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The Role of ApoE and its isoforms in Alzheimer’s disease

| March 14, 2015

Introduction
Research into the genetic causes of Alzheimer’s disease have progressed considerably and currently at least three different genes are known to be linked with AD pathogenesis. Of these the apolipoprotein gene E (APOE), an amino acid glycoprotein, is one of the main focus of research over the last decade or so and significant associations between one particular isoform of the gene and the onset of AD have been reported. This isomer, referred to as the ApoE 4 allele, has been implicated in AD pathogenesis while other forms of the gene the ApoE 2 and the ApoE 3 have been reported to have a protective effect against the onset of AD. Though research has confirmed this variable effect of the different forms of the ApoE gene, there is still a lack of concrete evidence as to the exact pathway and the mechanism by which ApoE 4 contributes to neurodegeneration in AD patients. A brief review of related research would provide us more insight into the various pathological actions of ApoE 4 and how these multiple factors could lead to a gradual cognitive decline which is symptomatic in Alzheimer’s patients.

The ApoE gene (Physiological Functions)
ApoE is an amino acid glycoprotein that is found mainly in the liver and the brain. In the brain, ApoE is predominantly secreted by the astrocytes. Two other forms of apolipoproteins namely ApoA-1 and ApoJ are also found in the brain. In vitro studies of these two genes have so far suggested that both of them exhibit a neuroprotective effect. Both these proteins bind to Aβ and therefore are thought to reduce Aβ aggregation in the brain which is a known marker of AD. However, in vivo studies have not replicated such results and so the protective roles of ApoA-1 and ApoJ are not yet clear. The ApoE supports cholesterol equilibrium by serving as a ligand during endocytosis of lipoprotein particles by LDL receptors. Research is divided in the opinion that the cholesterol released from ApoE mediated endocytosis process is actually used for improving the synaptic connections or the synaptic plasticity. Mice based in vivo studies have not attested to this synaptogenesis. (Kim et.al, 2009)

The ApoE isomers
Three single nucleotide polymorphisms of the ApoE gene are commonly identified in humans. These are the apoE2 (cys112, cys158), apoE3 (cys112, arg158), and apoE4 (arg112, arg158) respectively. (Kim et.al, 2009) The structural and functional aspects of the three isomers of ApoE are totally altered though they differ by only one or two amino acid positions. Studies have reported strong immune-reactivity of the human apoE4 gene on amyloid plagues and the apoE4 isomer has been associated with having a strongest risk factor for AD. Some studies such as (Bertram et al., 2007) and (Bertram et al., 2009) have attested to this role of the apoE4 isomer in AD. Bertram et al., 2009 for instance reported that people with a single copy of the apoE4 gene had a three fold increase in risk for AD, while the risk was 12 fold among those with a dual copy of the ApoE4 gene. The results from studies also suggest the possible role of some environmental factors as research based on some ethnic groups have revealed that the ApoE 4 gene does not have a significant effect on AD.

ApoE & Aβ Aggregation (Fibrillogenesis)
Several research studies have focused upon the potential role of ApoE 4 in causing Aβ aggregation and neuronal degeneration. This direct interaction between the ApoE 4 with beta amyloid is attributed in the pathogenesis of the disease. Therefore it was hypothesized that increased levels of ApoE 4 in the brain corresponded to increased formation and thickness of amyloid plaques. The findings from one comprehensive study by Tiraboschi et.al (2004) validated this hypothesis. The researchers in this study confirmed increased plaque density corresponding to increased levels of ApoE 4. (Kim et.al, 2009)

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Possible Pathogenic Mechanisms of ApoE in AD (Kim et.al, 2009)
This positive association was again confirmed by a more recent study by Reiman et.al (2009). The researchers of this study used an amyloid detecting agent such as Pittsburgh compound B (PIB) along with positron emission tomography to detect the levels of fibrillar Aβ in the subjects. The researchers then correlated this information among individuals with varied genetic risks. The results from the study clearly revealed that ApoE 4 homozygotes had the highest risk (91%) for developing AD while ApoE 4 heterozygotes had a moderate risk of 47% and finally ApoE 4 non carriers had only 20% risk of having AD. Furthermore, the mean age of onset of AD was also vastly different with ApoE 4 homozygotes at 64, heterozygotes at 76 and 84 years of age among non carriers. This longitudinal study clearly provides positive evidence connecting the ApoE 4 gene with a high risk for AD. Thus there is enough evidence that ApoE 4 gene may aid in fibrillogenesis and consequent cognitive decline. (Reiman et.al 2009).

