functions are relatively independent of nutritional status. The brain, arguably the noblest organ, must clearly remain on top of his game to give us a fair chance of survival. Shielded behind the Correspondence/Reprint request: Dr. Frédéric Calon, Molecular Endocrinology and Oncology Research Center, Centre Hospitalier de l'Université Laval (CHUL) Research Center, 2705 Laurier Blvd, Quebec, QC G1V 4G2, Canada. E-mail: frederic.calon@crchul.ulaval.ca

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blood-brain barrier (BBB), the brain is indeed separated from the rest of the body in terms of metabolic needs and immune defenses. We now know that it is not the case with polyunsaturated fatty acids (PUFA), because the BBB is fully permeable to circulating PUFA. By opposition to other types of nutrients, the amounts of PUFA ingested can impact concentrations of corresponding fatty acids in cerebral tissue. This is particularly the case for docosahexaenoic acid (DHA; 22:6 n-3, or cervonic acid). The fact that higher consumption of DHA is translated into higher brain concentrations is supported by a wealth of studies in animals and humans [2, 11, 13, 18, 31, 65, 83]. Dietary alpha-linolenic acid can provide a small fraction of DHA to the brain following enzymatic conversion in the liver, especially in a state of n-3 PUFA deficiency. However, it remains evident from rodent studies and even more vividly from human studies, that the mammal brain normally directly relies on dietary DHA or its close precursor, eicosapentaenoic acid [15, 29, 35, 76]. On the other hand, the brain is remarkably resistant to n-3 PUFA deprivation. Studies have shown that the brain has developed powerful adaptation mechanisms to retain DHA within cerebral tissues [12, 31, 65]. Therefore, the human brain, as noble an organ as it can be, still depends heavily on our dietary intake of DHA for its normal function.

Alzheimer’s disease Alzheimer’s disease (AD) remains the most common neurodegenerative disorder and the first cause of dementia in the elderly. Patients with AD suffer from profound intellectual failure, including deficit in language and memory. Accumulation of amyloid-β (Aβ) deposition, conversion of tau protein into an insoluble hyperphosphorylated form, synaptic defects, oxidative stress and abnormal inflammatory response are all closely associated with the progression of the disease, leading ultimately to neuronal death [27, 38]. Although there is considerable evidence supporting the amyloid cascade hypothesis, most clinicopathological data suggest that tau deposition and synaptic dysfunction better correlate with AD symptoms [4, 8, 36, 67, 68, 87, 93]. Although the importance of genetic factors underlying AD pathogenesis cannot be questioned, it is becoming increasingly obvious that environmental factors modify genetic risk. For example, monozygotic twins can be discordant for AD and, for those who eventually become concordant, the onset of AD can be delayed up to 15 years [33, 51, 80]. From a therapeutic standpoint, environmental factors are easier to manipulate than genetic factors [63]. Therefore, identification of key environmental factors required for the translation of an inherited susceptibility into full scale AD and understanding how these factors regulate AD pathogenesis could lead to the development of new preventive approaches.

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Animal models Replicating AD in an animal is obviously a very difficult endeavor. To that aim, several strains of transgenic mice have been produced and characterized in the last decades [39, 64]. It was found that mice overexpressing mutated forms of the gene for the human amyloid precursor protein (hAPP) develop amyloid deposits in their brain, along with assessable memory deficits [39, 50, 64]. Since the diagnosis of AD is dependent upon the histological visualization of both plaques and tangles, the group of Frank LaFerla has developed the triple-transgenic model (3xTg-AD), which expresses presenilin-1 (PS1M146V)knockin, APPK670N, M671L(swe), and tauP301L transgenes. This mouse line progressively develops both AÎ˛ and tau pathologies in AD-relevant brain regions as well as deficits in long-term synaptic plasticity and cognitive performance [9, 69-71]. The earliest signs of cognitive impairment were detected as early as 4 months in this transgenic mouse [9]. While 3xTg-AD mice are not a model of AD per se, they represent a model of AÎ˛ and tau pathologies appearing concurrently in the same animal, as it happens in humans developing AD. 3xTg-AD mice do not develop frank neuronal loss, however, which makes them rather a model of early AD. Other interesting models displaying aspects of AD neuropathology include aged dogs [82] and lemurian primates [10].

Spontaneous alteration of PUFA in transgenic animal models DHA accounts for more than 20% of total fatty acids in gray matter and is the most abundant polyunsaturated fatty acid (PUFA) in the brain [56, 83]. It is now well-known that DHA is essential for the maintenance of learning ability and memory in adult animals [31]. To determine whether DHA loss is characteristic of AD, post mortem levels of PUFA have been measured in the brain of AD patients. The mostly cited reports are those describing a reduction of DHA in individuals diagnosed with AD [61, 78, 90]. However, we and others have not found such significant difference [25, 54, 89], whereas a more recent analysis even reports increased DHA concentration in the frontal cortex of AD subjects [73]. These differences may probably be explained by methodology issues. First, most studies expressed fatty acids concentration in % of total fatty acids rather than in absolute terms [77]. Thus, global loss of fatty acid due to aging [91, 92] or absolute changes other fatty acids such as n-6 PUFA [89] may account for difference between studies. In addition, the distribution of n-3 PUFA in the brain is not homogenous [30, 78], and the exact region where fatty acids are measured is

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important. However, no gender effects were associated with brain n-3 PUFA levels. Finally, due to pre/post mortem metabolism of fatty acids as well as their limited stability during storage, the assessment of post mortem brain levels of PUFA remains a difficult task. To determine whether brain PUFA levels are sensitive to AD-like neuropathology, our research group has established the brain fatty acid profiles of 3xTg-AD mice compared to control littermates. In Figure 1, we show that the fatty acid profile within lipids from the frontal cortex of 12-month-old and 18-month-old 3xTg-AD mice is spontaneously unbalanced in favor of n-6 PUFA compared to n-3 PUFA. Such disequilibrium was also found in 16-month-old 3xTg-AD mice fed a different control diet [75]. In addition, a similar observation was recently reported in APPK670N, M671L mice when crossed with animals expressing mutant human PS1M146L [94]. Accordingly, increased levels of arachidonic acid (20:4 n-6) were detected in an hAPP model [85], whereas n-3:

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Figure 1. Decreased cortical n-3:n-6 PUFA ratio in 3xTg-AD mice. The n-3:n-6 PUFA ratio was reduced in the frontal cortex of 3xTg-AD mice compared to NonTg mice. All animals were fed a control diet containing linolenic acid as the only source of n-3 PUFA. The methodology has been described elsewhere [55]. Each dot represents an individual value and the horizontal line is the mean from 6 to 12 animals. Statistical analyses were performed using unpaired Student’s t test. Abbreviations: 3xTg-AD, triple transgenic mouse model of Alzheimer’s disease; NonTg, non-transgenic; PUFA, polyunsaturated fatty acids.

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n-6 PUFA ratios remained similar between Tg2576 mice and non-transgenic controls [18, 19]. Perhaps even more interestingly, Tg2576 and 3xTg-AD mice were shown to be more vulnerable to DHA depletion. In other words, exposing these animal models of AD to a low n-3 PUFA diet induced a larger decrease in brain DHA than in control littermates [19, 55]. Overall, these data suggest an important effect of the transgene-induced AD-like neuropathology on the accumulation of PUFA in membrane phospholipids of brain cells. Mechanisms such as increased turnover, decreased synthesis, phospholipase A2 dysregulation, or reduced incorporation of n-3 PUFA into cerebral phospholipids may be proposed [21, 22, 85]. Since PUFA are involved in synapse activity [7, 14, 19, 52, 84], this preferential accumulation of n-6 PUFA may play a role in cognitive deficits observed in AD models. Another aspect that warrants further clarification is the subcellular compartmentalization of PUFA within the brain [14, 16, 52]. Indeed, the selective loss of DHA in synapses is likely to have more direct effect on cognition than DHA located on glial or endothelial cells, for example.

Total fatty acid intake alters AD neuropathology in animal models High intake of fat is prevalent worldwide. An increased risk of developing AD has been seen in individuals exposed to high-calorie/high-fat diets [60, 74]. Studies in animal models of AD further evidence the deleterious effect of high caloric intake (based on saturated fat) on multiple markers of AD pathology, such as accumulation of Aβ and tau as well as drebrin loss [48, 55, 57, 58, 72]. Figure 2 shows the differential effects of a low n-3 PUFA diet and a high-fat diet on Aβ42 and tau in the cortex of the 3xTg-AD mice. The high intake of total fat had greater effect on Aβ42, while, in contrast, insoluble tau rather responded to n-3 PUFA deficiency. In accordance with these results, other groups have reported that dietary calorie restriction was associated with improved cognitive performance and reduced Aβ and tau pathologies in animals [41, 79]. Additional studies in hAPP models indicated that cholesterol intake can regulate brain Aβ metabolism [34, 49, 81]. Moreover, we recently reported that n-3 PUFA protected dopaminergic cells against MPTP neurotoxicity in a mouse model of Parkinson disease [13]. These data are consistent with the hypothesis that our modern lifestyle in terms of dietary fat consumption might have a negative outcome on the incidence and/or progression of neurodegenerative diseases.

PUFA alters AD neuropathology in animal models Besides the accumulation of tau and Aβ, AD is also characterized by a dramatic decrease of the postsynaptic protein drebrin, and of its mRNA

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Figure 2. High-fat diet accelerates the accumulation of Aβ42 and tau in the brain cortex of 3xTg-AD mice. Concentrations of Aβ42 and tau in homogenates from the parieto-temporal cortex of 3xTg-AD mice fed a diet with low-fat and low n-3:n-6 PUFA ratio or a diet with high-fat and low n-3:n-6 PUFA ratio, compared to a control diet. Aβ42 concentrations were determined using ELISA and Western immunoblotting, respectively [55]. Each dot represents an individual value and the horizontal line is the mean from 4 to 7 animals. Statistical analyses were performed using ANOVA followed by a Newman-Keuls post hoc test. Abbreviations: Aβ, amyloid-β peptide; Ctrl; control diet; O.D., optical density; PUFA, polyunsaturated fatty acids.

transcript [19, 26, 42, 46, 53, 88]. Such loss of drebrin was reproduced in DHA-depleted Tg2576 mice [19] and to a less striking extent in high-fat-fed 3xTg-AD mice [55]. Evidence for neuroprotective effects of DHA have also been demonstrated against accumulation of Aβ [59, 72], tau pathology [40] and cognitive deficits [19] in various transgenic animal models of AD, in agreement with earlier data gathered in rats receiving intracerebral infusion of Aβ42 [43-45]. While these studies highlight the importance of DHA, they also indicate that n-6 PUFA such as linoleic or arachidonic acid may exert deleterious effect on AD progression. For example, a study in young hAPP/PS1 transgenic mice shows an inverse correlation between brain n-6 PUFA content and cognitive performance [3]. From a mechanistic standpoint,

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both in vitro and in vivo argue in favor of a particular role of DHA is regulating cellular cascades involving phosphatidylinositol 3-kinase/Akt signaling [1, 19]. Series of data gathered in rodents also indicate that n-3 PUFA can enhance serotoninergic and dopaminergic neurotransmission, possibly contributing to their beneficial effect on animal cognition [20, 28]. The view that DHA might be protective in AD is also supported by a wealth of correlative epidemiological studies [5, 6, 37, 47, 62, 66, 86]. Consequently, underconsumption of DHA, which is commonplace in our modern society [62], stands as a potential modifiable risk factor of AD.

Conclusion Investigations in animal models of AD are in general agreement with the contention that high-fat, low n-3 PUFA diets can amplify the expression of markers of AD neuropathology in transgenic mice. We must be careful in our interpretation of these data in part because of differences in fatty acid metabolism between mice and humans. For example, absorption of dietary triglycerides, bioavailability of fatty acids ingested and their metabolism in the liver/brain differ in the two species. Moreover, the weight of food (g/kg) a mouse can eat per day is much higher than a human. Moving to animal species closer to humans might help extrapolating the data. Epidemiological data should also be interpreted with caution. Although based on human population, they suffer from the correlative nature of the analyses on which they rely. Indeed, high consumption of meals enriched in fat, particularly in n-6 PUFA, cannot be readily isolated from other variables and might rather be just a part of general life habits. From a scientific perspective, we may have enough preclinical arguments to launch intervention studies. Indeed, recent small randomized clinical studies suggest that n-3 PUFA can slow the decline in cognitive performance, but only in patients with very mild AD [23, 32]. A larger Alzheimer's Disease Cooperative Study (ADCS)-sponsored clinical trial is underway. However, to use DHA in a pharmaceutical or nutraceutical product, patentability issues and manufacturing challenges have to be overcome [17, 24]. Nevertheless, it can be speculated that adopting a healthy lifestyle, which includes reduced fat intake combined with high DHA, may provide a low-cost preventive strategy to alter the normal progression of neurodegenerative diseases. It may be difficult to prove the efficacy of DHA, but ongoing preclinical and clinical studies should shed some light on how manipulating dietary fat could be used to prevent AD.

Grant numbers and sources of support This work was supported by grants from the Canadian Institutes of Health Research (CIHR) (FC - MOP74443), the Alzheimer Society Canada

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(FC - ASC 0516) and the Canada Foundation for Innovation (10307). The work of FC was supported by a New Investigator Award from the Clinical Research Initiative and the CIHR Institute of Aging (CAN-76833).

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Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Recent Advances on Nutrition and the Prevention of Alzheimer’s Disease, 2010: 109-125 ISBN: 978-81-7895-470-7 Editors: Charles Ramassamy and Stéphane Bastianetto

7. Vitamin A and Alzheimer’s disease Valérie Enderlin and Véronique Pallet

Unité de Nutrition et Neurosciences - Université de Bordeaux Avenue des facultés 33405 Talence cedex, France

Abstract. To date, convergent data on the role of retinoic acid in the mature brain have established that this molecule, which acts as a hormone, helps to preserve cerebral plasticity by controlling dendritic spine density as well as hippocampal neurogenesis. A deterioration in cerebral plasticity seems to be at the base of the cognitive decline disease. Furthermore, the transcription of several genes, known as muted, in Alzheimer’s patients and whose transcripts are involved in the formation of senile plaques, are controlled by retinoic acid. As seen in other nutrients, aging leads to a lower production of retinoic acid; a phenomenon probably accentuated by the fact that Westem populations consume an insufficient amount of vitamin A (60% of the population has a consumption lower than the recommendations [1]). These two phenomena (i.e. level of consumption, the lack of activation of vitamin A) accompanied by important individual differences, would help to explain why some patients have an almost normal aging process, whereas others gradually develop cognitive disorders and then, the disease. A better understanding of the role of a collapse of the retinoid status in the genesis of Alzheimer lesions could, beyond the definition of a preventive nutritional strategy, open therapeutic perspectives, through the use of molecules targeting the nuclear receptors. Correspondence/Reprint request: Dr. Valérie Enderlin, Unité de Nutrition et Neurosciences - Université de Bordeaux, Avenue des facultés 33405 Talence cedex, France

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Introduction Retinoids, a family of compounds derived from vitamin A (Figure 1), have numerous important functions in many tissues, including: a role in vision, the maintenance of epithelial surfaces, immune competence, reproduction, and embryonic growth and development [2]. The majority of these functions (mechanisms underlying the extravisual functions) of vitamin A are performed by the vitamin A metabolite, retinoic acid (RA), which binds to receptors of the nuclear receptor superfamily, and regulates gene expression. It is well-known that retinoids, and particularly RA, play an important role during the normal development of the central nervous system (CNS) (see review by Maden [3]). Presently, the role of retinoids in the adult central nervous system is less conspicuous than their role in development, and has only recently attracted the attention of scientists. Some data suggest that the fine regulation of retinoid mediated gene expression seems fundamentally important for optimal brain functioning such as LTP, synaptic plasticity, learning and memory [4-9]. Recently, data from a number of studies have argued for the involvement of retinoid signaling in the etiology of Alzheimer's disease (AD) [10-12].

Figure 1. Chemical structures of retinoid family members.

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Vitamin A metabolism and transport (Figure 2) The capability for the de novo synthesis of compounds with vitamin A (retinol) activity is limited to plants and microorganisms. Therefore, higher animals must obtain vitamin A from their diet, either in the form of a preformed vitamin (the major dietary forms are long-chain fatty acid esters of retinol: retinyl ester or RE) or as provitamin carotenoids, such as β-caroten, α-carotene or β-cryptotoxanthin, found in plant-derived foods. Presently, it is recommended that 60% of the vitamin A intake is in the form of carotenoids (plant sources) and 40% in the form of retinols (animal sources). RE must be hydrolysed prior to intestinal absorption. The absorption efficiency is higher for preformed vitamin A (80-90%) than for carotenoids (50-60%). Carotene is converted to retinol in the intestinal mucosa via two enzymatic steps. Free retinol is reesterified in the mucosal cells by the enzyme lecithin:retinol acyltransferase (LRAT); the resulting RE are incorporated into chylomicrons and absorbed via the lymphatics (see review by Harrisson [13]). Under normal nutritional conditions, most of the vitamin A in an organism is stored in the liver (essentially as RE forms), partly in the hepatocytes and in higher amounts, as lipid droplets in the stelleate cells (also called Ito cells), from where vitamin A will be mobilized when needed [14]. RE are hydrolyzed prior to mobilization from the liver and free retinol is complexed to retinol binding protein 4 (RBP4). Approximately 95% of the retinol-binding proteins (RBP) circulate in plasma as a macromolecular complex with another transport protein, thyroxine hormone carrier transthyretin (TTR). Retinol is taken up by target cells through an interaction with the membrane receptor for RBP4 (STRA6). It then enters into the cytoplasm, where it binds to cellular retinol binding protein 1 (CRBP1) and, in a two-step process, is metabolized to alltrans retinoic acid (atRA), the active metabolite of vitamin A [2]. The rate limiting step in this process is the oxidation of retinol to retinal, and the final step, is the oxidation of retinal to retinoic acid (RA). Cytosolic Medium-Chain Alcohol Dehydrogenase (ADH), and more precisely ADH1, ADH3 and ADH4, are involved in the oxidation of all-trans retinol to alltrans retinal. The oxidation of retinol to retinal also appears to be catalyzed by members of the Membrane-Bound Short-Chain Dehydrogenase/ Reductase (SDR) family of microsomal enzymes, including RDH1, RDH5, RDH11, CRAD1, CRAD2, CRAD3 and retSDR1 [15]. The oxidation of alltrans retinal to all-trans retinoic acid is catalyzed by Retinal Dehydrogenase (RALDH), and more precisely by the isoforms 1, 2, 3 and 4. The catabolism of RA is an important mechanism for controlling RA levels in cells and tissues.

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Figure 2. The retinoid cascade.

