APOE genotype and chlorpyrifos: Examination of their roles in gut dysbiosis and influence on metabolites in the brain

Charlene Hoang

During birth to adulthood the gut microbiome can undergo a series of changes (Backhed et al., 2015), and dysbiosis of the gut microbiota is a result of diet changes, lifestyle changes, genetic factors and environmental factors and is a target of study for neurodevelopmental disorders like Alzheimer’s Disease. Guardia-Escote et al. (2020) explores the Apolipoprotein E (APOE) gene’s isoforms apoE3 and apoE4 –apoE4 is a genetic risk factor for Alzheimer’s Disease (Hersi et al., 2017) –and their suspected roles in modulating gut microbiota composition. They further explored dysbiosis and metabolite level changes in response to exposure to chlorpyrifos (CPF), a commonly used pesticide found at low levels in our diet, which was administered postnatally between days 10-15. Short-chain fatty acid (SCFA) levels and genomic differences of gut microbiota were examined across three transgenic mouse types –C57BL/6, apoE3-TR, and apoE4-TR at postnatal day 15. ApoE4-TR mice were found to have significantly reduced levels of A. muciniphilla, luteolibacter, and rubritaleo in response to CPF exposure and apoE3-TR mice were found to have increased levels of SCFAs including isovaleric acid and 4-methylvaleric acid. The results suggest that the gut microbiota can be altered substantially at early stages of development by both common genetic and environmental factors, and that alterations of metabolites like SCFA in the brain can lead to neurodevelopmental disorders later in life.

Keywords: Apolipoprotein E (APOE), chlorpyrifos (CPF), dysbiosis, short-chain fatty acid (SCFA), gut microbiota, Alzheimer’s Disease

Background and Introduction The symbiotic relationship between the gut microbiome, its composition, and humans are a major determinant of human health. Metabolism, immune system function, digestion, and even mental health are all affected by the gut microbiota, and when dysregulation or dysbiosis occurs human health can be influenced. Dysbiosis has been known to cause neurodegenerative diseases (Marizzoni et al., 2017) including Alzheimer’s Disease, dementia, and Parkinson’s Disease. Alterations in the gut microbiome can occur from diet changes (Makki et al., 2018), lifestyle changes, genetic factors and environmental factors like pesticide exposure (Yuan et al., 2019). The APOE gene exists in isoforms in the human body, of which the isoform apoE4 has been linked as a prevalent risk factor for Alzheimer’s Disease (Hersi et al., 2017). Normally, it exists as a gene important for lipid efflux and lipid transportation (Rebeck G. W., 2017) serving as a low-density lipoprotein receptor (LDLR) (Zhao et al., 2019) however, apoE4 has been linked to hallmarks of Alzheimer’s Disease such as accumulation of amyloid beta plaques (Munoz et al., 2018) and is a risk factor for the development of alpha synuclein derived Lewy Bodies (DLB) –a pathological hallmark of dementia and Parkinson’s Disease (Fyfe I., 2020). A quarter of the US population carries the apoE4 allele (Rebeck G.W., 2017) and of the patients suffering from AD, 50% are linked to this allele (Teter B., 2004), rendering almost 13% of the population susceptible to developing late onset AD and dementia. The genomic composition of the microbiota of individuals are also influenced by APOE genotype (Tran et al., 2019) and so the relative abundance of microbes can therefore determine differing metabolite production as well as interactions with CPF. Thus it is important to examine the role of APOE genetic backgrounds on gut microbiome susceptibility to CPF and its further implications on human health.

Chlorpyrifos are a commonly used pesticide that causes inhibition of acetylcholinesterase (AchE) (Burke et al., 2017) –an enzyme responsible for the catalyzation of acetylcholine which is a neurotransmitter involved in neuronal signaling. The gut microbiota is the most vulnerable to the toxicity of CPF and can cause dysbiosis (Liang et al., 2019) as CPF exposure is elicited through low doses during consumption of foods and is therefore in direct contact with the microbiome The authors administered CPF to postnatal mice at 10-15 days old orally to examine the effects it had on gut microbiome composition in mice of differing genetic backgrounds on day 15. Modulation of the microbiome depends on the genetic composition of the pre -existing gut, but changes from CPF can cause long lasting effects when done during development. The gut composition is associated with the production of SCFAs which are key signaling factors found in the brain. Acetate and butyrate play key roles as energy sources for the brain and can also provide neuroprotection for the brain however, levels of various SCFAs like isovaleric acid are to be monitored as overexpression can cause neurodevelopmental defects (Zhao et al., 2016).. The effects CPF have on gut microbiome composition and SCFA levels are therefore both indirectly and directly influencing human health. There is still a lack of direct connection between how alterations to the gut microbiome changes and affects brain development as well as neural behavior. However through this paper, the authors have tried to examine a link between vulnerabilities to environmental factors in developing gut microbiomes and brains of mice with varying genetic backgrounds and how such interactions can implicate future studies of neurodegenerative and neuropsychiatric diseases. The results of this paper suggests that there is more to be learned of about how genetic and environmental factors work together to modulate both the gut and the brain’s activities, and how predispositions and external factors can be therapeutically targeted in both treatment and prevention of neurodevelopmental and neurodegenerative diseases.

Major Results Baseline difference in relative abundance of microbes between different genetic backgrounds The phylum Verrucomicrobia was found at a generally higher level in apoE4 genotypes but levels had dropped to numbers relatively similar to that of apoE3-TR and C57BL/6 mice after CPF exposure (Fig 1).

