Injection of beta amyloid brain extract intravenously is shown to induce Alzheimer’s Like Disease in APPSwe/PS1dE9 mice A Critical Review

Nikol Digtyar

Alzheimer’s Disease is one of the most common neurodegenerative diseases. There has been much significant research in the field to determine whether Alzheimer’s Disease could be a transmissible disease. The spread of Alzheimer’s disease has been linked to seeding of β-amyloid protein plaques in the brain. However, the studies which were completed injected Human Alzheimer’s disease brain extracts intracerebrally into the hypothalamus and other corresponding brain regions. The following study was completed on transgenic APPSwe/PS1dE9 mice. There were three groups in the experiment. A control group was injected with brain extracts from a healthy male adult (HTC), the experimental groups were injected with brain extracts from two male human Alzheimer’s Disease patients (AD1, AD2). Both experimental groups were additionally subdivided. One subdivision was injected with the brain extract intracerebrally and the other was injected intravenously. Mice were then sacrificed 180, 270, and 360 days post-injection. The brain was then extracted and placed in formaldehyde and embedded in wax. Several histological analyses were completed on the tissue samples. β -amyloid was detected using anti-Abeta 4G8 antibody. The results of this study demonstrate that 180 days post intravenous injection of AD1 brain extract, mice began to develop β-amyloid plaques in the vasculature of the thalamus. This suggests that the β-amyloid brain extract was able to bypass the blood-brain barrier. The intracerebrally injected mice also showed similar results 360 days post injection.

Figure 1:A visual representation of the methods and major findwith C57BL/6 mice to produce heterozygous mice. APPswe/ PS1dE9 mice overexpress APP and have a mutated PS1 gene which contributes to Alzheimer’s like disease development (Malm T et. al. 2011). C57BL/6 are wild-type normal mice commonly used in a laboratory setting. These mice were injected with AD brain extracts both intracerebrally and intravenously. Their results showed that the mice injected intravenously had similar results to the mice injected with the intracerebral method. Furthermore, they showed that even at 180 days post intravenously injected the mice had β -amyloid plaques in the brain (Burwinkel M et. al. 2018). This is a new outlook on Alzheimer’s disease and the first paper to show that β -amyloid injected intravenously could enter the blood-brain barrier and include

ings of the research study. Alzheimer’s like disease in mice.

Introduction: Major Results:

Alzheimer’s Disease (AD) is a lethal neurodegenerative disorder which is characterized by severe dementia. Despite the vast scientific leaps in Alzheimer's research, there are still many unknowns and no definitive treatment nor cure. There have been precursor genes identified such as the amyloid precursor protein APP and others which increase the chances of acquiring AD (Lane, C., Hardy et. al. 2017). Other factors can also contribute to the development of Alzheimer’s Disease; however, they are not well understood or definitive. Alzheimer’s is a disease in which β-amyloid plaques and Lewy bodies accumulate within the areas of the brain responsible for memory such as the hippocampus (Lane, C., Hardy et. al. 2017). The disease slowly spreads throughout the brain causing atrophy, neuronal death, and is fatal. There is a seeding phenomenon that occurs in AD. Seeding is the deposition or “infection” of surrounding brain areas with the β -amyloid protein (Lane, C., Hardy et. al. 2017). There is still some debate in the scientific community concerning the notion of Alzheimer’s Disease transmission and the cascade of events which occur once amyloid plaques begin to accumulate. There has been research done in the past which consisted of injecting mice with brain extracts directly acquired from AD patients. The results of these numerous studies were varied based on their methods and criteria. The mice used for these experiments are generally transgenic amyloid precursor protein mice APP23 or tg2576. These mice overexpress the β -amyloid protein gene and with age acquire an increasing amount of beta-amyloid plaques in the brain. A study conducted by Michael D. Kane et. al. (2000) showed that intracerebral injection of brain extracts from AD patients did cause an accumulation of β -amyloid plaques (Kane MD et. al. 2000). Which does support the notion that seeding could significantly increase the chances of developing AD. Other studies have also been done on these mice with oral, intranasal, and intraocular administration of β –amyloid extracts. In these studies, only intracranial administration of AD brain extracts induced the development of β- amyloid plaques (Eisele YS et. al. 2009). A more recent study completed by Michael Burwinkel et. al. 2018 used a different approach to test the hypothesis that intravenous administration of AD brain extracts would be sufficient to induce Alzheimer’s like disease in mice (Burwinkel M. et. al. 2018). This study uses APPswe/PS1dE9 mice crossbred The histological study results for the following study showed that for both the experimental and the control group, mice developed β-amyloid plaques 360 days post-injection of the extract in the intracerebrally injected mice (Burwinkel M et. al. 2018). These findings are consistent with the transgenic mouse line which is predisposed to develop Alzheimer’s Disease with age.