Cellular retinoic acid binding proteins I and II (CRAPBI and II) are the cytoplasmic-binding proteins for RA. One function of these proteins may be to transport RA into the nucleus in order to mediate its effect by either inducing or repressing gene transcription by binding to specific nuclear receptors which function as transcription factors: RAR (whose ligands are the all-trans RA and 9-cis RA isomers) and RXR (whose ligand is the 9-cis RA

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isomer) [16]. The fact that RA activates nuclear transcription factors was discovered in 1987 [17, 18]. Τhe RA receptors belong to the same family as proliferators-activated receptors (PPAR), vitamin D receptors (VDR), thyroid hormone receptors (TR) and steroid receptors. In the presence of a ligand, the receptor switches in conformation and releases corepressors that would otherwise keep the gene repressed in the absence of a ligand. This is followed by a modification in the decompaction of chromatin structure, allowing the transcriptional machinery to gain access to the promoter and to initiate the transcription. The RA response element (RARE) binds the receptor dimmers [19]. Most often, RAR is dimerized to RXR, which has the particularity to act independently of the presence of its ligand [20]. RAR and RXR each have three genes (alpha, beta and gamma) and gene splicing significantly increases the number of variants [21]. RAR and RXR also have three isotypes each (α, β and γ), which are encoded by distinct genes. Thus, for each RAR isotype, there are several isoforms generated by differential promoter usage and splicing [22, 23]. The multiple RAR and RXR isotypes and isoforms are conserved in vertebrate evolution and display distinct spatiotemporal expression patterns in developing embryos and adult tissues, suggesting that each receptor performs a unique function [24]. Therefore, vitamin A and more precisely, RA via its specific nuclear receptors, play a critical role in a variety of essential life processes, including reproduction, embryonic development and modulation of the growth and differentiation of a wide variety of mammalian cell types. It has been suggested that RAR/RXR signaling regulates approximately 500 genes [2]. A much lower number was experimentally shown to be activated via the classical RARE driven pathway, whereas many cases of gene suppression have been described. Some authors have shown non-genomic modes of RA action. These rapid actions include the regulation of gap junctions [25], spinule formation in the retina [26], an effects on dendritic spines in the hippocampus [27]. Another pathway of action is the repression of AP-1 (Jun, Fos) activity, which involves RAR/RXR dimmers, but not RARE [28].

Vitamin A and the adult brain 1/ Retinoic acid signaling in the brain During brain development, vitamin A, and more precisely RA, plays a key role by regulating patterning, neurogenesis, neural specification, and neurite outgrowth. Recent studies have suggested that retinoids may also play an important role in the adult central nervous system [29-31]. In the adult brain, regions that exhibit RA signaling are regions of high neuronal

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plasticity i.e. the hippocampus, medial prefrontal cortex and retrosplenial areas [32]. Several lines of evidence point to the importance of RA in the functioning of the striatum and nucleus accumbens. It has been shown that (i) cellular retinoic acid and retinol binding proteins and high levels of nuclear retinoic acid receptors have been observed in these areas [33, 34] and (ii) the striatum presents the highest levels of RA [35]. Several authors have shown that the striatum synthesizes RA and contains much of the biochemical apparatus associated with RA responsiveness and metabolism [36-38]. Among the RA nuclear receptors, RARβ is the main isoform expressed in the mature brain [39]. Moreover, RXRβ and RXRγ are also expressed at high levels in the brain [40]. Thereby, the co-expression of RARβ and RXRβ/γ suggests that these receptors may contribute to specific functions in the central nervous system by modulating the expression of their target genes. Among the many genes whose expression is regulated by RA, there are those coding for their own nuclear receptors and those coding for neuron-specific proteins involved in many activities in the mature brain, e.g., synaptophysin, nerve growth factor, N-methyl-D-aspartate receptor, dopamine receptor 2, choline acetyltransferase, neurogranin, neuromodulin. Finally, retinoic acid and its receptors also regulate quite a lot of genes coding for proteins implicated in neurodegenerative processes, such as the APP protein and the tau protein. It is currently accepted that retinoic acid plays a dominating role in the preservation of cerebral functionality. Thus, it is of first importance to study the effects of the status in vitamin A, or in retinoic acid, in the adult brain, and more particularly during aging. Indeed, recent data have suggested that changes in retinoids are capable of producing alterations in neuronal target proteins and consequently may affect physiological maintenance processes in the mature brain [41]. Alterations of cerebral plasticity and memory deficits have been described in vitamin A depleted animals as described in a latest part. Moreover, it has been shown that RARβ and RARβ-RXRγ knockout mice display an alteration of LTP, as well as substantial performance deficits in a hippocampal-dependent spatial learning task [5, 42]. RARβ mutations, with either RXRβ or RXRγ, result in impaired locomotion typical of abnormal striatal function and possibly related to a decrease in the dopamine D1 and D2 type receptors in striatal neurons [43, 44]. Chronic ethanol consumption, which produces cognitive deficits, also induces disorders in RA biosynthesis; ethanol can induce RARβ and RXRβ/γ expression in vivo, and blocking RARβ activity was shown to reverse an alcohol-induced working memory deficit. Thus, the over-expression of brain RA nuclear receptors seemed to be involved in memory impairments observed during chronic

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ethanol consumption (Alfos et al., 2001). Astroglial-derivated RA may even be an important signal for neurogenesis in the dentate gyrus of the hippocampus [45]. Together, these results show that either nutritional or physiological situations involving modifications of the level of expression of the brain retinoid nuclear receptors lead to considerable neurobiological alterations and to mnemonic deterioration.

2/ Vitamin A and aging Age-related alterations in vitamin A metabolism, particularly plasma retinol concentrations, have been reported in both rats and humans [46, 47]. More recently, studies have shown that a moderate down-regulation of retinoid mediated transcription events occurs naturally with senescence. A lower abundance of RARβ and RXRβ/γ mRNA has been effectively observed in the whole brain and hippocampus of aged mice and rats. Generally, authors have attributed this decrease in nuclear receptor expression to a reduction in the bioavailability of RA associated with aging. This explanation seems to be confirmed by the fact that a significant decrease in retinol concentration was observed in the serum of aged rats. The administration of RA restores the age-related decrease in mRNA levels to their presenescence levels [4, 48, 49]. Finally, more recently, retinoid hyposignaling, as evidenced by a hypoexpression of retinoid receptors, has been reported in human peripheral blood mononuclear cells [50]. The hypoactivity of retinoid signaling observed in aged animals was associated with a decreased expression of genes encoding for neural proteins and implicated in synaptic plasticity: neuromodulin (GAP-43) and neurogranin (RC3) [51]. The age-related decreased expression of RC3 observed in mice was correlated with a LTP deficit and severe age-specific memory impairment. RA administration to aged animals restores the mRNA level of target genes involved in synaptic plasticity, and concomitantly alleviates both the hippocampal LTP and relational memory seen in aged mice [6]. Vitamin A supplementation was shown to counteract the aging-related hippocampal hypoexpression of GAP-43, as well as the short term/working memory deterioration and alleviated the longterm declarative memory impairment [42]. Ethanol consumption in aged mice also reversed the age-related hypo-expression in brain RARβ and the target genes RC3 and GAP-43 [52] and reduces a selective age-associated memory deficit in mice [53]. Together, these data suggest that a fine regulation of retinoid mediated gene expression is fundamentally important for optimal brain functioning during aging and for the maintenance of memory performances. This potential

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role of retinoids has been argued in works studying the functional consequences of a decrease in the bioavailability of vitamin A generated by a vitamin A deficiency (VAD).

3/ The vitamin A deficiency model VAD that is characterized by a reduced expression of brain-specific retinoid nuclear receptors in control animals results in a decline in the activity of two target genes involved in synaptic plasticity (Neurogranin/RC3 and Neuromodulin/GAP43) as well as in a selective behavioral impairment similar to that observed in aged mice [9, 39, 54-57]. In rats subjected to 14 weeks of VAD, there is a decrease in cell proliferation and neurogenesis concomitant to spatial learning and memory deficits. More importantly, these effects are reversed after four weeks of RA treatment [58]. Moreover, adult rats maintained on a vitamin A-free diet for 12 months developed a severe deficit in spatial learning and memory, and this cognitive impairment was fully restored when vitamin A was once again available in the diet [7, 8]. These few studies have revealed neurological alterations associated with VAD. Experiments using mouse models have provided evidence that, contrary to aged mice, the administration of RA to vitamin A-deprived animals failed to fully normalize the expression of RC3 and had no effect on relational memory [9]. Knowing that (i) RC3 is not only under the influence of retinoids [59], but is also regulated by thyroid hormones (whose active metabolite is triiodothyronine, T3) [60, 61] and (ii) in consideration of the close relationship between the activity and signaling pathways of retinoids and thyroid hormones previously described in VAD [62, 64], it has been suggested that thyroid disorders are involved in the inefficacy of RA to restore neurological alterations. Recent results have strengthened this hypothesis in that the alteration of the T3 signaling pathway associated with VAD has been shown to be a limiting factor that impedes RA from exerting its modulating effect [57]. The vitamin A deficiency model both highlights and describes the involvement of vitamin A signaling in cerebral plasticity and memory performance. Beyond its implication in the implementation of the cognitive deficits during aging, hypoactivity of the retinoid signaling pathway could also influence the late onset of Alzheimer’s disease (AD).

Vitamin A and Alzheimer’s disease AD is the most common cause of dementia in the elderly. This chronic neurodegenerative disease is characterized by the progressive deterioration of

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cognitive functions including memory, judgment, language skills, decisionmaking, orientation, etc. Clinical symptoms include alterations in neural plasticity (e.g. the loss of selective neurons and synapses), extracellular senile plaques containing amyloid-β peptide (Aβ) deposits, as well as intraneuronal neurofibrillary tangles. The involvement of RA in cognitive functions and neuronal plasticity has previously been described. Recently, data from a number of studies have argued for the involvement of retinoid signaling in the etiology of AD. On one hand, Goodman investigated the genetic links between RA signaling and AD since gene loci thought to be involved in AD were clustered around genes for CYP26 enzymes (cytochrome P450 enzymes involved in the catabolism of RA for controlling RA levels in tissues), RARα, RXRβ, RXRγ, CRABP-II, and RBP. On the other hand, a decrease in serum retinol levels has been revealed in patients with AD, and it has been hypothesized that a decrease in the availability of RA and a subsequent dysregulation of retinoid genes and their target genes contribute to late-onset AD [10, 65-67].

1/ Vitamin A transporters and AD Apolipoprotein E (ApoE), a transport protein for RE in chylomicron, is the major apolipoprotein in cerebrospinal fluid and has been identified as a major susceptibility gene in AD [68]. The ApoE ε2 allele, which is associated with a decreased risk of AD in humans and memory impairment in rats, has a better ability to carry retinoids [10]. In neuronal cells, the abundance of constitutively expressed ApoE is lower following RA treatment [69], while in rat primary astrocyte cultures, RA increases APOE secretion [70]. Levels of the lipocaline apolipoprotein D, another transporter protein of retinol in the CNS, are increased in the neurons of AD patients. RA regulates its expression.

2/ Vitamin A and neurofibrillary tangles Among the genes potentially regulated by RA, there is one gene that codes for microtubules-associated-protein-tau (MAPT) and which is preponderant in the formation of the neurofibrillary tangles [71-73].

3/ Vitamin A and β-amyloid (Aβ) (Figure 3) Aβ is a mixture of heterogeneous peptides derived from the amyloidogenic pathway, including two sequential endoproteolytic cleavages of the β-amyloid precursor protein (APP) catalyzed by two distinct enzymes

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Figure 3. Retinoid acid and the amyloidogenic pathway.

referred to as β- and γ-secretase [74], [75]. The β-site cleaving enzyme (BACE1) is highly expressed in brain tissue and colocalizes with intracellular Aβ production sites. The cleavage of APP by this enzyme results in the release of sAPPβ into the extracellular space. The subsequent cleavage of the fragment remaining in the membrane by γ-secretase, releases an intact Aβ peptide, a component of extracellular amyloid plaques. APP can also be processed by a non-amyloid pathway in physiological conditions. This secretary pathway includes the cleavage of APP by a putative α-secretase in the Aβ region, thereby precluding the formation of Aβ. This type of α-secretase activity has been attributed to the metalloproteinases ADAM9 and ADAM10 [76]. It had been shown that the disruption of retinoid signaling causes the deposition of β-amyloid in the adult brain [11]. These authors have documented an amyloid β deposition in the brains of the 1-year-old retinoid deficient rats. Similarly, vitamin A (or retinoid) inhibits and destabilizes preformed Aβ aggregates and consequently protects against plaque formation, probably via its nuclear receptors [77, 78]. To date, there are several biochemical data that support the involvement of vitamin A signaling in the formation of Aβ. Indeed, key steps of the amyloid production process

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are under the control of proteins whose expression is positively regulated by RA in vitro, including: APP, the β-site APP cleaving enzyme (BACE or β-secretase), presenilin 1 and 2 (PS1 and PS2), two of the complex γsecretase proteins (their up-regulation by ATRA might promote plaque formation), as well as ADAM10 [29, 79-81]. Interestingly, an in vitro study revealed that RA treatment increases the ADAM10 protein level much more than that of BACE, suggesting a shift in APP processing toward the α-secretase pathway in response to RA [82, 83]. The insulin degrading enzyme (IDE), a metalloprotease enzyme responsible for insulin degradation, has been shown to play a key role in Aβ peptide degradation both in vitro and in vivo, and is selective for the Aβ monomer. IDE has been observed in human cerebrospinal fluid (CSF). Its activity, levels, and mRNA are decreased in AD brain tissue and are associated with increased Aβ levels [84-87], suggesting that the modulation of IDE activity may alter the risk for AD. IDE contains a RARE response element in its promoter and the transcription of IDE is positively regulated by RA [88]. In order to consider the use of retinoid therapy for AD, it is necessary to establish the precise role of vitamin A and its receptors in vivo in the multiple processes involved in regulating plaque formation. As stated above, recent data have provided evidence that VAD was responsible for instigating an alteration of the neuronal plasticity as well as inducing cognitive impairment [9, 56, 58]. Additional data have revealed a vitamin A-deficiency-related dysregulation of the amyloidogenic pathway in the cortex of rats, which is known to be the first brain area altered by AD development: the first stage of Aβ deposition begins exclusively in the neocortex before expanding to other regions [89, 90]. Authors have shown that hypo-activity of the retinoid signaling pathway leads to (i) an increase in the APP770-751/APP695 ratio, which is an important clue to the etiology of amyloid deposition and also of plaque formation in AD patients [91], and (ii) a decreased expression of APP695, BACE and APP-CTF in whole brain and cortex of rats fed a vitamin A-free diet for 13 weeks. There is a longstanding controversy as to which APP transcript is up- or down- regulated in amyloidogenic diseases. Nevertheless, the loss of APP695, a brain-specific isoform, has been described in the cortex and hippocampus of AD patients [91, 92]. The phosphorylated forms of APP-βCTF and APP-β’CTF seem to play a determining role in CTF processing leading to neuritic plaque formation, by facilitating their own cleavage into Aβ by γ-secretase [93]. In this way, the reduced levels of phosphorylated β- and β’CTF, which occurred in the cortex when there was a vitamin A deficiency for an extended period of time, might be due to a rise in γ-secretase activity and thus, might reveal an increase in Aβ formation. Indeed, some studies have shown an inverse relationship between γ−secretase and α- and βCTF: (i) an elevation in

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the steady-state levels of α- and βCTF in PS1 gene knockout mice (a member of the γ-secretase complex) [94], and (ii) an accumulation of the γ-secretase product (γCTF) and a concomitant reduction in the levels of both α- and βCTF in different cell lines that over-express PS1 [95]. Moreover, the decreased amount of fragment αCTF (an α-secretase product generated during the physiological pathway) noted in the cortex of vitamin A-deficient rats might reflect an underlying neuropathic process, whereby APP695 is converted to βCTF and thus, Aβ, rather than to αCTF. In fact, such a process has already been described in AD patients [96]. This hypothesis was supported by recent reports suggesting a RA-associated shift in APP processing toward the alpha secretase pathway [81, 82]. Finally, very recently, several authors have reported results supporting all-trans-retinoic acid (ATRA) as an effective therapeutic agent for the prevention and treatment of AD. In amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic mice, the administration of ATRA induced a robust decrease in brain Aβ deposition and tau phosphorylation. This effect was accompanied by a significant down-regulation of tau phosphorylation [97]. These results complement earlier data indicating the involvement of retinoid signaling in the etiology of Alzheimer diseases and argue for the potent anti-amyloidogenic effect of vitamin A suggested, to date, by in vitro studies. Nevertheless, the underlying molecular mechanisms are incompletely understood.

Conclusion Together, these data suggest that a fine regulation of retinoid mediated gene expression seems fundamentally important for optimal brain functioning and argue that vitamin A has an important role, via its nuclear receptors, in the multiple processes involved in regulating plaque formation. In a perspective of a nutritional prevention of Alzheimer disease, it will be of first importance to better understand the involvement of the age-related hypoactivity of the vitamin A signaling pathway in the genesis of pathologic lesions. Besides retinoic receptors (RAR), the steroid/thyroid nuclear receptor superfamily includes other transcriptional factors relevant for neurodegenerative diseases. This is the case for peroxysome proliferator-activated receptors, which also form heterodimers with RXR in order to bind to their response elements (PPRE) and active gene transcription [98]. Currently, several studies have described a much closer relationship between RA and fatty acid signaling pathways, since the ability of several polyunsaturated fatty acids (PUFA) to specifically bind and activate RXR at supra-physiological levels has recently been shown [99, 100]. It is now well accepted that these nuclear

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receptors are master transcription factors which, by the means of a precise combination, orchestrate the maintenance of neurobiological properties which underpin memory processes. Moreover, it is known that these receptors are also sensors of vitamin or lipid content in the diet and control its metabolic response. It seems to be of first importance to consider vitamin A and fatty acid signaling pathways together, since it has now been established that the dietary intake of PUFA and, more importantly, the total intake of calories from fat leads to an accumulation of neurodegenerative markers [101]. Thus, it is possible to assume that modifications in the brain bioavailability of these modulators (RA or fatty acids) -via a lesser dietary content or by an age-related physiological decline in the capability to activate these modulators- may rapidly induce changes in the pattern of nuclear receptor expression, and consequently, lead to neurobiological deterioration and neurodegenerative processes.

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Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Recent Advances on Nutrition and the Prevention of Alzheimer’s Disease, 2010: 127-143 ISBN: 978-81-7895-470-7 Editors: Charles Ramassamy and Stéphane Bastianetto

8. Molecular mechanisms of homocysteine toxicity and possible protection against hyperhomocysteinemia Alexander Boldyrev Research Center of neurology, Russian Academy of Medical Sciences and M.V. Lomonosov Moscow state University, Moscow, Russia

Abstract. Homocysteine is a naturally occurring intermediate connecting metabolism of methionine and cysteine and, subsequently glutathione. Its normal level in mammalian blood does not exceed 10 µmol/l, whereas vitamin deficient diet, increased stationary levels of reactive oxygen species, as well as neurodegenerative and cardiovascular diseases are usually accompanied by an increased content of homocysteine in peripheral blood. An increase in homocysteine levels above 50 µmol/l may be a risk factor for recurrent heart attacks and that above 150-200 µmol/l is a cause for ischaemic stroke. Persistent hyperhomocysteinemia with homocysteine levels of 300 µmol/l or higher may induce mental deficiency. Molecular mechanisms of homocysteine toxicity are not fully understood as yet. In the review, novel data are presented which demonstrate that homocysteine and homocysteic acid, a product of its spontaneous oxidation, induce activation of NMDA receptors in the brain neurons accompanied by an increase in the levels of the reactive oxygen species and resulting in induction of apoptotic and Correspondence/Reprint request: Dr. Alexander Boldyrev, M.V. Lomonosov Moscow State University, Dept. Biochemistry, School of Biology, Room 141, Lenin’s Hills, 119992 Moscow, Russia E-mail: alexander.boldyrev@gmail.com

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(under long-term stimulation) necrotic cell death. Recent findings showed that NMDA receptors are encountered not only in neuronal cells but also in megakaryocytes, red blood cells, lymphocytes, neutrophils, and cardiomyocytes. Thus, the number of target cells of homocysteine is sufficiently increased, thereby underlying the importance of developing new strategies to protect the body from hyperhomocysteinemia. In a model of prenatal hyperhomocysteinemia in rats, we have demonstrated that the neuropeptide carnosine (a natural antioxidant and immune modulator) protects animals against homocysteine toxicity with no change in the blood homocysteine levels. A suggestion was made that carnosine interferes with NMDA receptors preventing their excitotoxicity.