Fig. 1. The phylum Verrcomicrobia

had significant baseline differences of relative abundance in apoE4 control mice compared to apoE3 and C57BL/6 mice

modulation is uniquely found in apoE4 genotype compared to apoE3 in response to CPF exposure (Fig. 2). ApoE4-TR control mice showed significantly higher levels of Verrucomicrobia, A.muciniphilla, luteolibacter, and rubritalea compared to apoE3 -TR and C57BL/6 mice indicating a baseline difference of rela

tive abundance of microbes between different genetic backgrounds. This indicates that genetic profiles influence the susceptibility of individuals to external factors as in this case where apoE4 carriers were more susceptible to effects of CPF on gut microbiota composition.

Fig. 3. Short-chain fatty acid level between control and CPF treated mice of (a) acetic acid, (b) propionic acid, and (c) butyric acid. Changes in all SCFAs were increased in CPF treated apoE3 mice.

Fig. 2. ApoE3, apoE4, and C57BL/6 control mice and

CPF treated mice’s relative abundance of (A) akkermansia,(G) luteolibacter, and (H)rubritalea Akkermansia muciniphilla’s heightened susceptibility to CPF are less abundant in control mice, but are increased after expo

exposure in apoE4 genotypes A.muciniphilla levels in apoE4 were most abundant amongst the three groups, with levels being almost 24 times that of apoE3 and C57BL/6 control mice. CPF treated apoE4 siderably diminished (Fig. 2A) suggesting that apoE4 genotype was more susceptible to effects of CPF during early development

SCFA levels in apoE3 genotype are increased after CPF exposure Important SCFAs including butyric acid and acetic acid were found at higher relative abundance after treatment with CPF in apoE3 mice (Fig 3A and 3B). Levels of SCFAs in apoE3 CPF treated mice were higher compared to control mice for butyric acid, acetic acid, isovaleric acid and 4-methylvaleric acid indicating that exposure to CPF increased the production of these SCFAs (Fig 4). These SCFAs are involved in brain signaling therefore alterations to levels by CPF can change brain function and behavior. Fig. 4. Isovaleric acid and 4-methylvaleric acid are SCFAs that

mice exhibited significant loss of A. muciniphilla as levels consure to CPF indicating CPF treatment could favor the release of less abundant SCFAs.

Discussion

ApoE4-TR mice exhibited naturally greater amounts of A.muciniphilla and greater loss after exposure to CPF as well. This microbe is a mucin degrading bacterium that is positively correlated with healthier individuals during early development as it promotes stronger immune defense and function. Individuals lacking this species are negatively correlated with obesity and diabetes, therefore the presence of this species is important for implication in early human development and later health. After exposure to CPF, A. muciniphilla experienced great loss indicating this species has a higher susceptibility to CPF and therefore apoE4 individuals are at greater risk of losing this species and its immune protective properties during development. These results indicate that there is a genetic and environmental factor that determines gut microbiota composition.

ApoE3-TR mice exhibited naturally higher levels of butyrate which acts as an inhibitor of histone deacetylases as well as having higher levels of acetate which acts as an energy source for the brain, therefore conferring higher neuroprotective properties compared to apoE4 genotype. After exposure to CPF, levels of these SCFAs increased inferring even higher neuroprotective properties in those with the apoE3 genetic profile after exposure to CPF. However, isovaleric acid and 4- methylvaleric acid levels also increased compared to control in are pesticide free or with pesticides. Along the way, they disease, then further research should be done with other

apoE3-TR mice. Higher levels of these less abundant SCFAs have been linked to individuals suffering from depression, another neurobehavioral disorder. This result further suggests that there is a link between genetic profiles, external factors, their affects on the gut microbiome, and its potential link to neurodegenerative diseases like Alzheimer’s disease. The results of this paper further support the idea that neurodegenerative diseases are multifactorial and that many factors act together to further potentiate the onset of certain developmental diseases. They suggest that early development and alterations to gut microbiome during this time can have major affects on later health, and that studying these effects can help formulate ideas of new therapeutic targets for major diseases like dementia and Alzheimer’s Disease. known genetic predispositions, or known environmental factors. This experiment can be replicated with not only neurosuch as diabetes, developmental, immunosuppressive diseases

Critical Analysis Neurodegenerative diseases like AD are influenced by many factors ranging from genetic to environmental and can be caused by gut microbiota composition. These neurodegenerative diseases, although occurring later in life, can be of higher risk to certain individuals because of early development of gut microbiota composition. As seen in the results apoE4 CPF treated mice, susceptibility to such toxins are greater in mice carrying said genotype indicating that genetic factors and environmental risk factors work together to alter gut microbiota compositions and therefore human health later on in life. The decrease in A. muciniphilla negatively correlates with health complications like diabetes and obesity later on in life and well as diminishing immune protective properties, and with apo4 being a prevalent predisposition for 50% of AD patients. This leads us to believe that early and recurring exposure to pesticides commonly found in food can cause an even higher risk of AD development in those carrying the E4 genotype. In apoE3 individuals, CPF exposure induces higher neuroprotective properties, however increases in less abundant SCFAs have also been correlatalterations from external factors paired with genetic profiles lead individuals to higher susceptibility of neurodevelopmental and health issues. The authors should further investigate the neurodegenerative properties that are a result of changes in the gut microbiota and SCFA production from CPF exposure in genetically predisposed individuals, namely to Alzheimer’s Disease.

Future Directions

As stated above, further investigation will allow for future therapeutic directions in terms of treatment and prevention of neural developmental diseases like AD. Experiments should be done using genetically predisposed people like carriers of the apoE4 genotype following their lives and diets that should monitor microbial composition as well as SCFAs levels in the plasma, and record behavior and mental health. If the results of these experiments show that common pesticide use increases the risk even more in almost 13% of the US population for neurodevelopmental diseases like AD, then this could further implicate the use of pesticide in wide spread farming practices. However if results fail to show an increased risk of degenerative and neurobehavioral disease, but other disease ed with depression suggesting again that early gut microbiome

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