However, the AD1, AD2 mice showed the deposition of β-amyloid plaques in the vasculature of the thalamus (Figure 2). This was not observed in HTC mice which were injected with brain extract from a non-AD patient. The methodology of injecting the extract directly into the brain has been tested in other studies. In a 2000 study conducted by Michael D. Kane researchers injected βAPP transgenic mice with brain extracts from human patients with AD, unilaterally into the neocortex and hippocampus ( Kane MD et. al. 2000). Their results revealed that 5 months post-injection, mice developed β–amyloid plaques in the injected areas as well as some of the vasculature. Therefore, the findings from the study done by Michael Burwinkel et. al. 2018 are consistent with the results of the previous research.

A key finding of the 2018 study however is the result of injecting the mice intravenously using the tail vein. The results for the intravenous injection of brain extracts of both AD1 and AD2 showed a substantially higher amount of plaques located in the vasculature of the thalamus when compared to the control at 180 days post-injection (Figure 3). They also found increased pathology in the cortices in the intravenous group. These results show that there is a significantly higher amount of β-amyloid plaques in mice even 180 days post-injection when compared to the intracerebral group at 360 days. However, both the hippocampal and cortical regions both at 180 and 270 days post-injection did not show significant amyloid angiopathy.

component of Alzheimer’s disease. With further understanding and research, the information uncovered in this study could contribute to preventing the transmission of β-amyloid plaques in human patients.

Figure 2:A)Intracerebral Injection of HTC (non-AD extract) hippocampus and thalamus 360 days after injection. B) Intracerebral Injection of AD1 (AD + extract after injection). C)&D) Increased magnification showing the deposition in the vasculature of the thalamus (Burwinkel M. et. al 2018).

Critical Analysis: The study uncovered some new concepts concerning the transmission of β-amyloid in mice models. In order to fully understand these findings, further research needs to be completed. The intravenous injection did result in β -amyloid plaques in the vasculature of the brain however it is poorly understood how and why this occurred. A question that the authors should investigate is how and why the human β -amyloid brain extract was able to pass through the blood-brain barrier, which has not been observed before. A research paper conducted by Eisele YS et. al. 2009 showed that oral, intravenous, intraocular, and intranasal administration of human AD brain extract did not show angiopathy or plaques within the brain. This is completely controversial to the study completed in 2018. However, the 2009 study used APP23 transgenic mice while the 2018 study used APPSwe/PS1dE9, C57BL/6 crossbred mice.

Figure 3:A) Intravenous Injection of HTC (non-AD extract) control, hippocampus and thalamus at 180 days after injection. B) Intravenous Injection of AD (AD + extract) at 180 days after injection. C) Thalamus after AD injection with higher magnification D) Thalamus after AD injection higher magnification. (Burwinkel M. et. al 2018).