Introduction 1. Homocysteine as a risk factor for cardiovascular and neurodegenerative diseases The total level of homocysteine (HC) in blood plasma of healthy donors amounts to 10-12 µmol/l and slightly increases with ageing [1]. Before the pubertal period, the HC content in blood of children is about 5 µmol/l (irrespective of the gender). Later on, this value increases to 6-7 µmol/l, being slightly lower in girls than in boys. In blood of adults HC levels reach 10-12 µmol/l, being slightly higher in men than in women [2]. An increase in HC blood levels with age is usually accompanied and followed by a decrease in the renal function with higher accumulation of HC in men than in women, depending on specificity of hormonal metabolism. HC easily involves in red/ox reactions and its spontaneous oxidation results in homocysteic acid (HCA) accumulation [1] so that in the blood stream it exists both in its oxidized form (homocysteic acid, HCA) and in a complex with cysteine or proteins, with its bound form prevailing (accounting for over 70 %). The term “total HC” corresponds to all constituents including several homocysteine derivatives, and protein bound HC. Excess of HC is considered to be a risk factor for a number of pathologies. The term “hyperhomocysteinemia” is used when the HC content exceeds 15 µmol/l. A level of 15-30 µmol/l corresponds to mild, 30-100 µmole/l – to moderate, and more than 100 µmol/l – to severe hyperhomocysteinemia [1, 3]. Common causes of hyperhomocysteinemia typically include: renal impairment induced by several pathologies [4], deficiency of vitamins involved in HC metabolism [5, 6], overloading with dietary methionine or excessive accumulation of reactive oxygen species (ROS) in tissues [7] as well as deficiency of enzymes, which promote HC metabolism (see below). In some cases, normalization of blood HC levels may be achieved by diet modulations. Adherents to dietary restrictions believe that one of the positive effects of caloric restriction prolonging the life span is a decrease in methionine supply [8].

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Apparent disposition to hyperhomocysteinemia is encountered in smokers [9] or cafe amateurs [10]. Regular alcohol consumption reduces folate and vitamin B12 status and increases HC levels in blood of healthy donors [11]. In the second half of the XX century, homocysteinuria syndrome was described as related to cystationine-β-synthase deficiency [12]. Homocysteinuria is usually accompanied by thromboembolia, progressive cardiovascular diseases and mental deficiency [13]. Moreover, an increase in blood HC was demonstrated to induce atherosclerosis because HC stimulates an atherogenic action of cholesterol [12, 13]. Thus, hyperhomocysteinemia is one of the pathogenic factors of atherosclerosis [14, 15]. It also provokes myocardial infarction [16], cerebral stroke [17], and complicates diabetes [18]. Severe hyperhomocysteinemia was shown to result in brain convulsions and dementia [19], as well as may contribute to Alzheimer’s disease (AD) [20]. Recent studies showed that genetically provided deficit of methylenetetrahydrofolate reductase and coherent accumulation of HC in blood is a risk factor of AD in human population [21]. In AD patients, plasma and brain HC levels were found to be increased significantly [22, 23]; the same was noted for the cerebrospinal fluid [24]. Statistical analysis showed that with plasma HC levels above 14 µmol/l there is a two-to-five-fold increase in the risk factor of AD as compared with that in the age-matched controls [25, 26]. Therefore, hyperhomocysteinemia is often considered as a predictive factor for cognitive impairment in the elderly [24, 27] and for AD [28], although there is debate of whether hyperhomocysteinemia is a risk factor for, or a risk marker of AD [29]. HC may also play a provocative role in neurotoxicity of amyloid-β peptides, and the presence of amyloid-β in the brain of AD patients was shown to be exacerbated by HCA [30]. Both HC and HCA may increase sensitivity to toxic effects of amyloid-β peptides directly affecting their conformation and inducing the formation of neurotoxic β-fibrils [30, 31]. Currently, it is widely accepted that HC is a risk factor for cardiovascular and neurodegenerative diseases [7, 21, 22], although the molecular grounds of homocysteine toxicity are not fully understood. The present review is devoted to analysis of molecular mechanisms of HC toxicity.

2. Neurotoxic effects of homocysteine A toxic effect of HC and its derivatives on the cerebellum granule cells was described about a decade ago and it was suggested to be realized via glutamate receptors [32-34]. The most expected candidate could be glutamate receptors activated by N-methyl-D-aspartic acid (NMDA), the so-called

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NMDA receptors. Their activation results in a significant rise in intracellular Ca ions and severe accumulation of ROS inside the neurons [35]. An increase in ROS levels could be suggested as an immediate reason of the toxic effect, for both superoxide dismutase and catalase proved to be strongly protective [32]. Moreover, the neurotoxic effect of HC was prevented by both MK-801 (an irreversible non-competitive antagonist of NMDA-receptors) and αmethylcarboxyphenylglycine, which is known to inhibit metabotropic Group I glutamate receptors, whose effect is associated with an activation of phospholipase A2 and phosphatidyl inositole-3-phosphate induced mobilization of intracellular calcium ions from the sarcoplasmic reticulum [36]. It was found that another antagonist of Group I metabotropic receptors, LY 367385, which also partially protects neurons from toxic effect of HC could completely prevent neuronal death induced by HC, once added in a combination with МК-801, whereas agonist of metabotropic Group I receptors, t–ADA induced neurodegeneration [37]. Thus it was concluded that both ionotropic and metabotropic glutamate receptors are involved in neurotoxic effects of HC. It was found recently that one of the consequences of incubation of hippocampal slices with HC is inactivation of intracellular protein phosphatases and subsequent hyperphosphorylation of neurofilaments which resulted in cytoskeleton impairments [38]. This phenomenon was found to be an important cause of damaging the neuronal membrane. Nevertheless, HC should be considered rather a weak neurotoxin. In the in vitro experiments, its cytotoxic effect appears at concentrations higher than 1 mmol/l and induces cellular necrosis [37]. A similar neurotoxic action may be achieved by glutamate at only 2 times lower concentration [32]. Some HC derivatives, like HCA, which is considered to be an endogenic neurotoxin [39] may have a much stronger neurotoxic effect than HC [40, 41]. Sensitivity of neurons to HCA is sufficiently higher than that to HC and at 10-100 µmol/l concentration (moderate hyperhomocysteinemia which is characteristic of AD patients), HCA activates NMDA receptors [42, 43, 44]. HC and HCA render a similar effect on an increase in calcium and ROS levels inside the neurons, the latter being suppressed by both Nacetylcysteine (cell membrane penetrating antioxidant) and BAPTA-AM (cell membrane penetrating Ca-buffer) [45]. The authors concluded that HC or HCA induced a calcium signal preceding intraneuronal ROS accumulation. ROS signal rather quickly (after 1-3 hrs) induces externalization of phosphatidylserine on the neuronal membrane [36, 44], which corresponds to initiation of the apoptotic process.

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It was demonstrated that an intraventricular injection of either NMDA or HCA to two-week-old rats induced long lasting convulsions accompanied by massive apoptotic death of neurons in several regions of the brain; the NMDA receptor antagonists act as anticonvulsants [43]. Thus, HC and HCA induce an excitotoxic effect influencing preferably the NMDA receptors, which is easy to explain in terms of similarity of their structures:

3. Discovery of NMDA-receptors in the non-neuronal cells It is clear now that glutamate receptors diversely spread in several tissues. Metabotropic glutamate receptors associated with G-proteins present in various cells and even ionotropic glutamate receptors belong not only to the brain. Several years ago, NMDA receptors were described in rodent and human lymphocytes [46, 47] and it was found that their activation with NMDA induces calcium [48] and ROS [47, 49] accumulation. Biological significance of these receptors in immune competent cells is not fully understood but the levels of their expression were found to depend on the functional state of the cells. Incubation of lymphocytes in vitro with phytohaemagglutinin (lymphocyte activator of plant origin) rapidly increases the portion of cells, which express NMDA receptors [49, 50]. Under in vivo conditions, inflammatory factors also increase the number of lymphocytes expressing these receptors [51]. Simultaneous presence of ionotropic and metabotropic receptors on the membrane of activated lymphocytes makes these cells close to neuronal cells where interaction between receptors of these classes is an intrinsic mechanism of regulation of cellular function [52]. It is possible that the

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interaction between metabotropic and ionotropic receptors regulates efficiency of the cytokine synthesis. Such a conclusion was made when γ-interferon accumulation was measured by IL-2 activated lymphocytes expressing both ionotropic (NMDA activated and AMPA activated) and metabotropic Group I glutamate receptors [47]. It was found that neither NMDA, nor glutamate affect γ-interferon accumulation in the native (nonactivated) cells and the level of the cytokine synthesis is neglected. Once incubated with IL-2 the cells start to accumulate γ-interferon, and this process is inhibited in the presence of NMDA but stimulated in the presence of glutamate [49]. The authors interpreted these data in such a way that ionotropic receptors of NMDA class suppress and metabotropic Group I receptors stimulate the cytokine production [49]. Thus, glutamate may play a role of both a neuromediator (in the neuronal system) and immunomediator (in the immune system) [53]. Recently, a similar situation was described in other immune cells of rodents possessing phagocytic properties, neutrophils. Neutrophils isolated from peripheral blood of intact animals were free from NMDA receptors, whereas they have a number of adenosine receptors regulating their immune function [54]. Adenosine receptors of A1 class affect phagocytosis and those of A3 class affect degranulation, whereas A2 adenosine receptors control the cAMP levels, thus regulating neutrophic activation [54-56]. Hence, the result of activation of adenosine receptors of the neutrophils would depend on the species of receptors involved in realization of the signal. For example, an interaction between A1 or A2 adenosine receptors with Fcγ receptor is described in the literature [56]. Activation of A1-receptors switches on Fcγ receptors and results in ROS generation, whereas activation of A2 receptors switches off Fcγ receptors and suppresses ROS accumulation [57]. Interrelations between A1 receptors and NMDA receptors are also noted on the postsynaptic membrane of the neuronal junction where activation of A1 suppresses the NMDA dependent ionic fluxes [58]. At the same time, on the presynaptic membrane, adenosine suppresses the glutamate release in the synaptic cleft, whereas glutamate via activation of NMDA receptors stimulates adenosine release in synaptic cleft [59]. Revealing NMDA receptors in lymphocytes [47-49] suggests the presence of similar mechanisms of interaction between the glutamate and adenosine receptors in these cells. We have found recently that lymphocytes and neutrophils isolated from an inflammatory region of rats expressed NMDA receptors on their membrane contrary to the intact cells which are predominantly free from them [51, 60]. The functional significance of this phenomenon is still obscure but one can suggest that they may regulate ROS production during cell

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activation. Especially important is that HC can stimulate ROS production in these cells presumably through NMDA receptors [60, 61]. With this in mind, identification of NMDA receptors in megakaryocytic cells (platelet precursors) [62, 63] and in cardiomyocytes [64] where they can regulate Cafluxes across the outer membrane is very noticeable. Thus, we conclude that NMDA receptors are involved in intracellular signaling not only in the brain but also in a broad variety of other tissues.

4. Effect of homocysteine on red and white blood cells The above data concerning the capability of the immune competent cells to express NMDA receptors makes it interesting to evaluate the effect of HC and HCA on their function. Recently, we have analyzed the effect of HC and HCA on both cytokine producing cells (lymphocytes) and phagocytic cells (neutrophils). It was found that in lymphocytes, these receptors are similar to those in neurons by their ability to increase calcium and ROS accumulation [36, 46, 47] and by the effects of HC and HCA [65]. Effective concentrations of HC are similar to those found in moderate and severe hyperhomocysteinemia (100-500 µmol/l) and effects are quickly manifested: even a 1-to-3-hour incubation is enough to demonstrate the initial steps of cellular death via apoptosis or necrosis. As we noted earlier, neutrophils isolated from peripheral blood of intact animals are practically free from the functionally active NMDA receptors and HC does not affect the ROS levels. It can however stimulate ROS production when the cells are stimulated by fMet-Leu-Phe (fMLP), which itself brings about the so-called “respiratory burst”. This phenomenon is known to depend on specific fMLP receptors [66], which are unlikely to interact with HC (HC does not affect the neutrophil function in the absence of fMLP) and modulation of the respiratory burst by HC requires its interaction with other receptors. The role of HC target may be played by adenosine receptors [67] virtually interacting with HC, thus resulting in stimulation of ROS production [61, 68]. Inhibitory analysis showed that A2 receptors are involved in HC effect resulting in over-production of ROS by neutrophils [61]. Interestingly, HCA renders a weaker effect on neutrophils than HC does, whereas it affects neuronal NMDA receptors more effectively than HC. Thus, adenosine receptors demonstrate higher affinity to HCA whereas NMDA receptors demonstrate higher affinity to HC. An ability of neutrophils to express NMDA receptors during activation by inflammatory cytokines suggests involvement of these receptors in ROS production by these cells. Actually, HC stimulates the respiratory burst of

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such neutrophils and this effect is sensitive to MK-801 [60]. Thus, HC hyperactivates the NMDA receptors of both the neuronal and immune competent cells. It shows that hyperhomocysteinemia is a factor exhausting the functional activity of both the neuronal and immune systems. Recently, a strong hemolytic effect of HCA was described consisting in acceleration of red blood cells hemolysis induced by several unfavourable factors [69]. Preincubation of RBC with HCA increased the rate of acidic hemolysis and decreased the lag-period. Such action can be easily understood, taking into consideration possible presence of NMDA receptors on the erythrocytic membrane [69, 70]. Their activation can induce the Caentry into red blood cells and modify their functional properties. A possible toxic effect of HC on the platelets is also noteworthy. A high blood level of HC stimulates platelets aggregation, which may be associated with increased ROS production and damage of vascular endothelial cells [71, 72]. These effects at least partially can be a result of HC effect on NMDA receptors recently described in megacaryocytes [62, 63].

5. Possibilities to protect the body from toxic effects of homocysteine As we discussed, impaired HC metabolism results in its accumulation in blood stream and this accompanies a number of pathologies [7, 73]. High levels of blood HC may be not an immediate cause of a pathological state but their consequences. In any case, a toxic action of HC will aggravate the development of disease revealing neurological symptoms. Thus, hyperhomocysteinemia is associated with serious complications, impelling to the search of protectors against HC toxicity. An important aspect of hyperhomocysteinemia consists in toxic effects of HC on the female body in pregnancy. HC easily penetrates from the maternal blood into the fetus to render a teratogenic/fetotoxic action. HC levels in the maternal blood were found to have a negative correlation with the body weight of the newly-born infant [74]. Hence, all the above-described toxic effects of HC may induce pregnancy complications [75, 76]. A commonly accepted means to compensate hyperhomocysteinemia is a systemic increase in dietary vitamins (B6, B12, folic acid). In some cases, however, especially when an increase in HC levels is induced by genetically provided deficiency of enzymes of its metabolism dietary regulation is not sufficient. That is why it is important to devise a special strategy to defend the body from HC toxicity. Because accumulation of ROS and misbalance in cell signaling in NMDA receptorâ&#x20AC;&#x2122;s expressing cells is one of the features of

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HC toxicity administration of drugs modulating properties of the receptors may have undesirable side effects. On the other hand, the use of natural metabolites able to regulate the intracellular ROS levels and preserve viability and functional activity of the cells could be effective. In order to develop such approach we turned the attention to neuropeptide carnosine (β-alanyl-L-histidine) which is a specific constituent of excitable tissues being able to protect the brain neurons against oxidative stress [77, 78]. Carnosine prevents neuronal death induced by excitotoxic effects of NMDA in vitro [79, 80]. It is characterized by extremely low toxicity [81, 82] and ability to penetrate the blood-brain barrier after administration into blood stream [83]. Excess of carnosine is quickly cleaved by serum carnosinase preventing effects of possible over-loading [84, 85]. Carnosine was also found to protect the brain under stroke conditions in vivo [86, 87]. The described recently favourable effect of carnosine on red blood cells under acidic hemolysis aggravated by HCA [69] suggested that this compound possesses a diversity of properties besides its antioxidant activities. In order to estimate possible protection of the body from toxic effects of HC, we used a recently developed model of prenatal hyperhomocysteinemia induced by over-loading of pregnant rats with excess of methionine (1g/kg body weight daily) [88, 89]. Thus the prenatal development of the brain is implemented under stable hyperhomocysteinemia (in our case, blood HC levels increased from 5.9 ± 1.8 to 33.0 ± 3.9 µmol/l) [89]), resulting in a decrease in the average number of neonatal rats in the litter and a marked decrease in their body weight (Table 1). Table 1. Characteristic of pups in the progeny of several groups of rats (р1 estimates the statistically significant difference from group 1, and р2 – that from group 2), from [90] with permission. Groups of animals

Number of families

Average number of newborns in the litter 12 ± 2

Weight, g (10 days old)

Group 1 (intact animals)

Group 2 (methionine administration)

7±1 p1<0.05

18.9±0.5

13 ± 2 p1>0.05

24.1±0.6 p2<0.05

Group 3 (methionine+ carnosine . administration)

23.3±0.4 p2<0.05

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Animals whose prenatal and early postnatal development was under stable hyperhomocysteinemia were tested using the Morris’ water test [91]. An efficiency of searching for the platform in a water pool was estimated after preliminary training. The test demonstrated that the animals in the methionine-treated group were characterized by substantially poorer memory (Table 2). The period of search the platform in the water pool for these animals was several times longer, the swimming velocity was somehow less (not statistically significant), and duration of search in the central area of the pool (where the platform was located) amounted to only 7 % of the total time (in the control group, this time was 20 % of the total search period). Moreover, analysis of the cerebellum neurons of the animals showed desensitization of the NMDA receptors to NMDA, HC or HCA [90]. The group-three animals treated with carnosine (taken with drinking water at a daily dose of 100 mg/kg bogy mass) simultaneously with methionione substantially differed from group-two animals and by some parameters were similar to the control group. As can be seen from Table 1, pregnancy was more successful and the number of neonates in the litter, as well as their weight was similar to that for intact animals. Moreover, they were more successful in the learning test (Table 2). Finally, suspension of the cerebellum neurons isolated from these animals, revealed the lowest percent of dead cells found and the mean fluorescence of viable neurons (reflecting the intracellular ROS levels) was lower than that of the neurons from the cerebellum of the group-two animals. All these data demonstrated that systemic administration of carnosine turned out protective against HC toxicity and had improved the conditions for the foetus development in spite of a similar high content of HC in the blood Table 2. Learning of the animals of 3 groups (designed as in Table 1) obtained from Morris’ water test (from [90] with permission). Parameter registered Time to reaching the platform, s Average rate of swim, m/s Duration of swimming in the central area of the pool (% of the total period)

Group 1 (n=18)

Group 2 (n=18)

Group 3 (n=18)

20 ± 7

140 ± 18 p1<0.01

45 ± 6 p2<0.01

0.24 ± 0.02

0.18 ± 0.02

0.25 ± 0.04 p2>0.05

20 ± 7

7±5 p1<0.01

35 ± 5 p2<0.01

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(because carnosine did not decrease the blood HC levels) [90]. Consequently, a protecting effect of carnosine manifested itself not in metabolism of homocysteine but in suppression of its toxic action. It is not clear so far, how carnosine acts in the in vivo conditions – does it suppress affinity of NMDA receptors to HC, prevent accumulation of ROS, or use other (still unknown) mechanisms of protection?

6. Carnosine improves therapeutic outcomes in patients with neurodegenerative diseases All the data characterizing efficiency of carnosine as a natural brain protector in animals led us to a conclusion that it might be appropriate to use it for treatment of neurodegenerative diseases in man. At the end of this Chapter, the first experience is presented related to this new approach. Discirculatory encephalopathy (DE) is characteristic of chronic subacute expansion accompanied by increased levels of blood HC [17]. A double-blind placebo-controlled trial comprised a total of 42 patients (both men and women) diagnosed with chronic DE [92]. Carnosine was included into basic (symptomatic) therapy, given at two doses (either 0.75 or 2.0 g, daily), with the treatment duration amounting to 21 days. At the end of the trial, some neurochemical and neurological characteristics of the patients before and after the treatment were compared. The basic therapy, as well as its combination with low doses of carnosine turned out practically ineffective in improvement of either the somatic or psychological state of the patients. However, a combination of the basic therapy with carnosine taken at a higher dose (2.0 g per day) was characterized by noticeable effects. Resistance of blood lipoproteins to Fе2+-induced oxidation increased and the rate of acidic hemolysis of red blood cells decreased. At the same time, the ability of leucocytes to generate (in vitro) the zimozane-induced respiratory burst was significantly accelerated. All these data demonstrated that carnosine improved the antioxidant defence system, as well as the ability of the immune system to withstand extrinsic factors. A conclusion on the cognitive function of the brain was made based on analysis of the induced potentials P300 from the electroencephalogram of patients. Sufficient dynamics of P300 potentials was only noted in the basic therapy patients treated with a high dose (2.0 g per day) of carnosine. Only in this case (not in the case of basic therapy alone or in its combination with low doses of carnosine) the latency of P300 peak decreased from 378 ± 21 msec before treatment to 345 ± 12 msec after treatment (p < 0.05) and the number of low-amplitude spikes fell from 60% to 27% (p < 0.01). This demonstrated a positive effect of carnosine on the brain cognitive function of DE patients

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[92]. It is noteworthy that such an effect of carnosine was also demonstrated in spite of pronounced hyperhomocysteinemia in patients. This again supports a point of view that the carnosine’s effect is based on prevention of HC toxicity rather than on its metabolic neutralization. Parkinson’s disease. Parkinson’s disease (PD) is a chronic neurodegenerative disease characterized by progressive loss of dopaminergic neurons in the substantia nigra pars compacta. The exact aetiology of PD remains obscure, but what we clearly know is that the disease is of a multifactorial nature, including both environmental and genetic factors, which result in development of oxidative stress in specific areas of the brain. One of the factors accompanying PD development is pronounced hyperhomocysteinemia [93]. A conventional protocol of PD treatment consists of replacing lost dopamine with its agonists including L-DOPA therapy, administration of MAO B or catechol-o-methyltransferase inhibitors, and other factors aimed at facilating symptomatic improvement, not altering the course of disease. For this reason, treatment of PD patients is accompanied by several side effects that render the treatment nearly ineffective [94]. Therefore, it is important to develop complex treatment of PD including in the protocol antioxidants and/or neuroprotectors [93, 94]. Several recent publications illustrate a positive effect of carnosine used as an additive to basic therapy of PD patients [95, 96]. A pilot clinical trial was composed of 36 patients with trembling-rigid and trembling manifestations of PD to be compared with 20 apparently healthy donors comprising a control group. Basic therapy consisted of DOPA-containing drugs Madopar or Nacom, dopamine receptor agonists Pronoran or Mirapex and uncompetitive antagonist of NMDA receptors, amantadine at individually selected doses depending on the state and severity of clinical manifestations. For half of the PD patients carnosine was prescribed at a daily dose of 1.5 g. The treatment lasted for 30 days and preliminary data are presented below. After basic treatment, the baseline level of neurological symptomatic of patients decreased from 38.9 ± 2.5 to 32.5 ± 2.0 points (measured by the Unified Parkinson’s Disease Rating Scale, UPDRS) which corresponded to a 16.4 % improvement. Combining basic treatment with carnosine decreased the symptomatology to 24.9 ± 2.1 (36% improvement), which was at least 2 times better. Thus, carnosine included in the protocol of treatment as an additional component significantly improved the neurological state of the patients. In the carnosine-treated group, an improvement of the locomotor system (rigidity of extremities, and upper-limb movements) amounted to 32 – 38% (p < 0.05) compared with the basic-therapy group which correlated well with improvement of one of the most important clinical signs of parkinsonism – hypokinesia. The authors noted that the so-called “every day activity” was also

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improved significantly more in the carnosine-treated patients, which gave them ability for more independent self-service [96]. No any negative side effect was detected. A decrease in the neurological symptomatology of PD patients correlated with a decrease in blood serum carbonyl levels and an increase in resistance to oxidation of lipoproteins in blood plasma, as well as in restoration of SOD activity (measured in red blood cells), with the increment of SOD restoration being in a correlation (r = 0.654) with a neurological symptomatic decrement. The authors concluded that a combination of carnosine with basic therapy of PD patients may be a reasonable way to improve the PD treatment and to decrease possible toxic effects of over-loading of DOPA containing drugs. Alzheimerâ&#x20AC;&#x2122;s Disease. Alzheimerâ&#x20AC;&#x2122;s disease is another example of a neurodegenerative disorder complicated by an oxidative stress and increased levels of HC in blood of patients [22, 27, 28]. In the available literature we found no information concerning carnosine used to treat AD patients; however, based on the above data such an approach seems very reasonable.

7. Conclusion In conclusion, hyperhomocysteinemia is one of serious risk factors for cardiovascular and neurodegenerative diseases, as well as other pathologies in which various manifestations of oxidative stress are involved. We have shown that the molecular basis for toxicity of homocysteine and its derivatives consists in over-activation of the NMDA class specific glutamate receptors. A wide distribution of these receptors in a number of tissues including the brain, heart, and immune competent cells makes HC toxic for many tissues. Successful attempts to use naturally occurring compounds to protect the body from HC toxicity might find a favourable application in present-day clinical protocols. I am thankful to Academician Z.A. Suslina, Professor S.N. Illarioshkin and Professor I.A. Ivanova-Smolenskaya (Research Center on neurology, Moscow, Russia) for organization of clinical trials and Drs. B.A. Kistenev, M.Yu. Maximova, V.V. Gnezditsky, G.Ch. Bagyeva and M.A. Loskutnikov for highly professional participation in clinical trials. The work is supported by RFBF Grant #06-04-49675 (Russia).

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Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Recent Advances on Nutrition and the Prevention of Alzheimer’s Disease, 2010: 145-154 ISBN: 978-81-7895-470-7 Editors: Charles Ramassamy and Stéphane Bastianetto

9. Catechins and resveratrol as protective polyphenols against beta-amyloid-induced toxicity: Possible significance to Alzheimer’s disease Stéphane Bastianetto, Yvan Dumont and Rémi Quirion Douglas Mental Health University Institute, Department of Psychiatry, McGill University, 6875 Blvd LaSalle, Verdun, Québec, H4H 1R3 Canada

Abstract. Polyphenols have recently received particular attention because of their possible preventive role in the incidence of agerelated cognitive deficits and neurological disorders. Recent evidence from epidemiological studies has shown that beverages or food enriched in polyphenols are related to a low risk of dementia such as Alzheimer's disease (AD). These findings are in accordance with animal and cell culture studies indicating that catechins (or flavanols) and resveratrol display neuroprotective abilities or reverse cognitive deficits. Using cultured rat hippocampal neuronal cells, we investigated the neuroprotective abilities of various catechin ingredients of green tea and red wine against toxicity induced by free radicals and betaamyloid (Aß) peptides, in respect to the possible deleterious role of these agents in age-related neurological disorders. Our studies, along with those obtained by other groups, indicated that these beneficial effects appeared to be only partly attributable to their well-known antioxidant activities, but also to their ability to directly Correspondence/Reprint request: Dr. Stéphane Bastianetto, Douglas Mental Health University Institute, 6875 LaSalle Boulevard, Verdun, Québec, H4H 1R3, Canada. E-mail: stephane.bastianetto@douglas.mcgill.ca

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inhibit cell death produced by Aß peptides, and to modulate intracellular signaling pathways and gene expression associated with cell death/survival as well. We summarize in this review the purported beneficial effects of polyphenols in human as well in in vitro and animal models of neurotoxicity. We will then focus on the possible mechanisms underlying their neuroprotective effects and finally discuss possible clinical implications and the possible role of polyphenols as therapeutic agents.

Abbreviations Aβ, β-amyloid; ADDLs, Aβ-derived diffusible ligands; AD, Alzheimer’s Disease; bis-ANS: 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonate; DCF, dichlorofluorescein; EC, epicatechin; ECG, epicatechin gallate; EGC, epigallocatechin; EGCG, epigallocatechin gallate; EGb 761, gingko biloba extract; PKC, protein kinase C; ROS, reactive oxygen species; ThT, Thioflavin T.

Introduction Alzheimer’s disease (AD) is the most common form of dementia and is mainly characterized by specific types of lesions, called amyloid plaques and neurofibrillary tangles with abnormally phosphorylated tau [1]. The principal component of amyloid plaques consists of aggregated forms of amyloid (Aß) that may play an important role in neuronal degeneration. The relative contribution of the various forms (soluble dimers, small oligomers and fibrils) of Aß to neuronal death is still uncertain [2, 3]. Hence, the weak correlation between fibrillar Aß accumulation and neurological dysfunctions observed in AD suggests that aggregation of Aß fibrils is not fundamental to the neurodegenerative processes occurring in AD [2]. Moreover Aß deposits may also develop in cognitively normal elders [2]. On the other hand, recent findings indicate that soluble monomers and oligomers of Aß - including Aβderived diffusible ligands (ADDLs), may represent the most important pathologic species [3]. Preliminary analyses have revealed abundant soluble oligomers in AD patients, consistent with the notion that oligomers precede senile plaques development and may be linked to cognitive impairments [4]. Since available drugs are not able to significantly stop the progression of AD, it has been proposed that the inhibition of soluble/insoluble forms of Aß may be an appropriate strategy to block or even reverse the progression of the disease. There is much evidence that consumption of fruits, vegetables, green tea and red wine (in moderation) reduces the risk of developing age-related neurological disorders such as stroke, AD and Parkinson’s disease [5-9]. Polyphenols present in high amounts in fruits, vegetables and tea likely

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contribute to their beneficial effects. In support of this hypothesis, a 5-year follow-up study reported that regular consumption of polyphenols was inversely linked to a risk of dementia [10]. Moreover, using cell cultures of transgenic mice model of AD, the tea-derived flavan-3-ol such as epigallocatechin gallate (EGCG) and the stilbene resveratrol have been reported to protect neurons from Aß toxicity [11-19] and modulate tau pathology [20]. Moreover, moderate consumption of Cabernet Sauvignon, which contains both catechins and resveratrol and a standardized ginkgo biloba extract, a polyphenols-derived natural extract were able to attenuate deficits of spatial memory in 14-months old transgenic mice (Tg2576), that develop Aß plaques [21,22]. Finally, a recent animal study indicated that long-term green tea administration may prevent age-related learning and memory decline by modulating the transcription factor cAMP-response element binding protein (CREB) in the hippocampus [23]. Various hypotheses have been proposed to explain this inhibitory action on Aß-associated events. Ono et al. (2009) have shown that various polyphenols were able to inhibit amyloid fibrils and destabilize fibrillized forms of Aß [24], suggesting that they could be viewed as therapeutic agents for the treatment of Aß-associated diseases [17,25]. Moreover, resveratrol, promoted the intracellular degradation of Aß by a proteasome-dependent and secretases-independent mechanism [26] whereas EGCG may modulate the nonamyloidogenic -secretase proteolytic pathway [15]. We review here the interaction of polyphenols with soluble and insoluble forms of Aß peptides, and their neuroprotective abilities. We then discuss the identification and characterization of specific binding sites for polyphenols in the rat brain and their possible relevance to the neuroprotective action of these molecules.

Materials and methods Mixed hippocampal cell cultures and experimental treatments Hippocampal cell cultures were prepared from E19 fetuses obtained from Sprague-Dawley rats. Animal care was according to protocols and guidelines of the McGill University Animal Care Committee and the Canadian Council for Animal Care. Mixed (glial/neuronal) hippocampal cells were obtained as described in detail elsewhere [12]. Measurement of cell survival/death was performed as described in detail elsewhere, using MTT and Sytox® green assays, respectively [12]. Briefly, 6-day old cells were exposed to fresh solutions of either Aß25-35 (25µM) or Aß1-42 (15µM) for 24 hours, in the presence or absence of different drugs.

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Measurement of soluble and insoluble forms of Aβ 4, 4′-dianilino-1,1′-binaphthyl-5,5′-disulfonate (bis-ANS) is a fluorescent probe that has been shown to evaluate amounts of soluble forms of Aβ [27]. Briefly, the physiological fragment Aβ1–42 (15µM) was incubated for 30 min at room temperature in the presence of different polyphenols. Bis-ANS fluorescence (excitation = 360 nm, emission = 485 nm) was then measured by dilution of 100µl aliquots to a final volume of 300 µl with citrate buffer (30 mM, pH 2.4) containing bis-ANS (25µM), using a fluorescence multiwell plate reader (Bio-Tek Instruments® Inc.). The thioflavin T (Th-T) fluorescence method was performed to determine amyloid fibril formation, as previously described [17]. Briefly, a fresh solution of Aß1-42 (15µM) was incubated at 37°C for 24 h in phosphate-buffered saline (pH 7.4). After incubation, a 100µl aliquot of solution was added to a final volume of 300 µl of phosphate buffer (50mM, pH 6.0) containing 5 µM Th-T in the presence of different drugs. Th-T fluorescence was evaluated using a fluorescence multiwell plate reader (excitation and emission wavelengths of 450 and 485 nm, respectively).

Binding assays and receptor autoradiography In brief, binding assays were initiated by adding membrane preparations in a solution of Krebs containing [3H]-resveratrol, and competitors as described earlier [28]. Saturation experiments were performed at room temperature in the presence of increasing concentrations of [3H]-resveratrol, whereas competition binding experiments were performed in the presence of 20 nM [3H]-resveratrol and various competitors (10-10 to 10-4 M). Non-specific binding was determined in the presence of 100 µM resveratrol [28]. Quantitative receptor autoradiography was performed as described previously [29].

Results Catechins gallate esters protected hippocampal cells against Aß– induced toxicity The neurotoxic effect of Aß25-35 was reduced, in a dose-dependent manner by a treatment with green tea extract (5-25 µg/ml), as well as a black tea extract (5 µg/ml) which contains 80% of condensed products of catechins. The neuroprotective action of green and black tea extracts was shared by the most abundant green tea flavan-3-ols gallate esters, also called catechins gallate esters, known as epigallocatechin gallate [EGCG], and to a lesser

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extent by epicatechin gallate (ECG), which represents approximately 5% of the total extract [30]. In contrast, non-gallate forms of catechins such as epicatechin (EC) and epigallocatechin (EGC) were ineffective. The Sytox® green assay revealed that the extent of neuronal death was increased in the presence of Aß1-42, as compared to the vehicle-treated control group. The green (25 µg/ml) and black (5 µg/ml) tea extracts were able to completely block cell death produced by Aß1-42, as did EGCG (10 µM) and the ginkgo biloba extract EGb 761 (100 µg/ml), a well-known standard natural extract prescribed for the treatment of cognitive disorders in AD patients [31]. Finally gallic acid and tannic acid - a polymer of gallic acid molecules also displayed neuroprotective action, data summarized in Table 1.

Resveratrol protected cells against Aß-induced neurotoxicity Treatment with 20 µM Aß peptides (Aß25−35 or Aß1−42) caused nearly 40% cell death and that was dose-dependently reduced in the presence of resveratrol (15−40 µM), with a maximal effect at 25 µM. A pre-treatment with the PKC inhibitor, GF 109203X, but not MAP kinase (PD98059) and PI3 kinase (LY294002) inhibitors significantly blocked the neuroprotective action of resveratrol against Aß25−35-induced neurotoxicity. Moreover, the role of the PKC- isoform was confirmed by Western blot that resveratrol induced the phosphorylation of PKC and abolished the inhibitory effect of Aß25−35 on phosphorylation of PKC- at the same range of concentrations, it protected hippocampal neurons. Among other stilbenes tested in our model, piceatannol, a natural resveratrol analog, was the most potent, whereas the other stilbenes known as trans-4-stilbenemethanol, transtilbene and diethylstilbestrol were ineffective (Table 1).

Effects of polyphenols on soluble/insoluble forms of Aβ We investigated the effect of catechins and stilbenes on both soluble and insoluble forms of Aβ1-42 using the bis-ANS and Th-T fluorescence assays, respectively. The bis-ANS assay revealed that EGCG and piceatannol reduced Aβ1-42-induced increased fluorescence, whereas resveratrol did not modify bis-ANS fluorescence. An incubation with Aβ1-42 increased Th-T fluorescence by 30-75 fold relative to control; the effect was diminished in the presence of neuroprotective polyphenols including EGCG, resveratrol and piceatannol. In contrast, EC and EGC were ineffective. Results are summarized in Table 2.

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Table 1. Summary of the effects of catechins, phenolic acids and stilbene derivatives against toxicity induced by Aß peptides (Aß25-35, Aß1-42) in rat hippocampal cell cultures. Treatment Green tea extract Black tea extract Epigallocatechin gallate Epicatechin gallate Epigallocatechin Epicatechin Tannic acid Gallic acid Resveratrol Piceatannol Trans-4-stilbenemethanol Transtilbene Diethylstilbestrol

Neuroprotection + + + + + + + + -

Taken from [11, 12, 19, 26]

Table 2. Summary of the effects of catechins, phenolic acids and stilbene derivatives on soluble and fibrillar forms of Aß1-42. Treatment Green tea extract Black tea extract Epigallocatechin gallate Epicatechin gallate Epigallocatechin Epicatechin Gallic acid Tannic acid Resveratrol Piceatannol Trans-4-stilbenemethanol Transtilbene Diethylstilbestrol ND: not determined Taken from [12, 19, 26]

Soluble forms of Aß1-42 + ND + -/+ ND + ND ND ND

Fibrillar forms of Aß1-42 + ND + -/+ + + + + -

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Identification of [3H]-resveratrol binding sites We next identified and characterized [3H]-resveratrol binding sites in the rat brain subcellular fractions. Significant [3H]-resveratrol binding was detected in plasma membrane, and to a lesser extent in nuclear fraction. Binding to the PII fraction was significantly reduced by pretreatment with trypsin or boiling, suggesting that specific [3H]-resveratrol binding sites are particularly abundant in the plasma membrane. Scatchard transformation of isotherm saturation binding experiments suggested that [3H]-resveratrol specifically binds to a single class of sites, with an apparent affinity of 220 ± 80 nM in the PII fraction [28]. Quantitative autoradiographic studies revealed that specific [3H]-resveratrol binding sites are the most abundant in the choroid plexus and subfornical organ, and to a lesser extent in other regions such as the hippocampal formation and the cortex [28]. We evaluated the ability of various analogs of resveratrol and catechins to compete for specific [3H]-resveratrol binding in PII fraction. Interestingly, EGCG and (-)-epicatechin gallate (ECG) are most potent to compete for specific [3H]resveratrol binding with Ki values of 45 and 25 nM, respectively, whereas resveratrol was found to be less potent (Ki = 102 nM). In contrast, the non-neuroprotective polyphenols including EC, EGC, trans-4stilbenemethanol and diethylstilbestrol were inactive (Ki > 10000nM). Most importantly, the apparent affinities of a series of analogs of resveratrol and catechins for [3H]resveratrol binding correlated well (r = 0.74) with their neuroprotective abilities against Aβ25-35-induced toxicity in primary hippocampal cells, suggesting their functional relevance [28].