Conclusion/ Discussion: The major findings of the study supported previous studies on intracerebral administration of AD brain extracts. The researchers found increased amyloid angiopathy in the injected areas of the brain and the surrounding vasculature. The findings exclusive to this study showed that intravenous injection also induced amyloid angiopathy in the thalamus and vasculature of the brain. This was a key finding of the study which has not been observed before. A study was conducted in 2009 by Yvonne S Eisele et. al. in which researchers administered β -amyloid extracts orally, intravenously, intraocularly, and intranasally (Eisele YS et. al. 2009). Their results showed no β-amyloidosis in the mice. However, this study used the APP23 transgenic mice in comparison to the crossbred APPSwe/ PS1dE9, C57BL/6 mice which did yield results in the 2018 study. The results of the study are especially significant because before this time other studies were unable to demonstrate the amyloid extracts can pass through the blood-brain barrier and transmit to the vasculature of the CNS in the brain. These findings could aid researchers in understanding the transmissible There are various animal models of human neurodegenerative diseases and the differences in the altered genes of the mice can lead to different results in studies. In the APP23 mouse model, the APP gene is the amyloid precursor protein which is composed of β-amyloid and an amino acid peptide (Van Dan D et. al. 2005). This is an autosomal dominant mutation that is identified in some Alzheimer's disease families with early-onset pathologies (Van Dan D et. al. 2005). This mouse model is neuron-specific and expresses a promoter which leads to overexpression of a human β-amyloid precursor (Van Dan D et. al. 2005). These mice are usually bred

effects of intravenous injection. The study shows that βwith C7 wild-type mice to achieve a heterozygous transgenic mice model. As the mice age they do develop β-amyloid plaques in the hippocampus and neocortex (Van Dan D et. al. 2005).

On the contrary, the APPSwe/PS1dE9 mouse model includes the APP mutation as well as a PS-1 mutation. PS1 is a protease catalyst and when mutated or inhibited can increase the development of β-amyloid plaques (Malm T et. al. 2011). In addition, this mouse model is not neuron-specific and can affect the CNS and allow for β-amyloid accumulation in other areas of the brain such as the parenchyma of the brain and the vasculature (Malm T et. al. 2011). Research has also shown that this mice model does not spontaneously develop β-amyloid plaques within the brain even at 30 months of age (Prado, M. A., Baron, G. 2012). Intracerebral injection of β -amyloid in these mice showed the acceleration of amyloidosis in the brain in other studies (Rosen, R. F. et. al. 2012). Therefore, this may be the reason for the lack of results and evidence of intravenous injections affecting the formation of β - amyloid plaques in the brain in past studies.

Future Directions: A possible future direction and experiment which could be explored by future researchers is to determine the cascade of events that occurs after a venous injection of the AD brain extract is administered. Viral GFP tagging of the β - amyloid could be used to determine which pathways the transmission of β -amyloid extract is taking to reach the CNS and the vasculature of the brain. Cultures of the AD brain extracts can be transfected and combined with fluorescent GFP in culture and then be administered intravenously to the test animals. Imaging techniques and other histological studies could also be used to track the progression and the path that the extract takes, from entering the vein of the tail to crossing the bloodbrain barrier. Instead of sacrificing animals 180 days postinjection, histological studies can be done earlier on nerves and vasculature of the mice near the injection site to determine that path the β-amyloid takes prior to reaching the brain. In particular, it is important to understand how β-amyloid is able to pass the blood-brain barrier and reach the brain vasculature only 180 days post-injection into the tail. It is difficult to hypothesize what results such experiments would lead to. This is due to the notion that this discovery is fairly recent and would be particularly difficult to track, and infer by which path this particular prion-like molecule would take to reach the CNS and the brain. Future research should focus on determining this pathway and how it is able to go through the blood-brain barrier, as this knowledge would provide great insight into understanding the underlying aspects of β -amyloid plaques and their destruction of brain areas. With this knowledge, possible treatment for prevention and isolation of transmission from one area of the brain to the surrounding regions could be obtained. With newer technology being invented finding a treatment or cure could be possible in the future.

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