Discussion We have demonstrated that resveratrol and catechins gallate esters derived from fruits, vegetables and beverages (e.g. red wine, green tea) protect hippocampal cells against the neurotoxic action of Aβ. These data are in accordance with previous findings reporting neuroprotective action of polyphenols derived from grape seeds and tea extracts in various in vitro and animal models of toxicity [11-19]. It also suggests that regular consumption of polyphenols may attenuate the accumulation of Aβ peptides that contributes to the process of neurodegeneration occurring in AD [1-3]. Resveratrol, an active silbene from grapes, was shown to concentrationdependently protect against Aβ-induced toxicity in cultured hippocampal neurons. The mechanism(s) involved in the neuroprotective effects of resveratrol likely include PKC as evidenced by the inhibitory action of the potent PKC antagonist GF 109203X and the stimulation of the phosphorylation

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of this enzyme by resveratrol, suggesting that the PKC pathway plays a major role in the neuroprotective properties of resveratrol in our model. Other mechanisms may also be involved since resveratrol was shown to promote proteasome-dependent intracellular Aβ degradation [26] and inhibit Aβ fibrils formation and amyloid plaques [18,32], in accordance with our findings. The strong neuroprotective effects of tea extracts against Aβ toxicity are likely to be explained by the presence of catechins found as monomers and dimers (i.e. theaflavins) in green and black teas respectively [30]. Among catechins tested here, EGCG, and to a lesser extent ECG, displayed strong neuroprotective activities, in accordance with previous studies [15, 16]. Similar protective effects were also observed with theaflavins that are almost exclusively present in black tea [30]. These findings suggest that neuroprotective activities of catechin gallate esters depend on the esterification of the pyran hydroxyl group of catechins by gallic acid – a phenolic acid present in tea and red wine. In support of this hypothesis, gallic acid and tannic acid –a polymer of gallic acid significantly blocked both toxicity and fibrils formation produced by Aβ peptides [17]. We subsequently investigated the ability of these polyphenols to modulate the formation of soluble and fibril forms of Aβ1-42. Globally, polyphenols with neuroprotective actions tend to inhibit Aβ fibrils and to a lesser extent, soluble forms of Aβ, whereas non-gallate forms of catechins were ineffective. These data agree with previous findings reporting antiamyloidogenic and fibril-destabilizing activities of these polyphenols [25, 33]. Among them, EGCG appeared to be the most potent polyphenol and was able to inhibit Aβ-derived diffusible ligands (ADDLs), suggested to mediate the neurotoxic effects of Aβ1-42 [3]. Taken together, these data suggest that the neuroprotective action against Aβ-induced neurotoxicity may be due, at least in part, to their inhibitory action on Aβ fibrils/oligomers formation. Finally, binding and autoradiographic studies revealed the existence of specific polyphenols binding sites in the rat brain, in particular in the choroid plexus. Structure-activity data support the hypothesis that these specific binding sites may be responsible for the neuroprotective actions of polyphenols. In summary, our results demonstrate that the neuroprotective action of catechins gallate esters and resveratrol is partly due to their interaction with intracellular kinases and their inhibitory action on Aß oligomers and/or fibrils formation. We have also shown the presence of specific [3H]-resveratrol binding sites at the level of the plasma membrane in the rat brain. These findings support the hypothesis that regular intake of polyphenols derived

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from red wine, tea, fruits and vegetables may delay or even prevent age-related neurological disorders such as Alzheimerâ&#x20AC;&#x2122;s disease, and suggest that polyphenols may be considered as possible neuroprotective agents.

Acknowledgements This work was supported by research grants from the Canadian Institutes of Health Research (CIHR) to RĂŠmi Quirion.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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21. Rezai-Zadeh, K., Arendash, G.W., Hou, H., Fernandez, F., Jensen, M., Runfeldt, M., Shytle, R.D., and Tan, J. 2008, Brain Res., 1214, 177. 22. Stackman, R.W., Eckenstein, F., Frei, B., Kulhanek, D., Nowlin, J., and Quinn, J.F. 2003, Exp. Neurol., 184, 510. 23. Wang, J., Ho L., Zhao, Z., Seror, I., Humala, N., Dickstein, D.L., Thiyagarajan, M., Percival, S.S., Talcott, S.T., and Pasinetti, G.M. 2006, FASEB J, 20, 2313. 24. Li, Q., Zhao H.F., Zhang Z.F., Liu Z.G., Pei X.R., Wang J.B., Cai M.Y., and Li Y. 2009, Neuroscience, 159, 1208. 25. Ono, K., Yoshiike, Y., Takashima, A., Hasegawa, K., Naiki, H., and Yamada, M. 2008, J. Neurochem., 87, 172. 26. Porat, Y., Abramowitz, A., and Gazit, E. 2006, Chem. Biol. Drug., 67, 27. 27. Marambaud, P., Zhao, H., and Davies, P. 2005, J Biol. Chem., 280, 37377. 28. LeVine, H. 3rd 2002, Arch. Biochem. Biophys., 404, 106. 29. Han, Y.S., Bastianetto, S., Dumont, Y., and Quirion, R. 2006, J. Pharmacol. Exp. Ther., 318, 238. 30. Dumont, Y., Fournier, A., and Quirion, R. 1998, J. Neurosci., 18, 5565. 31. Del Rio, D., Stewart, A.J., Mullen, W., Burns, J., Lean, M.E., Brighenti, F., and Crozier, A. 2004, J. Agric. Food. Chem., 52, 2807. 32. Le Bars, PL. 2003, Pharmacopsychiatry 36 Suppl 1, S44-9. 33. Riviere, C., Richard, T., Quentin, L., Krisa, S., Merillon, J.M., and Monti, J.P. 2007, Bioorg. Med. Chem., 15, 1160. 34. Ono, K., Hasegawa, K., Naiki, H., and Yamada, M. 2004, Biochim. Biophys. Acta., 1690, 193.

Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Recent Advances on Nutrition and the Prevention of Alzheimer’s Disease, 2010: 155-168 ISBN: 978-81-7895-470-7 Editors: Charles Ramassamy and Stéphane Bastianetto

10. Neuroprotective effects of oxyresveratrol from fruit against neurodegeneration in Alzheimer’s disease 1

Raymond Chuen-Chung Chang1,2,3, Jianfei Chao1,4, Man-Shan Yu1 and Mingfu Wang4

Laboratory of Neurodegenerative Diseases, Department of Anatomy, LKS Faculty of Medicine The University of Hong Kong, Hong Kong SAR; 2State Key Laboratory of Brain and Cognitive Sciences The University of Hong Kong, Hong Kong SAR; 3Research Centre of Heart, Brain, Hormone and Healthy Aging, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR 4 School of Biological Sciences, The University of Hong Kong, Hong Kong SAR

Abstract. Many countries are stepping into aging society. The increasing proportion of elderly in population is in high risk of having more aging-associated diseases. Alzheimer’s disease (AD) is the most common age-associated disorder, characterized by progressive dementia. Nowadays, 30 million people have suffered the disorder. However, no cure is applied in clinic except symptom-relieving strategy. Epidemiological studies have shown the reverse association between consumption of nutritional diets rich in anti-oxidants and developing neurodegenerative diseases. Therefore, healthy aging should be put forward by introducing healthy life style, in which good nutrition plays a key role. Oxyresveratrol (OXY) is a natural hydroxystilbene present in mulberry fruit (fig.1), or fruits of several trees belonging to Artocarpus genus. It has been found as tyrosinase inhibitor. Correspondence/Reprint request: Dr. Raymond C. C. Chang, Rm L1-49, Laboratory Block, Faculty of Medicine Building, Department of Anatomy, LKS Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Hong Kong. E-mail: rccchang@hkucc.hku.hk

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Recently, increasing lines of evidence have demonstrated its neuroprotective effects on age-associated diseases such as AD and stroke. Apart from conventional antioxidant property and the permeability to blood-brain-barrier, we focus on reviewing the anti-inflammatory activity, cell signaling regulation property, as well as its bioavailability in vivo and in vitro. Findings enlighten that nutritional supplement, such as fruits containing OXY may retard aging or benefit healthy aging, which is helpful to reduce the risk of neurodegeneration in elderly population.

Introduction Dementia and Alzheimer’s disease (AD) In the next few decades, with the increasing longevity in population, most Western and Asian countries are stepping into elderly society. Meanwhile, a ‘silent epidemic’ is spreading with various problems from elderly community [1]. Dementia has been one of the major public health problems in both developed and developing countries. The prevalence of senile dementia increases dramatically from 0.8% in the 65–69-year-old groups, and to 28.5% in populations of 90 years and older [2]. In developed countries, the prevalence rate of dementia among people aged ≥ 65 years has been reported to be around 5–6%. Among all types of dementia, AD is the commonest form, with a prevalence of 0.4% in women and 0.3% in men aged 60–69 years, rising to 11.2% in women and 10% in men over 80 years [3]. Systematical investigation has revealed that the chronological prevalence of AD increased significantly from 1980 to 2004 in China. As the most major subtype of dementia in China, the pooled prevalence of AD for the population aged 60 years and older is 1.6% [4]. In the process of aging, the brain appears more and more vulnerable to oxidative stress, which has been reported to be the major culprit of the most commonly age-related neurodegenerative disease, AD. The onset and manifestation of AD would significantly decrease the quality of life in human, along with psychiatric and behavioral problems (e.g. depression, delusions, misidentifications), and cognitive deficiency that results in aphasia (impairment of language), amnesia (loss of memory), agnosia (inability to recognize) and apraxia (incapability in motor behaviors). These deficiencies would gradually destroy daily life, such as difficulties in using telephone, dressing, driving, eating and toileting. Mental emptiness and loss of controlling whole body functions will eventually occur in the last stage of the disorder [5]. Risk factors leading to the development of AD include genes like the APP gene, the presenilin genes 1 and 2, the α2-macroglobulin gene and the apolipoprotein E-4 gene [6-8], as well as a series of factors concerned with

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daily life, such as smoking, alcoholism, diet, high LDL cholesterol, depression, hypertension, even low level of education [9].

Generation of reactive oxygen species (ROS), oxidative stress and AD Physiologically, ROS are normal intrinsic metabolites as utilizing O2 by most life organisms. The narrow definition of ROS refers to oxygen free radicals and nonradical reaction products, including superoxide radical anion (O2˙¯), hydroxyl radical (HO˙), hydroperoxyl radical (HO2˙) and hydrogen peroxide (H2O2). As normal metabolites, ROS have been documented for mediating intracellular signaling and redox regulation to keep cell homeostasis [10; 11]. ROS are reported to play roles in normal biochemical processes involving some growth factors, cytokines, hormones, as well as neurotransmitters [12-14]. On the other hand, organisms have developed defense systems to scavenge excessive ROS or quench them by transforming ROS into less active or safe products. The intrinsic antioxidant defense systems are operated in enzymatic and nonenzymatic ways, and the cellular antioxidants consist of (1) enzymes that can interact with ROS; (2) enzymes that can catalyze the generation or regeneration of cellular antioxidants [15]; (3) metal chelators, inhibiting reactions (Fenton reaction) catalyzed by metal and therefore preventing the generation of free radicals; and (4) lowmolecular weight antioxidants such as glutathione (GSH), NADH, carnosine, uric acid, melatonin, belirubin. Besides endogenous anti-oxidants, several kinds of low-molecular weight antioxidants (e.g. phenolics, ascorbic acid, carotenoids, quinones, tocopherols) can be intaken by dietary supplement [16]. In this context, keeping stable redox status within life body is essential to be healthy, even in the process of healthy aging. Once the cellular balance is destroyed by severe burdens of ROS, the inability of intrinsic antioxidant defense systems, or both of the situations, oxidative stress occurs subsequently. Under oxidative stress, the excessive production of ROS may directly damage proteins, lipids, carbohydrates, DNA and even cellular molecules involved in antioxidant defense systems. In addition, normal cellular signaling events may be interfered and dysregulated by excessive ROS. Increasing lines of experimental or clinical studies have proposed oxidative stress as the major pathological cause of a series of human diseases, such as cancer, cardiovascular diseases, diabetes, as well as age-associated immune and neurodegenerative disorders (AD, Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS)) [17; 18]. Among all cases, the central nervous system (CNS) seems to be particularly vulnerable to ROS. High consumption of total body oxygen (about 20%) by

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the brain, high content of unsaturated lipids, iron accumulation in brainspecific regions but relatively deficiency in iron-binding proteins, as well as the limited ability of regeneration for brain tissues, may account for the vulnerability of brain to pro-oxidant environment [5].

Antioxidants and AD Although the exact etiology of AD remains unknown, increasing lines of evidence have demonstrated the crucial role of oxidative stress in the pathogenesis of AD. Reports of oxidative damage in AD include lipid peroxidation, decreased unsaturated fatty acids, increased iron, aluminum, and higher levels of the most frequent oxidative modification of nucleic acid, 8-hydroxyguanosine (8-OHG) in AD brains [19; 20]. Increased ROS are proposed to be produced by activated microglia that surround most senile plaques, AÎ˛ deposition in conjunction with binding of redox-active transition metals, protein modification by advanced glycation and lipoxidation end products (AGEs and ALEs), mitochondrial abnormalities such as reduction in mitochondrial electron transport and mutation in cytochrome c oxidase genes [21; 22]. A number of studies have compared the antioxidant concentrations between AD patients and controls, and find that the concentrations of antioxidants like Vitamin A, C, or E are decreased to different extents in the plasma, cerebrospinal fluid and brains of AD patients. A series of epidemiological studies have suggested the inverse association between dietary antioxidants intake and the development of AD or cognitive impairment. The dietary antioxidants mainly include Vitamin A, C, and E, red wine, tea and curry [16; 23-25]. The limitation for these reports is that only few of them are study from randomized double-blind clinical trials. Debate about the relationship between antioxidants intake and AD onset and disease progression become difficult to be resolved. This shifts the focus of a group of scientists to investigate healthy lifestyle to promote healthy aging with interventions in diet and exercise [26; 27]. A number of laboratories have demonstrated significant neuroprotective effects of berry (blueberry [28; 29], strawberry [30], cranberry [31], mulberry [32]) fruit polyphenols, dietary flavonoids [33], phenolic apple extracts [34], spinach extracts [30], and / or grape supplements [35]. Among various kinds of polyphenols, we here mainly focus on introducing biological and pharmacological activities, especially its neuroprotective effects, of oxyresveratrol (OXY, a natural stilbene analogue to resveratrol (RES)). Stilbene is a class of antioxidant compounds sharing the same chemical skeleton, which is a diarylethene, a hydrocarbon consisting of an trans/cis

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Figure 1. Picture of mulberry fruit. The color of the fruit changes from white to dark red along with maturity difference.

ethene double bond substituted with an phenyl group on both carbon atoms of the double bond. The name stilbene was derived from the Greek word stilbos, which means shining. Many stilbene and their derivates (stilbenoids) are present naturally in plants (dietary fruits or herbs). The most widely investigated stilbene compound is resveratrol (3,5,4â&#x20AC;&#x2122;-trans-trihydroxystilbene, RES). It is found to be a stilbene phytoalexin in plants such as grapes, peanuts, berries and pines [36]. RES is synthesized in these plants to counteract various environmental injuries such as UV irradiation and fungal infection. RES is reported to be one of the active agents in Itadori tea, which has been used as a traditional medicine mainly for curing heart disease and stroke in China and Japan [37]. Since epidemiological studies have reported the inverse association between moderate consumption of red wine and the incidence of coronary heart disease, RES as the major ingredient in red wine has stimulated investigations on its cardioprotective activity which is due to its free radical scavenging property [38]. Among the wide range of biological and pharmacological activities, RES has been received increasing attention for its chemopreventive effects. It is reported to inhibit the growth of several tumor cell lines, such as leukemic, prostate, colonic, breast and esophageal

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cells generally through inhibiting tumor initiation, promotion and progression. Besides, RES has been demonstrated to inhibit platelet aggregation and lipid peroxidation, attributing to inhibit the activity of cyclooxygenase and hydroperoxidase. In recent years, studies on the activity of RES have extended to animal models of CNS disorders or injury, such as AD [39; 40], PD, HD, cerebral ischemia, as well as traumatic brain injury (TBI). Increasing numbers of report have shown that acute chronic treatment of RES exhibits neuroprotective effects against colchicine [41] 3-nitropropionic acid [42] or trauma [43; 44] -induced cognitive and motor impairment, as well as hippocampal neuron loss. The underlying mechanisms mainly attribute to the antioxidant activity of RES to reduce the related oxidative stress, including reducing the elevated malondialdehyde (MDA), lipid peroxidation, nitrite, nitric oxide (NO) and xanthine oxidase (XO) levels, increasing the depleted GSH level and succinate dehydrogenase activity in the brains of rats [39]. In addition, RES administration elicits neuroprotective effects on cerebral ischemia-induced [45] neuron damage, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced [46] motor coordination impairment, hydroxyl radical overloading, and neuronal loss through free radical scavenging activity.

Biological and pharmacokinetics studies on oxyresveratrol (OXY) However, recent studies have found that RES is not the most effective agent in regard with some biological and pharmacological activities. Investigations on differential bioactivities of OXY (2,4,3’,5’-transtrihydroxystilbene) have provided increasing lines of evidence on this point. From the chemical name of OXY, we can figure that OXY has an extra hydroxyl group compared to RES, making it be a readily hydrogen-donor, which accounts for its notable anti-oxidant activity. OXY can be found in heartwood or fruits of Artocarpus heterophyllus, Artocarpus lakoocha, Artocarpus gomezianus, Artocarpus dadah, wood extracts or fruits of mulberry trees (Morus australis, Morus alba L.), fruits of Melaleuca leucadendron, rhizome of Smilacis chinae, as well as Egypt herb Schoenocaulon officinale. Water-soluble OXY is known for its antiviral [47], hepato-protective [48] activities [49]. OXY reduces production of Aβ by inhibiting β-secretase (BACE1) [50]. We then mainly review its activities in tyrosinase inhibition, anti-inflammation, neuroprotection and pharmacokinetics.

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Tyrosinase inhibitor The first bioactivity of OXY that has been found is that OXY is a potent tyrosinase inhibitor [51; 52]. Tyrosinase is a widely distributed enzyme in nature, catalyzing the rate-limiting step for biosynthetic pathway of melanin pigments. It exists in many organisms with slightly different forms. In animals, the deposition and distribution of melanin pigments determine the skin color. Abnormal accumulation of melanin pigments is responsible for hyperpigmentations including melasma, freckles, and senile lentigines [53; 54], while depigmenting agent such as kojic acid, can afford satisfactory improvement to subjects. Kim et al. [55] have demonstrated that OXY exhibited potent tyrosinase inhibition activity in order of OXY >> kojic acid ≌ RES > rhapontigenin ≌ 3,5-dihydroxy-4’-methoxystilbene regarding the IC50 values. OXY showed a 45-fold stronger inhibitory effect on mushroom tyrosinase activity than RES. They further demonstrated that OXY works through noncompetitive inhibition of tyrosinase activity rather than suppression of both mRNA expression and protein synthesis of tyrosinase. Kojic acid is able to chelate copper at the active site of tyrosinase, which is regarded as its underlying mechanism of inhibiting tyrosinase activity. The analogue of OXY, RES has shown antioxidant activity by chelating copper in several biological systems [56]. Consequently, authors proposed that the potent inhibition on tyrosinase activity of OXY may be attributed to the similar ability of chelating copper.

Anti-inflammatory activity Reports have documented the in-vitro and in-vivo anti-inflammatory effects of OXY isolated from Artocarpus heterophyllus [57], Artocarpus dadah [49], or mulberry wood. Mori Cortex is the dried root bark of Morus alba L., and has been widely used as an antitussive, antiphlogistic, antiinflammatory and diuretic herb in China, Japan and Korea. Chung et al. [58] have isolated mulberroside A and OXY from Mori Cortex extracts, the two compounds exhibit an in vivo anti-inflammatory effect on carrageenininduced paw edema in rats, suggesting these compounds as the active components of Mori Cortex. They further investigated the underlying mechanism of the action by examining the effect of OXY on NO production and prostaglandin E2 (PGE2) biosynthesis in lipopolysacchride (LPS)activated RAW 264.7 macrophage. The production of NO and PGE2 have been implicated in the process of inflammation. Results exhibited that OXY could significantly inhibit the production of nitrite, PGE2, the expression of inducible nitric oxide synthase (iNOS) and NF-κB activation. In contrast to

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the in vitro study, Mouihate et al. [59] found that OXY blocked specifically LPS-induced hypothermia but had no significant effect on fever in rats. They show for the first time that OXY targets specifically TNF-α independent of IL-6 production or IL-6-activated STAT-3 signaling pathway [59]. However in the in vivo model, OXY did not alter the immune activation of NF-κB transcription factor in the liver; it did not affect the cyclooxygenase (Cox-2, a key enzyme in the production of inflammatory prostaglandin), and induction in the organum vasculosum of the lateral terminalis (OVLT) / preoptic area (POA). The conflicting results may be attributed to the fact that inflammatory responses in vivo are much more complex than that in vitro. The cytokine specific property of OXY may suggest that it can target TNF-α production at phases of transcription, translation or secretion. All these anti-inflammatory effects may have implication as neuroprotective agent in AD.

Neuroprotective effects OXY has been reported to exert neuroprotection in Aβ (25-35)-induced neurotoxicity in cortical culture neurons, 6-hydroxydopamine (6-OHDA)induced neurotoxicity in SH-SY5Y cells, as well as in vitro and in vivo models of transient cerebral ischemia. In cultures, Aβ or Aβ peptide fragments can induce cell death and render neurons vulnerable to excitotoxicity and oxidative damage. N-methyl-Daspartate (NMDA) receptor (a glutamate receptor subtype) modulation induced by glutamate release, sustained elevations of intracellular Ca2+ concentration, and oxidative stress are proposed to be involve in mechanisms of Aβ-induced toxicity. It has been reported that OXY (effective dosage range 1-10 µM) isolated from Smilacis chinae rhizome can significantly inhibit 10 µM Aβ (25-35)-induced neuronal cell death by attenuating the elevation of cytosolic Ca2+ concentration, glutamate release into medium and ROS generation [60]. 6-OHDA is a neurotoxin widely used in PD culture model to induce oxidative stress to neurons [61-64]. Its capability of being transported into dopaminergic neurons via dopamine transporter and subsequent induction of free radical-mediated oxidation was proposed to be responsible for its neurotoxicity. Our group [32] revealed that dietary OXY (1-50 µM) elicited potent neuroprotective effects on 6-OHDA-triggered neurotoxicity by attenuating release of LDH and caspase-3-like activity, with a wider effective window compared to RES (1-10 µM). We further elucidated that OXY was able to pass through cell membrane (by HPLC analysis) and acted as an intracellular ROS scavenger. Furthermore, we demonstrated that the inhibition of 6-OHDA-activated JNK pathway and increase in cytosolic

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levels of SIRT1 may account for neuroprotective effects of OXY. Our results have demonstrated the potential of OXY as an ideal neuroprotectant against neurodegenertation in PD. Neurotoxicity initiated by overstimulation of NMDA receptors and subsequent influx of free Ca2+ leads to an intracellular cascade of cytotoxic events. Ca2+-dependent depolarization of mitochondria may contribute to oxidative stress in neuronal injury through production of ROS. NMDA neurotoxicity models have also been widely used to elucidate cellular responses to brain ischemia in vitro. Ban et al. [65] have demonstrated that Smilacis chinae rhizome (30 and 50 mg/kg, orally) prevented cerebral ischemic injury in rats induced by 3-h middle cerebral artery occlusion (MCAO) and 24-h reperfusion. They further showed that over a concentration range of 10-50 Âľg/ml Smilacis chinae rhizome protected neurons against 1 mM NMDA-induced cell death, increases in Ca2+ concentration and ROS generation. They proposed that OXY and RES may be responsible for the effects of Smilacis chinae rhizome, since among six compounds isolated from Smilacis chinae rhizome, OXY and RES exerted neuroprotective effects similar to that of Smilacis chinae rhizome. In addition, a recent investigation also showed that an intraperitoneal application of a dose of 10 mg/kg OXY was sufficient to significantly provide neuroprotection against MCAO-induced brain infarct volume. The maximal protective capacity was at a dose of 20 mg/kg, since no further neuroprotection was seen by increasing the dose to 30 mg/kg. The cytochrome c release, immunohistochemical staining for caspase-3 as well as labeling of apoptoticDNA were also found to be reduced after OXY treatment [66]. The authors proposed that the neuroprotective effects by OXY may be attributed to a combined effects of both its anti-oxidative and anti-nitrosative activities [67]. The anti-oxidant and neuroprotective activities of OXY attract an interesting research direction about whether OXY can reduce the toxicity triggered by elevated homocysteine (Hcy). Normal level of Hcy is involved in numerous methyl group transfer mechanisms, such as reactions targeting DNA, RNA, proteins, phospholipids and neurotransmitters. While elevated Hcy (> 14 Âľmol/L) in plasma, namely hyperhomocysteinemia (HHcy), has been reported to be an independent risk factor in systemic vascular diseases [68], cognitive impairment [69], dementia (including AD) [70-74] and PD [75; 76]. The major mechanisms underlying toxicity of HHcy may involve oxidative stress [77-80] because of the pro-oxidant property of excess amino acid, leading to endothelial damage or apoptosis proved in several cell lines [81-83]. Experimental studies have demonstrated that Hcy and its derivatives (homocysteic acid and homocysteic sulfinic acid) may induce excitotoxicity by stimulating NMDA receptors and damaging neuronal DNA, in cultured neurons of hippocampus [84]. In addition, in vitro studies show that HHcy induced by a diet lacking folate, or direct

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infusion of Hcy may not exert direct neurotoxicity in mice, but might enhance the toxicity induced by neurotoxins, such as kainite [85] or 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) [86]. In recent years, some studies have reported elevated plasma Hcy levels in PD patients treated with L-3,4-dihydroxyphenylalanine (L-Dopa) [87], or even early PD patients [88]. Besides, HHcy in PD is reported to be related to B-vitamin status [89] and genetic factors [90; 91]. Although the question whether L-Dopatreated PD patients with HHcy are at higher risk to develop vascular diseases and cognitive impairment remains to be solved, counteracting elevated Hcy in PD patients by effective agent (e.g. catechol-O-methyltransferase inhibitors, currently used in PD treatment) or dietary intake (folate and B-vitamins) should be part of therapeutic strategy, at least as improvement in general metabolic balance. Since OXY has exhibited significant anti-oxidant capacity and neuroprotective potential, observations above motivate us to explore whether OXY or related dietary supplement can reduce neurotoxicity induced by HHcy in vitro, in animal models, or in L-Dopa-treated PD patients.

Pharmacokinetics study Early pharmacokinetics study [92] reported that, when Mori Cortex extracts were administered orally to rats, the bioavailability of mulberroside A was only about 1%. Most mulberroside A was converted into OXY and transported into the circulating blood, and the absorption ratio of OXY was estimated at about 50%. Recently, a sensitive and simple HPLC method [93] has been developed and validated for the determination of OXY and RES in rat plasma. After orally administered with 1 g/kg S. china extract (an equivalent to 180 mg/kg OXY and 80 mg/kg RES). Results showed that the two stilbenes were rapidly absorbed into the body fluid from the gastrointestinal tract (the tmax was 15 min) and they could still be detected in the plasma at least 360 min after oral treatment. In vivo microdialysis [94] in the striatum showed that OXY could penetrate, to a low extent, the blood-brain barrier (BBB) in control animals, while microdialysis samples from animals that were subjected to MCAO displayed strongly increased OXY levels (more than six-fold) in the infarct region as compared to shamoperated rats, suggesting that OXY exerted neuroprotection by directly penetrating into the BBB.

Conclusions Taken together, water-soluble OXY is widely present in fruits of Artocarpus plants, mulberry, and several herbs. OXY is reported to be more

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effective in anti-oxidation, anti-inflammation and neuroprotecion than widely investigated RES. Dietary OXY or fruits containing OXY may be good choices for constructing healthy lifestyle or dietary strategies promoting healthy aging. Moreover, OXY exhibits high bioavailability after oral administration to rats, and it can penetrate BBB to exert neuroprotection directly in transient ischemia model. However, no in vivo study has investigated its neuroprotective effects in AD or PD. To develop much more effective neuroprotectant in AD or PD, further animal work deserves exploring for OXY.

Acknowledgement The work from this laboratory about research in oxyresveratrol is partly supported by University Strategic Research Theme on Drug Discovery, The University of Hong Kong.

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Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Recent Advances on Nutrition and the Prevention of Alzheimer’s Disease, 2010: 169-185 ISBN: 978-81-7895-470-7 Editors: Charles Ramassamy and Stéphane Bastianetto

11. Medicinal and nutraceutical uses of wolfberry in preventing neurodegeneration in Alzheimer's disease 1

Raymond Chuen-Chung Chang1,2,3, Yuen-Shan Ho1, Man-Shan Yu1 and Kwok-Fai So1,2,3

Laboratory of Neurodegenerative Diseases, Department of Anatomy, LKS Faculty of Medicine 2 Research Centre of Heart, Brain, Hormone and Healthy Aging, LKS Faculty of Medicine 3 State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong Pokfulam, Hong Kong SAR, China

Abstract. In recent years, more people embrace complementary and alternative medicine as a possible mean for disease prevention. While wolfberry is considered to be a medicinal herb, it is also a common ingredient in oriental cuisines. Recently, there are many different lines of wolfberry products as dietary supplements in North America, South Africa and Europe. Chemical analysis suggests that wolfberry is composed of polysaccharides, carotenoids such as β-carotene, zeaxanthin, lutein, different vitamins and some trace elements. Owing to its multi-nutritional values, it would not be surprising that wolfberry exhibits a wide array of beneficial effects. Wolfberry has been reported to have anti-cancer and immune-modulating properties. More importantly, wolfberry is well-known for its anti-aging effects. Some studies have shown that wolfberry can ameliorate age-related conditions including glaucoma, hypertension and diabetes. These protective Correspondence/Reprint request: Dr. Raymond Chuen-Chung Chang, Rm. L1-49, Laboratory Block, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong SAR, China E-mail: rccchang@hkucc.hku.hk

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effects actually agree with its traditional use in oriental medicine and as a dietary supplement. Some laboratories have suggested that wolfberry provides its protection via its anti-oxidative effect as it composes high content of β-carotene and zeaxanthin. Recent research suggests that wolfberry elicits protective effects against neurodegeneration in Alzheimer’s disease (AD). Studies have shown that wolfberry extract containing mainly polysaccharides can reduce neurotoxin β-amyloid peptideinduced neurodegeneration in vitro. Wolfberry can also protect neurons against some risk factors in AD. The role of wolfberry in AD prevention as well as in anti-aging deserves more exploration, and use of wolfberry may pave a new road in early intervention of AD pathogenesis.

Introduction Complementary and alternative medicine (CAM) has become more popular in recent years. According to a survey released in 2004 by the National Center for Complementary and Alternative Medicine, 36% of adults in the United States are using some forms of CAM. Among these people, 19% of them have used natural products and the expenses on these products are about 5 billion US dollars [1]. More herbal products have become available as dietary supplements and wolfberry is one of these examples. Wolfberry is the common name of the fruits Lycium barbarum or Lycium chinense, which are two very closely related species. It belongs to the family Solanaceae and it can be found in many regions of the world. There has been a long history for using wolfberry as a medicinal herb in Asian countries. In China, where it is named as Gouqizi, wolfberry has been used as a “yintonifying herb” and is believed to be beneficial to the eye, kidney and liver. People believe that the fruit is highly nutritional and has an important therapeutic role. More importantly, it has been regarded as anti-aging in Chinese pharmacopoeia. For a long time, wolfberry has been used to treat various diseases including diabetes and glaucoma in oriental medicine. As wolfberry has a bright red appearance and sweet taste, it is also a common ingredient in food dishes, soup, and even snacks. Wolfberry is commonly sold as a dried fruit and it can be directly eaten without further processing. When it is used as medicinal herbs in oriental medicine, the dried fruit (Fig. 1) is usually boiled in hot water for hours with other herbs to obtain maximum effects. It can also be added into soup and tea to provide a pleasant flavor. As people are placing more attention on health food products, wolfberry juice, extract or essence capsule have gained increased popularity. While manufacturers always claim that these products have multiple benefits, there is usually insufficient data to support their use as health-promoting supplements. The purpose of this review is to summarize the medicinal and

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Figure 1. Dried wolfberry found in the market. It appears red in color with white seed inside.

nutraceutical beneficial effects of wolfberry, and discuss its possible therapeutic effects on the prevention of neurodegeneration in Alzheimer’s disease (AD).

Alzheimer’s disease - diverse pathology and multiple risk factors AD is a progressive neurodegenerative disease which can eventually lead to dementia in elderly. The classical symptoms of AD include memory impairment, language deterioration and visuospatial deficits [2]. These symptoms, however, may occur years or decades after the onset of pathological changes [3]. Evidence from brain imaging studies indicates that brain atrophy and neuronal dysfunction precede the onset of cognitive impairment [4; 5]. Therefore, prevention or slowing down the onset of AD may be more effective than reversing the disease process [6]. Different mechanisms and hypotheses have been proposed to explain the pathological changes and clinical symptoms of AD. It is generally accepted that protein misfolding, aggregation, beta-amyloid (Aβ) peptide accumulation and tau accumulation in neurofibrillary tangles (NFT) play important roles in AD development [7]. Based on the pathological characteristics of the disease, several targets have been suggested for disease-modifying therapy. Current development on disease-modifying agent for AD focuses on production of Aβ and tau protein [8]. While Aβ and tau are involved in the initial stage of AD, it is possible that secondary pathological cascades such as inflammation and oxidative stress can occur and cause further damage to brain [9; 10].

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Therefore medication or nutritional intervention targeting these secondary changes may also be helpful in slowing down disease processes. Apart from these pathological changes, AD is also believed to be associated with a number of risk factors such as hyperhomocysteinemia, diabetes, midlife hypertension, cardiovascular diseases, hypercholesterolemia and viral infections [11-17]. Although the exact linkage between AD and these risk factors remains unclear, pharmaceutical or nutritional interventions that minimize these risk factors may be benefit to AD prevention. In view of the diverse pathology and multiple risk factors, effective disease-modifying agent should be multifunctional and targets different aspects during AD progression.

General effects of wolfberry Wolfberry belongs to the “Yin-tonifying” herbs in traditional Chinese medicine. In Chinese pharmacopoeia, it is listed as an upper class herb, implicating that it has multiple beneficial effects but little side effects. The fruit is used to provide a general tonic effect and is especially beneficial to kidney, liver and eye. Although it has a good reputation for vision improvement, its effects is not limited to the eye. It helps to restore body homeostasis (balance “Yin” and “Yang”) and strengthen body energy. Interestingly, this idea is supported by a recent clinical study, which suggests that daily consumption of wolfberry juice for 2 weeks increases subjective feelings of general well-being and improves gastrointestinal functions [18]. Wolfberry is also famous for its anti-aging properties. Up to date, there is no research in rodents to investigate if taking wolfberry can enhance longevity. However, increasing lines of evidence suggest that wolfberry can improve diverse aging-associated conditions as follow: Zhang et.al. (2005) have reported that polysaccharides from wolfberry can have anti-tumor effects on hepatoma QGY7703 cell line probably by inducing cell cycle arrest in S phase and increasing intracellular calcium to induce apoptosis [19]. Another study suggests that wolfberry extract can inhibit hepatocarcinoma cell proliferation and stimulate p53-mediated apoptosis [20]. Degeneration of organs is also involved in aging processing, and the degenerative processes may be attenuated by wolfberry. In a study of glaucoma, an aging-associated eye disease, oral feeding of wolfberry extract protects rat retinal ganglion cells from ocular hypertension-induced damage [21]. Wolfberry can provide beneficial effects on different organs and body systems. Traditional literatures of oriental medicine also describe its antiaging effects. Therefore, our laboratory hypothesizes that wolfberry can be a potential therapeutic agent for the prevention of neurodegenerative diseases such as AD. More importantly, findings from our group support the idea that

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wolfberry has multi-target effects on neurodegeneration of AD. The results imply that wolfberry can protect brain cells from neurodegeneration of AD by modulating different AD-related pathogenesis [22-24]. We will discuss these effects in this review.

Chemical compositions of wolfberry and their beneficial effects Wolfberry is made up of a group of components including polysaccharides, betaine, zeaxanthin, beta-carotene, groups of vitamin, 19 kinds of amino acid and trace minerals. Many of these components are helpful in maintaining body health and some of their benefits are listed as follows.

Polysaccharides It is believed that the polysaccharides of wolfberry, also named as Lycium barbarum polysaccharides (LBP), are the active components responsible for its various biological activities, especially the immunemodulating and anti-oxidative functions [25]. LBP is not a single compound but a group of proteoglycans made up of monosaccharides. The carbohydrate content of wolfberry is found to contain arabinose, rhamnose, xylose, galactose, glucose, glucoronic acid, galacturonic acid and mannose [23; 26]. This composition is not fixed. By using different extraction methods, such as using water or alcohol or alkaline solutions, LBP can be purified and isolated into different sub-fractions [22]. Studies have found that these LBP can have diverse biological activities and they may affect cell signaling pathways [22; 27]. The beneficial effects of LBP will be further discussed in the later part.

Zeaxanthin Apart from LBP, zeaxanthin is also abundant in wolfberry. Zeaxanthin is an oxygenated carotenoid and is found specially located in the human macula [28]. Findings indicate that zeaxanthin is beneficial to eye and can protect the eye against age-related macula degeneration and aged-related cataract formation [29; 30]. Zeaxanthin can be obtained from fruits and vegetables [31]. However, its bioavailability is varied from different food. In wolfberry, zeaxanthin presents as an esterified from, zeaxanthin diaplmitate. Its content varies depending on the grade and source of the berry [32]. There are several advantages of using wolfberry as the source of zeaxanthin. Firstly, zeaxanthin content in wolfberry is very high. It has been reported that the content of

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zeaxanthin in wolfberry can be as high as 30 mg/100g of the material [33], which is much higher when comparing to that found in egg yolk (210 Âľg/yolk). Secondly, wolfberry intake does not affect plasma lipid levels. When taking egg yolk to supplement zeaxanthin, the plasma low density lipid (LDL)-cholesterol level increases and this would increase the risk of heart diseases [32; 34]. Thirdly, the bioavailability of zeaxanthin from wolfberry is good and can directly increase plasma zeaxanthin concentrations after intake [35]. Fourthly, studies have shown that zeaxanthin diaplmitate from wolfberry can have hepatoprotective effects. In a rat model of hepatic fibrosis, oral feeding of the compound can slow down collagen deposition and reduce the activities of aspartate transaminase and alkaline phosphatase in serum [36]. These unique characteristics of wolfberry regarding to its zeaxanthin content enable the fruit to act as a medicinal herb as well as a highly nutritional food.

Betaine Wolfberry contains 0.797-1.18% of betaine [37]. Betaine is important for maintaining the health of cardiovascular system. It works with other nutrients such as folic acid, vitamin B6 and B12 to break down homocysteine, a substance which can increase the risk of atherosclerosis and hence heart attack [38]. Recent studies have suggested that betaine supplementation may protect liver cells from ethanol-induced steatosis [39]. A pilot study also suggested that betaine may provide biological and histological improvement in patients with nonalcoholic steatohepatitis [40].

Beta-carotene Wolfberry also contains a high content of beta-carotene. Every 100 g of wolfberry contains 19.6 mg of carotene [41], which is even higher than that in carrot (about 5.6 mg/100 g) [42]. Beta-carotene is the most common form of carotene. It is a precursor of vitamin A and can provide strong antioxidative effects. A recently randomized trial suggested that long-term betacarotene supplement might provide cognitive benefits [43].

Wolfberry as a potential disease-modifying agent for prevention of AD As mentioned in the previous section, LBP from wolfberry is responsible for many biological functions. Recent findings have shown that these polysaccharides may have protective effects against AD-associated conditions

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[22-24]. We summarize these effects in two categories: direct protective effects and indirect protective effects. Direct protective effects refer to actions that target on neurons. Indirect effects, which can also be considered as global effects, refer to those that target AD risk factors.

Direct protective effects against Aβ-peptide neurotoxicity Base on the hypothesis that wolfberry can provide protection against neurodegeneration in AD through multiple mechanisms, our laboratory has conducted a comprehensive research on the polysaccharides of this fruit. We found that neurons pretreated with LBP reduced neurotoxicity induced by Aβ-peptide [23]. It is generally accepted that Aβ-peptide and its aggregation are related to AD progression [44]. Experiments have shown that Aβ-peptide can induce apoptosis, tau phosphorylation and activation of microglia [45]. Surprisingly, this protective effect is only provided by the polysaccharide fraction of wolfberry. We have examined other fractions from wolfberry including betaine and β-sitosterol, none of them provide protective effects against Aβ-peptide neurotoxicity [22-24], suggesting that LBP is the active component for neuronal protection. Moreover, LBP provides an additional advantage as it has a wider effective and safety dosage when comparing to a well-known Western neuroprotective medicine, lithium chloride (LiCl) [23; 24]. The ability of LBP to protect neurons against Aβ toxicity enables it to be a potential candidate for AD prevention. The mechanisms mediated by LBP for its protective effects against Aβ remains unclear. Our data suggested that LBP can inhibit some proapoptotic signaling pathways, including the c-Jun N-terminal kinase (JNK) and double-stranded RNA-dependent protein kinase (PKR) in Aβ peptide neurotoxicity [23; 24]. If we extract LBP by a different solvents, such as an alkaline solvents, the resulting LBP shows different properties and can stimulate the survival Akt signaling pathway [22]. This kind of changes for the biological effects may reflect conformational changes of LBP during extraction.

Indirect effects of wolfberry Although wolfberry can protect neurons against Aβ peptide-induced neurotoxicity, research shows that its beneficial effects are not limited to this neurotoxin. By modulating AD-related pathogenesis and reducing pathological changes in the associated risk factors, wolfberry may provide its beneficial effects in a holistic approach.

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Attenuation of glutamate excitotoxicity Glutamate is the most abundant excitatory neurotransmitter in the mammalian central nervous system. It is released from the presynaptic vesicles and binds to specific postsynaptic glutamate receptor (e.g. N-MethylD-Aspartate (NMDA), Îą-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA), kainate and some metabotrophic glutamate receptors) to trigger depolarization and action potential. Many studies have shown that glutamate receptors play important roles in AD pathogenesis [47-50]. The expression and functions of glutamate receptors can be impaired by AÎ˛ peptide [51; 52]. It has been shown that NMDA receptor antagonist can reduce loss of hippocampal neurons induced by injection of AÎ˛1-40 [53], which further supports the involvement of glutamate excitotoxicity in AD development. One of the AD therapeutic strategies is targeting these receptors. Since blockage of glutamate excitotoxicity is beneficial for AD treatment, our laboratory investigated the protective effects of LBP against glutamate-induced neuronal damage. We found that LBP could reduce necrotic and apoptotic cell death in cortical neurons treated with glutamate or NMDA. Its protective effect is comparable to memantine, which is a NMDA receptor antagonist. This is a good indication as memantine is a currently approved drug for AD treatment based on its effect on glutamate toxicity [54]. Our data also suggest that LBP can attenuate glutamate-activated JNK. Future research will be focused on the change of electrophysiological properties of neurons treated with LBP to find out its protective mechanisms. The protective effects of LBP against glutamate toxicity further support its potential role in preventing neurodegeneration in AD [54].

Protection against diabetes Diabetes mellitus (DM) is a common and devastating health problem in the elderly. Epidemiological studies suggest that it is a risk factor for a number of diseases including AD. In a population-based cohort study, it is found that patients with adult onset diabetes mellitus have significant increased risk of AD [55]. Similar results were also found in another population-based cohort study, the Honolulu Asia Aging Study [56]. In this study, it suggests that type 2 diabetes together with the presence of APOE epsilon4 allele is associated with increase prevalence of AD. Several mechanisms are proposed to explain the association between AD and DM. It is generally believed that DM, being an atherogenic risk factor itself, can cause stroke and vascular change that is related to vascular dememtia. DM

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may exacerbate AÎ˛-neurotoxicity by advanced glycation end-product [57], insulin resistance [58] and impaired glutamate receptors functions [59]. Since it has been documented that a better glycemic control in DM patients may result in cognitive improvement [60], agents that can provide hypoglycemic effects or reduce DM-related pathology may provide some benefits to AD prevention or treatment. Several studies have found that wolfberry has hypoglycemic effects or can attenuate hyperglycemia-induced damage. In one study, researchers have shown that feeding LBP can reduce blood glucose level, serum total cholesterol and triglyceride concentrations in alloxan-induced diabetic or hyperlipidemic rabbits [61]. Since hyperglycemia pathology is often associated with oxidative stress, some studies also investigate if wolfberry or LBP can attenuate the associated oxidative damages. It has been reported that LBP treatment for 4 weeks can reduce blood glucose level, DNA damage, malondialdehyde (MDA) and nitric oxide (NO) in serum of fasting rats with non-insulin dependent diabetes mellitus (NIDDM). It is proposed that LBP may have anti-oxidant properties which account for its protection against hyperglycemia-induced DNA damage [62]. Similar results are also found in another study, which shows that LBP can restore abnormal oxidative index to near normal level in blood, liver and kidney of streptozotocin-injected rats [63]. Apart from these, LBP can also ameliorate insulin resistance in a rat model of NIDDM. It is suggested that LBP can increase cell-surface level of GLUT4, improve GLUT4 trafficking and intracellular insulin signaling [64]. As wolfberry can normalize blood glucose level and reduce DM-associated pathological changes and DM is a risk factor for AD, it may help preventing AD and improve cognitive functions by targeting this risk factor.

Protection against high cholesterol level Elevated plasma cholesterol levels are also considered as another risk factor for AD. This is supported by in vivo and in vitro studies. In animals, it has been shown that cholesterol rich diet can increase brain intraneuronal AÎ˛ level, and this effect can be reversed by returning the animals to normal diet [65; 66]. In vitro data also show similar results. In one study, a cholesterollowering drug has reduced AÎ˛ production in hippocampal and mixed cortical neurons [67]. These data have suggested that cholesterol plays an important role in AD development. Currently, cholesterol-reducing drug statin is being investigated for its protective effects in AD. In a case-control study, statin users have shown a 39% lower risk of AD relative to non-statin users. This association between the use of statin and AD is affected by the presence of certain chronic medical conditions including hypertension, ischemic heart

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disease and cerebrovascular disease [11]. It is still uncertain if cholesterolreducing strategy is protective in AD prevention. Nevertheless, maintaining appropriate low serum cholesterol level is beneficial to health as there is a close relation between serum cholesterol level and atherosclerosis, a risk factor for many diseases. Wolfberry may have hypolipidemic effects. There is a report showing that wolfberry water extract and its crude LBP extract can reduce serum total cholesterol and triglyceride level in hyperlipidemic rabbits. In the same time, researchers have found that there is an increase in high density lipoprotein cholesterol levels after wolfberry feeding [61]. Oral feeding with wolfberry also dose-dependently attenuates the level of total cholesterol, total triglyceride and hepatic cholesterol in experimental hyperlipidemic mouse, while no adverse effect on liver and kidney function can be observed [68]. Further studies are required to demonstrate the effect of wolfberry on maintaining serum cholesterol level. Its potential hypolipidemic effect deserves more investigation.

Attenuation of hyperhomocysteinemia neurotoxicity Homocysteine is a non-essential sulfur-containing amino acid that is derived from methionine metabolism. Its metabolism depends on folate, vitamin B6 and vitamin B12. Elevated plasma homocysteine is associated with cardiovascular disease and stroke [69; 70]. Recent studies suggest that hyperhomocysteinemia is a risk factor for AD [71, 17]. In fact, homocysteine can induce a direct neurotoxicity to neurons. It has been reported that homocysteine can activate NMDA receptors to cause excessive influx of calcium ions and generation of reactive oxygen species (ROS) [72]. An oxidized metabolite of homocysteine, homocysteic acid, may also induce intraneuronal accumulation of AÎ˛1-42 [73]. Rats with elevated plasma homocysteine are also found to have tau hyperphosphorylation, and this change in tau pathology may link to alteration in protein phosphatase 2A activity [74-77]. As hyperhomocysteine is one of the important risk factor leading to AD, we will investigate whether wolfberry can protect neurons against this kind of toxicity.

Protective mechanisms of wolfberry Antioxidant effects Wolfberry provides beneficial effects in different body systems. Its benefit is not limited to the nervous system, it can also protect the liver [20; 78], the eyes [21], and the kidney [63]. In many reports, wolfberry or its

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polysaccharides are shown to provide strong anti-oxidative effects. Wolfberry can provide protection against oxidative damage induced by hydrogen peroxide [79; 80]. It has been shown that treatment with LBP protects mice testicle cells from oxidative stress-induced DNA damage [80]. The antioxidative properties of wolfberry may be due to its direct free-radical scavenging effect [81; 82] or its ability to increase anti-oxidant enzyme activities [83; 84]. LBP administration can reduce exhaustive exerciseinduced oxidative stress in rats, and it increases anti-oxidant enzyme activities [83]. Wolfberry also protects hepatocytes from carbon tetrachloride (CCL4). This toxin can decrease the activities of glutathione reductase and glutathione peroxidase in hepatocytes. After wolfberry treatment, the hepatocytes can maintain a relatively normal level of these enzymes [84]. Oxidative stress is suggested to be involved in normal aging process as well as many degenerative diseases [85; 86]. In an aging-induced oxidative stress study, reports have shown that aged-mice have decreased superoxide dismutase, catalase, glutathione peroxidase activities and total antioxidant capacity. Feeding LBP to rat can restore the aged-induced changes in these anti-oxidant enzymes and its anti-oxidant activity is comparable to vitamin C [87]. In a human study, dietary consumption of wolfberry for 10 days also significantly increases superoxide dismutase activity in elderly [88]. Since increased levels of oxidative stress in the brain have been considered to be the important factor mediating AÎ˛ toxicity, anti-oxidative effects of wolfberry can provide protective effects to neurons. Although wolfberry can act as anti-oxidant, its functions and neuroprotective mechanisms is certainly not simply due to its anti-oxidative effects. The immune-modulation ability of wolfberry may also account for its multiple beneficial effects on the CNS and other body systems.

Providing immune-modulation effects It has been known that modulation of the immune system can help fighting against AD and even improve the learning ability [89; 90]. It has been recently proposed that systemic infections and inflammation affect chronic neurodegeneration [91-93]. Therefore, it is important to investigate whether wolfberry can modulate body immunity or inflammatory responses. In fact, wolfberry is found to have immune-modulation effects. This property is contributed by the high content of LBP in wolfberry rather than other components like zeaxinthine and carotene [25]. LBP has been recently demonstrated to stimulate murine bone marrow derived dendritic cells (BMDC) maturation as reflected by the co-expression of I-A/I-E, CD11c and secretion of IL-12 p40 [26]. Purified wolfberry polysaccharides also show diverse immune

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stimulating effects. LBP3P can increase macrophage phagocytosis and interleukin-2 (IL-2) mRNA expression level in transplantable sarcoma S180bearing mice [94]. This polysaccharide also increases the expression of IL-2 and tumor necrosis factor-alpha (TNF-α in human peripheral blood mononuclear cells [95]. Another report has also shown that wolfberry can increase IL-2 receptor expression in isolated human peripheral lymphocytes [96]. LbGp4 can stimulate splenocytes proliferation in mice, which is associated with upregulation of nuclear factor κB (NF-κB) and activator protein 1 (AP-l) [97]. It is clear that wolfberry demonstrates immune-modulation effects on the whole body immune system. However, not many study has been focused on its effects on the CNS. Whether the changes in the peripheral immune systems induced by wolfberry would contribute to its neuroprotective properties remains unclear, this certainly would be an interesting area for further exploration.

Adverse effects and drug interaction of wolfberry At present, there is no report showing that intake of wolfberry would cause any side effect. This is agreed with the general thought that traditional Chinese medicine (TCM) has fewer side effects than single pure drug used in Western medicine [98]. Its characteristics of diverse biological effects and little adverse effect have also been described in ancient Chinese pharmacopoeia. Despite the safe nature of wolfberry, it is still possible for it to interact with other medications. There are two reports showing that wolfberry may interact with warfarin. In the first report, a patient who received warfarin therapy developed an elevated international normalized ratio (INR) after consumption of wolfberry tea. INR is used for the measuring of prothrombin time and hence blood clotting tendency. The increase in INR indicates an increased chance of bleeding. In vitro studies suggested that wolfberry may slightly inhibit the metabolism of S-warfarin through CYP2C9, the isoenzyme responsible for its metabolism [99]. The second report showed similar observation, in which a patient taking warfarin experienced 2 episodes of INR elevation after wolfberry-containing herbal tea consumptions. Since impairment of warfarin metabolism would lead to excessive bleeding, it has been suggested that patients should avoid taking warfarin together with herbal products including wolfberry [100].

Concluding remarks Wolfberry and its polysaccharides have been shown to exhibit multifaceted protective effects against Aβ neurotoxicity. Based on current evidence on wolfberry and its components, we propose that this fruit can provide protection through direct and indirect approaches (Fig. 2). Wolfberry can protect neurons directly by attenuating Aβ-induced neurotoxicity. Through targeting at certain

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Wolfberry as disease-modifying agent for AD Nurture Kidney

Nurture Liver

Wolfberry ↑ Moderate immune response

↓ Oxidative damages

Disease-modifying Agent

↓ β-Amyloid toxicity

↓ Glutamate toxicity

↓ Cholesterol level

↓ Hyperhomocysteine toxicity ↓ Diabetic pathology

Figure 2. Multifaceted protective effects of wolfberry against neurodegeneration in AD. Wolfberry has a long history for being used as medicinal herb in oriental medicine. It acts as a tonifying herb to nurture the kidney and liver, which may account for its anti-aging properties. It can also modulate body immune response and act as anti-oxidant. These may be responsible for its neuroprotective effects against several conditions related to AD pathogenesis. Wolfberry is able to attenuate Aβ peptide toxicity and glutamate excitotoxicity. It can also reduce pathological changes cause by high cholesterol level and diabetes mellitus. All of these beneficial effects enable wolfberry to become a potential disease-modifying agent for the prevention or treatment of AD.

AD risk factors, it may prevent or ameliorate devastating neurodegeneration process in an indirect approach. The neuroprotective mechanism of wolfberry is not completely elucidated, but its ability to suppress pro-apoptotic signals, reducing oxidative stress and regulating the immune system may account for its biological benefits. Besides polysaccharides, wolfberry also contains a number of components such as zeaxanthine, betaine and beta-carotene. The high content of these compounds in wolfberry further support its role in the prevention of aging-associated diseases. Although wolfberry is a potential herbal dietary supplement for the prevention of neurodegeneration in AD, most of its research has been carried out in cell culture or animal models. Its effects on human need further investigation. Its long-term safety and its interaction with other medicine also deserve proper address.

Acknowledgement The work done by author’s laboratory is partly supported by HKU Alzheimer’s Disease Research Network and Azalea (1972) Endowment Fund. YSH is supported by a postdoctoral fellowship from The University of Hong Kong.

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References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

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Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Recent Advances on Nutrition and the Prevention of Alzheimer’s Disease, 2010: 187-205 ISBN: 978-81-7895-470-7 Editors: Charles Ramassamy and Stéphane Bastianetto

12. Nutraceutical modulation of the aging process and age-related diseases – What the worm has taught us Marishka K. Brown and Yuan Luo Department of Pharmaceutical Sciences, School of Pharmacy, Center for Integrative Medicine Department of Medicine, University of Maryland, Baltimore, MD 21201, USA

1. Abstract. Increased subsets of elderly persons in the total populations of many countries have suffered from age-related diseases in the past few decades. Diseases that at one point in time seemed rare are amplified in aged individuals. Sedentary lifestyles and unhealthy diet choices have also exacerbated these conditions. It is well known that a healthy diet and moderate exercise improves the overall quality of our physical being and improves mental functions. Recent reports have associated instances of Alzheimer’s disease (AD) among obese individuals, and suggest that abnormalities in insulin signaling in the brain maybe the underlying cause. The link between insulin signaling and aging has been illustrated in the invertebrate models Drosophila melanogaster and the nematode Caenorhabditis elegans. In C. elegans, there are several mutations involving the insulin/insulin-like growth factor 1 (IGF-1) signaling pathway that will either accelerate or delay the aging process. There are also several models that express the human amyloid beta peptide, one of the pathohistological hallmarks associated with AD. These models have been indispensable for nutraceutical studies. Studies in our laboratory have revealed that in C.elegans, nutraceuticals such as Correspondence/Reprint request: Dr. Yuan Luo, 20 N Pine St. PH501, Baltimore, MD 21201, USA E-mail: yluo@rx.umaryland.edu

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the Ginkgo biloba extract EGb 761 and a green tea constituent epigallocatechin gallate (EGCG) increase the wormâ&#x20AC;&#x2122;s life span and stress resistances. EGb 761 has also been shown to delay age-associated tissue degeneration and alter serotonin (5-HT) sensitivity in C. elegans. Evidence suggests that there is substantial crosstalk between the insulin and 5-HT signaling pathways. This chapter will discuss studies that utilize C. elegans as a system to elucidate the relationship between nutraceuticals, the aging process and their effects on amyloid beta toxicity. The chapter will also illustrate what has been learned from C. elegans is likely to be applied to mammalian systems.

2. Introduction Many countries around the globe are experiencing an increase in their elderly populations, which has elevated interest in the scientific community, not only in the normal aging process, but in age-related diseases as well. Although the maximum life span has significantly increased in most developed nations in the past several decades, due to sanitation requirements and vaccine development, there is still much work to be done on increasing the health span of individuals. The old argument that debates the quality of life versus quantity still persists, but a diagnosis that continues to persevere throughout is the benefits of healthy lifestyle choices. It is well known that a healthy lifestyle, which includes a balanced diet and moderate exercise, improves the overall quality of your physical well being. However, recent scientific evidence has demonstrated that these choices/modifications can also increase cognition and other mental functions[1]. There is a clear linear relationship between unhealthy lifestyles, obesity and diabetes mellitus. Research is now correlating dysfunctions in insulin signaling to certain types of dementia, including Alzheimerâ&#x20AC;&#x2122;s disease (AD) [2-4]. Although more than a century has pasted since the first diagnosed case of AD, we are still far from finding a cure for this neurodegenerative goliath. As the elderly populations steadily rise in many developed nations, the prevalence of AD also continues to rise and is projected to reach proportions that will strain health care systems throughout the globe. Though the etiology of this disease is still unknown, scientific evidence has reported that the instances of dementia are increased in obese individuals [5]. Genetic studies performed in the nematode Caenorhabditis elegans have given important insight into gene regulation of life span and health span. These studies have demonstrated that life span in the worm can be regulated by several different mechanisms, including, caloric restriction, reduced insulin/ (insulin-like growth factor) IGF-1 signaling and serotonin (5HT) receptor modulation [6-9], which control life span either by accelerating or delaying the aging process. Many natural compounds, such as the ginkgo biloba extract EGb 761 and several of its active components, epigallocatechin gallate (EGCG) and Îą-lipoic acid all affect the life span and/or health span of the worms [10-13] which in turn, could delay or even prevent the development of agerelated neurological disorders.

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3. Obesity and AD Since many conditions that are associated with obesity are also associated with aging, research in recent years has focused on understanding the role of insulin signaling in cognitive process and how dysfunctions in insulin metabolism contribute to age-related pathologies, in particular AD [2, 14]. These relationships have become even more important in the present society where conditions that directly involve insulin regulation, such as obesity and diabetes, have dramatically increased [15-18]. A recent clinical study shows that intranasal administration of insulin modulates Aβ and facilitates memory without affecting fasting plasma glucose or insulin levels in early AD patients [19]. Evidence has also demonstrated that in aging individuals, insulin receptors in the brain become less sensitive to insulin [20, 21], an event which possibly reduces synaptic plasticity in the brain [4]. There is evidence that this reduction in insulin sensitivity, which is due to aging and exacerbated by diabetes and obesity, decreases the clearance of Aβ from the brain [4, 22-24]. Also, insulin and amyloid beta protein are in direct competition for the insulin degrading enzyme (IDE) [22, 23] in the brain. This activity can affect the hippocampus, a brain region that is well known for its role in cognition. Another study reported that obesity-related leptin had the ability to modify Aβ levels both in vitro and in vivo by reducing beta secretase levels [25]. This evidence has made many researchers postulate that Aβ deposition would be increased because of excessive levels of insulin. Many studies have demonstrated that manipulation of insulin can affect cognitive process and Aβ levels that are detected in cerebrospinal fluid [26, 27]. Although there is a fair amount of evidence that supports the role of increased insulin levels in AD, this only indicates a correlative relationship. The evidence does not implicate excessive insulin in the causation of the disease; rather this condition may compound and amplify the known pathologies.

4. A worm model for studying aging, insulin & serotonin (5-HT) signaling Since the introduction of C.elegans into the laboratory setting [28], the area of worm research has exploded. The majority of the research has focused on genetic studies that have lead to many insights into the complex and illunderstood aging process. The discovery that metabolic health can regulate the worm’s life span highlights the importance of healthy lifestyle choices [7, 8, 29]. Years ago it was reported that a high-calorie diet shortened the life span of the worm, but animals that had their calories restricted lived much

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longer [30, 31]. Also, mutant worms that have their normal feeding behavior genetically disrupted live 20-40% longer [8] than their wild type counterparts. These studies, yielded similar results when they were later preformed in the fruit fly drosophila and mice [32-34]. There are at least 38 insulin-like peptides that are encoded by the C. elegans genome that act as either agonist or antagonist to DAF-2, which is the only insulin receptor found in the animals [6, 35-37]. The use of mutant worms elucidated the relationship between the insulin/IGF-1 and 5-HT pathways and their regulation of life span and health span in the worm. The wormâ&#x20AC;&#x2122;s body fat content is controlled by metabolic activity and feeding behaviors; both of which have afforded researchers the opportunity to use this model in the identification of genes that control feeding/satiety, fat metabolism and nutritional uptake [31]. The neuromodulator 5-HT controls a variety of behaviors in invertebrates and vertebrates alike. Several behaviors in C.elegans are affected by 5-HT which include: locomotion, pharyngeal pumping ,egg-laying and feeding [3842]. Also, 5-HT receptors have recently been found to antagonistically modulate life span in the worms, probably through the insulin/IGF-1 pathway and independent of caloric restriction [9]. This work was confirmed by another laboratory that also used the worm model, although this group antagonized the receptors in a pharmaceutical approach, instead of utilizing mutant worms [43]. Worms that are deficient in the serotonergic biosynthetic enzyme tryptophan hydroxylase (tph-1) are defective in behaviors and metabolism that is normally coupled to the ingestion of food, such as decreased mating and egg retention [44-46]. These worms also accumulate larger stores of fat than their wild type counterparts, although their feeding rates are reduced [45]. Despite the fact that exogenously applied 5-HT increases the feeding rates, it potently decreases fat accumulation in the worm [42]. When 5-HT is applied exogenously, it modulates the worms behaviors in a similar fashion as changing the levels of food [45] i.e., decrease in locomotion and increase in egg-laying [38, 39]. Prior evidence has demonstrated that both the tph-1 and the daf-2 mutants have a tendency to developmentally arrest at the alternate dauer larvae stage and accumulate excess fat, and deletion mutations of daf-16 suppresses these phenotypes [45, 47, 48]. Research has also shown that the DAF-2 insulin/IGF-1 receptor which regulates DAF-16, the FOXO transcription factor, is targeted by 5-HT to affect stress responses [48]. In mammals, 5-HT influences the synthesis, release, and sensitivity of insulin as well as many other hormones that regulate the appetite and metabolic homeostasis. Evidence has shown that dysregulation in metabolism is partially due to the decrease in insulin-like neuroendocrine signals and that the activity of the serotonergic system in metabolic regulation and obesity is similar in the worm and mammalian models [45, 48].

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5. Modeling amyloid-beta toxicity in the worms Different animal models have been constructed over the years to try to mimic the pathogenesis of AD. Although none have been totally successful, there are several ways humans disease states can be partially mimicked in the worm. The first model is by 1) knocking out (mutants) or knocking down (RNA interference) a worm gene that is homologous to the human gene that is known to be involved in a certain disease 2) targeting a biological process in the worm that reproduces certain aspects, whether cellular or molecular, that are involved in that particular disease mechanism and 3) expressing a human gene in the worm that can induce a disease-related phenotype [31, 49]. The transgenic worm strains that carry human Aβ (1-42) were all constructed in the laboratory of Dr. Chris Link [50-53]. These worms were made by injection of the minigene construct, unc-54/Aß-(1-42) in their gonads, in conjunction with the dominant marker rol-6 gene [53, 54]. The transgenic progeny obtained exhibit a non-sinusoidal, roller movement phenotype due to the rol-6 marker. So, expression of the Aβ gene causes the animals to rotate along their longitudinal axis [50, 54], and later there is a progressive paralysis [53, 55]. This paralysis is attributed to the accumulation of intracellular Aβ deposits in the C. elegans muscles. From this construct, two strains CL2006 and CL4176, along with their respective vectors were identified. CL2006 is cultivated at 20°C, the same as wild type animals, throughout the duration of their life span, and Aβ-(1-42) is continuously expressed. CL4176, on the other hand, is maintained at the lower permissive temperature of 16°C, without the expression of Aβ [50, 52]. Aβ is expressed in this strain, when the worms are upshifted to the non-permissive temperature of 23°C [52]. This mechanism of temperature inducibility was explained by Link et al [52] and is caused by mutations in the mRNA surveillance (smg) system of the worm. In CL4176, smg-1 is inactivated, and this allows for mRNA translation of the human Aβ-(1-42) transgene at the non-permissive temperature. CL2355 is the strain that express human Aβ in the neurons. Although it exhibits wild type movement because there is no insertion of the rol-6 marker, there have been other phenotypes characterized for this strain [56]. The advantages of using C. elegans for drug combination studies are: 1) highly conserved biochemical pathways, cell signaling and stress response between worms and human; 2) well established mutants linking biological pathways with pathological phenotypes; 3) the small size, ease of maintenance, genetic amenability and rapid generation time makes evaluation of numerous animals on microtiter plates feasible. Several examples illustrated the power of C. elegans in screening for new drugs [57-59]. The

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genetic target of drugs such as fluoxetine was identified in worm mutants [60]. As stated by Dr. WR Schafer [61]:”Drugs have historically been effective tools for investigating how worm neurons work; worm’s neurons may prove equally effective for investigating how drugs work”. Intriguingly, data obtained from three AD-relevant models; Aβ cell culture, Aβ worm behaviors and hippocampal slices exposed to non-fibrillar oligomers called Aβ-derived diffusible ligands or ADDLs, support each other for nutraceutical studies. For example, among the EGb 761 constituents tested, ginkgolide J (GJ) enhanced phosphorylation of cyclic AMP response element-binding protein (CREB) in Aβ expressing cells [62], reduced Aβinduced paralysis in CL4176 Aβ-muscle expressing worms [56] and restored ADDL-evoked LTP defect [63].

6. Insulin/ IGF-1 like signaling, heat shock and modulation of proteotoxicity Substantial experimental evidence has demonstrated the regulatory effects of insulin signaling and heat shock factors (HSF) in the aging process [64, 65]. Heat shock factor 1 (HSF-1) has been reported to have roles in the developmental process, stress responses and the circadian rhythm cycle [6668]. Reports have shown that when the expression of HSF-1 was increased, the worms’ life span was extended 40% and this extension required daf-16 [64]. Recent studies are now focusing on these factors and their relationship to protein misfolding or aggregation: proteotoxicity. DAF-16 and HSF-1 transcriptomes both result in the expression of a variety of chaperons, leading researchers to hypothesize that protein folding plays a key role in life span and aggregate-associated proteotoxicity [65, 69]. In a paper published by Cohen et al, the relationship between the insulin signaling pathway and Aβ was examined. It reported that Aβ worms that were grown on daf-2 RNAi showed a significant extension in life span and delay in paralysis in comparison to their vector control groups [69]. This study summarized that protein toxicity is highly dependent on the aging process and that daf-16 and hsf-1 are both required for the reduced insulin signaling mediated alleviation of Aβ (1-42) in the body wall muscles of the worm [69]. Dietary restriction functions through a pathway that is genetically distinct from the insulin/IGF1 like signaling pathway. Bacterial food deprivation or BD was found to suppress age-associated paralysis in the Aβ (1-42) worms through an hsf-1 dependent mechanism [70]. A study conducted in our laboratory demonstrated that a protective heat shock dramatically delayed paralysis in the Aβ worm strain (Figure 4). More importantly, we found that the heat shock is not

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associated with Aβ transgene expression, but with a reduction of Aβ oligomeric protein [71]. Although direct regulation of HSF-1 by the insulin/IGF-1 receptor has not been established and many molecular interactions have not been elucidated, the indirect evidence from these intermingling factors, suggest that altering one may modulate the other [65, 69, 72]. With the obesity epidemic reaching epic proportions, particularly in the United States [16], the public is now looking to nutraceutical interventions as potential treatments for weight management.

7. C. Elegans as a model organism for screening of nutraceuticals The current literature has expounded on the virtues of the roundworm C.elegans in pharmacological studies. With its tiny size, adult hermaphrodites are ~1mm in length, to its ease and low cost of cultivation, extremely short generation time and life span; it is not surprising that this model organism has branched beyond the primarily genetic studies. The latest trends in C.elegans research are leaning toward the drug discovery arena. Since about half of the worm’s genome is homologous to the human genome, this makes targeting specific genes to study and treat fairly easy. Pharmacological modulation of targets that have been correlated with obesity, depression and Alzheimer’s disease (AD) are all being tested in the worm. Altering the activity of a specific gene can be a fundamental readout of the genes function [31]. This approach has aided us in our understanding of the general aging process, but it will probably be less effective in a multi-factorial disease such as AD; in particular the sporadic and most common form, which unlike the familial version of this disorder, has no solid genetic links.

8. Epigallocatechin-3-gallate (EGCG) Green tea has seen a boost in its popularity in the Western Hemisphere over the past few years because of its beneficial effects on health. Catechins, the polyphenolic plant metabolites that are common in the drink, and EGCG, which is the most abundant and active component, has become mainstream. Now EGCG can be found in a variety of commercial products ranging from fortified supplements to facial creams. EGCG has several pharmacological and biological properties. These include free radical scavenging activity, antioxidant activity, iron-chelating capabilities and attenuation of lipid peroxidation, due to various forms of radicals [73]. Researchers have reported that EGCG can rescue PC12 cells and protect rat hippocampal cells

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against amyloid β-induced neurotoxicity in a dose dependent manner, [74, 75] and attenuate Aβ-induced toxicity in cultured hippocampal neurons [76]. EGCG has been demonstrated to have potent antioxidative properties in vitro and is also displays biphasic action, e.g., it acts as a prooxidant or antioxidant depending on the cellular environment and concentration [77, 78]. Although our laboratory did not find an increase on life span at the concentrations used in this particular study; we did find that EGCG delayed the age-related slowing of sinusoidal movement (Figure 1A) [11], which probably delays severe muscle degeneration termed sarcopenia.

9. α-lipoic acid Thioctic acid, more commonly known as α-lipoic acid (LA), has been shown to increase insulin sensitivity when administered orally in patients diagnosed with type-2 diabetes [79]. LA is synthesized in a host of organisms that range from bacteria to humans and is a natural cofactor in the pyruvate dehydrogenase complex, where it will bind acyl groups and transfers them from one complex to another [80]. LA also exhibits networking capabilities, because of its capacity to regenerate endogenous antioxidants [80]. LA has been reported to extend the life span of the fruit fly Drosophila [81] and C.elegans [11], in a daf-16 dependent manner. Our laboratory found that the chemotaxis index, which decreases in an age-dependent manner in the worms, was enhanced in older animals that were fed with LA. Also, combined treatment with EGCG showed a markedly positive additive effect (Figure 1B) [11]. Although LA and EGCG both exhibit favorable effects on age-dependent declines of the worms’ behavior, it is obvious that their mechanism of action is different.

10. Ginkgo biloba extract EGB 761 & ginkgolide J The ginkgo biloba leaf extract EGb 761 has been at the forefront of many studies involving complementary and alternative medicine [12, 82-85]. Our laboratory reported many pharmacological effects of EGb 761 in the C.elegans model. It increased the worm’s median life span, by 8%, with the flavonoid fraction exhibiting a much higher median life span increase of 25% [10]. EGb 761 also dramatically increased the worm’s resistance to the oxidative stressor jugalone [10] and directly attenuated elevated levels of reactive oxygen species in the Aβ worms [86]. We concluded through these findings that EGb 761 extended the worm’s life span through alleviation of oxidative stress, which is probably the most prominent aging theory and has been around since the middle of the twentieth century [87, 88]. This conclusion, although valid, may

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Drug Treatments Figure 1. (A) Locomotive assay in wild type C. elegans fed EGCG or LA at day 12 or day 16 of age. Worms were scored as A, if they moved spontaneously and without the aid of touch stimulus. Worms were scored B, if a touch stimulus was needed in order for the worms to move. Worms were scored as C, if they only moved their head or tail and it required a touch stimulus. (B) Effects of EGCG, LA, or their combination, on the chemotaxis index (CI) in adult day 5 C. elegans. For the chemotaxis assays, 1 µl attractive odorant diacetyl were spotted near one edge of the plate plus 1 µl of sodium azide (1 M) to paralyze the worms which arrived at the odorant. After 1 h, worms that reached the odorant were quantitated. The Chemotaxis Index (CI) was defined as (the number of worms at the attractant − the number of worms at the control spot) / the total number of worms. * p<0.05, ** p<0.01, *** p<0.001 [11].

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not be the complete story. EGb 761 has been shown to reverse the age-related loss of 5-HT1A receptors [83]. Studies conducted in our laboratory demonstrated that worms that express the human Aβ transgene in the neurons (CL2355) show dysfunctions in serotonergic transmission [56]. This was demonstrated by their defect in two characteristic 5-HT controlled behaviors: the chemotaxis behavior, which activates input from several sensory, and interneurons and translates them into a motor output [89] [90] and in their hypersensitivity to 5-HT. When exogenous 5-HT is applied to wild type animals, it causes a decrease in their locomotor behavior [60, 91]. However, the animals that express Aβ in the neurons (CL2355) were defective in these responses [56]. Both of these behavioral defects were reversed in worms that were fed with EGb 761 (Figure 2A and B). Although no clear mechanism of action has been defined for the effects of EGb 761 on serotonin transmission in the worm, there are several scenarios that could be possible: 1) Response to 5HT could be affected by the transgene expression, 2) Acetylcholine (ACh), which negatively regulates 5-HT in the worms, could be decreased or 3) there could be direct or indirect blockage of 5-HT reuptake by Aβ expression [56]. Several of the single components of EGb 761 are pharmacologically active on their own. EGb 761 suppresses Aβ deposition and its single component Ginkgolide J (GJ) alleviates paralysis induced by Aβ expression in the transgenic CL4176 worm strain (Figure 3B) [56]. RNA interference of the serotonin receptor (SER-1), which has been shown to increase thermotolerance, UV –related stress and life span in the worms [9, 92] was knocked down in Aβ worms that paralyze at non-permissive temperature. These worms were also fed Ginkgolide J, to determine if GJ functions in the serotonergic pathway (unpublished data). Although GJ and SER-1 each delayed paralysis separately, there was no combinatorial effect (Figure 3B). The unique biological properties of the ginkgolides are attributed to their unique “cage skeleton” structure. Other compounds such as Congo red and/or curcumin [93], may share structural similarities with the ginkgolides, which may explain why all exhibit affinity for amyloidogenic conformations. Minuet differences in the chemical structure of the ginkgolides can have varying effects their biological activity. Ginkgolide J is able to inhibit the deleterious effects of Aβ on LTP impairment of CA1 hippocampal slices [63, 94]. Although some may question the validity of using C.elegans as a model system because a direct line cannot be drawn from different phenotypes to human pathological behaviors, several basic biochemical processes are conserved between these species. We do not expect C.elegans to answer the complexity of disease states of higher organisms, but we do expect them to give us insight into basic gene functions that can provide clues for novel therapeutic interventions.

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Figure 2. (A) Chemotaxis behavior in neuronal Aβ strain, CL2355, was reduced compared with the transgenic control strain CL2122 (no Aβ strain). Feeding with EGb 761 alleviated this descent in the transgenic strain, but not in the control strains (n=4; *p<0.05). (B) Serotonin hypersensitivity in CL2355 is normalized by EGb 761 and its constituents. Strain CL2355 were fed with EGb 761 (100µg/ml), vehicle (Ctrl; 100µg/ml), ginkgolides (GA, GB, GC, GJ) (10µg/ml each), BB (10µg/ml), quercetin (QC) (10µg/ml), kaempferol (KA; 10µg/ml) L-ascorbic acid (VC) (100µg/ml), and CR (139µg/ml). The worms were collected at 36 h after temperature upshift to 23°C. 5-HT sensitivity assay was conducted in 96-well plate containing 200µl of 1mM 5-HT solution. Percentage of active worms is calculated as follows: number of the active worms (after placed into serotonin solution for 5 min and still kept moving for 5 s)/total worms. (n=3; **p<0.01) [56].

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Figure 3. Effect of EGb 761, its constituent GJ and ser-1 (RNAi) on Aβ-induced paralysis in muscle Aβ strain CL4176 (A) Representative images of Aβ-induced paralysis in the transgenic CL4176 strain. CL1175, vector control (no Aβ) untreated (Control; CL1175, open circles), and in the transgenic CL4176 strain (muscle Aβ strain) fed with (EGb 761; closed circles) or without EGb 761 (Control; CL4176, open squares) at 36 h after temperature upshift. (B) Time course of paralysis assays in CL4176 with serotonin receptor (SER-1) knocked down by RNA interference; ser-1(RNAi) (purple circle) fed with Ginkgolide J (GJ) (green squares) or a combination of ser-1(RNAi) and GJ (blue squares). Worms were grown for 38 h at 16°C followed by upshifting the temperature to 23°C to induce the transgene expression. The paralysis was scored at 60 min intervals.

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Figure 4. Schematic of EGb 761, EGCG and α-lipoic acid treatment and the effects they have on worm responses.

Nutraceuticals may exhibit disease modifying effects by abrogating the deleterious effects induced by amyloid beta toxicity and neurofibrillary tangles (NFTs). The green tea component EGCG was shown to promote the nonamyloidogenic pathway by increasing α-secretase cleavage of the amyloid precursor protein, APP [95] and reducing potentially toxic sarkosyl-soluble phospho-tau isoforms [96]. α-lipoic acid was found to improve learning and memory retention in a transgenic mouse model of cerebral amyloidosis associated with AD [97]. Utilizing nutraceuticals to target insulin signaling and the serotonergic system could possibly provide novel therapeutic strategies to ameliorate dysfunctions which are associated with both the aging process and the development of AD.

Acknowledgement Studies in our laboratory are supported by NIH National Center for Alternative and Complementary Medicine (R01AT001928). Marishka Brown is supported by Diversity Supplement 3R01 AT001928-04S1.

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