Aβ Clearance by ApoE
Some studies have shown that ApoE by binding with soluble Aβ promotes the cellular uptake and ingestion of the ApoE –Aβ complex by endocytosis. However, isomer specific results for this ApoE facilitated cellular uptake of Aβ are still awaited. Research has also focused on the possible clearance of Aβ from the brain via the blood brain barrier. However, there is growing evidence that ApoE 4 might actually hinder or reduce this clearance of Aβ. Deane et.al (2008), a study based on mice reported that the clearance rate of ApoE 4 –Aβ complex is much lower than that for the ApoE2 and ApoE 3 complexes. Other recent studies such as Bell et.al (2007) and Ito et.al (2007) that focused on studying the Aβ clearance in humans found that the clearance rate along the Blood Brain Barrier (BBB) was significantly lower for the ApoE 4 –Aβ complex compared to that of simple Aβ peptides. These studies offer enough evidence that ApoE 4 might actually increase the fibrillogenesis by directly affecting the Aβ metabolism in the brain. By reducing the clearance rate and promoting aggregation ApoE 4 gene definitely poses an increased risk factor for AD.

ApoE and Neuronal Inflammation
Several studies have reported that ApoE has marked anti inflammatory properties which explain the surge in their production in the aftermath of an injury. These studies hypothesize that ApoE is needed for maintaining the cholesterol homeostasis, and in particular for increasing the availability of cholesterol for neuronal repair and improving the synaptic plasticity subsequent to brain injury. (Slezak & Pfriege, 2003 ). However studies have also reported differences in neurite growth and synaptic plasticity post neuronal insult in animal studies based on the genotype. One transgenic mice study found that neuronal growth and synaptogenesis were markedly lower among ApoE 4 transgenic mice compared to ApoE3 transgenic mice. (White et.al, 2001) An extensive review of the neuroprotective effects of the various ApoE isomers by (Cambon et al, 2000) clearly showed differential effects between the isomers. Most of the studies reported that ApoE3 promoted synaptic plasticity and neurite growth. However similar positive results were not witnessed in the case of ApoE 4 alleles with some studies even reporting the negative effects of ApoE 4 on synaptic plasticity and neuronal growth. Colton et.al (2004) found that the anti-inflammatory effects vary drastically between the ApoE isomers. The researchers studied this variability in anti-inflammatory response triggered by the ApoE 3 and ApoE4 isomers by using in vivo experiment conducted on ApoE knockin mice. By studying Lipopolysaccharide (LPS) mediated inflammatory responses the researchers observed the differences between the isomers. The inflammatory response was much greater in the ApoE 4 knockin mice when compared to the ApoE3 Knockin mice. These studies suggest that the anti inflammatory response maybe less active in ApoE 4 and this might lead to neuronal damage as witnessed in AD patients. (Kim et.al, 2009)
Conclusion
The review of studies has revealed that the ApoE isomers have differential effects on the Aβ metabolism in the brain. While the ApoE 2 and ApoE 3 isomers have been reported for their protective effects the ApoE 4 allele clearly seems to contribute to the AD pathogenesis. Results from recent fibrillar PET imaging studies suggest a clear positive correlation between the ApoE 4 allele and the density of amyloid plaque formation. It is clear that by slowing down clearance of Aβ as well as contributing to its aggregation in the brain, the ApoE 4 allele increases the susceptibility of a person for AD. It is also possible that the ApoE 4 also lowers the protective function by increasing the neuronal inflammatory response and by hindering neurite growth. More studies are necessary to understand the complex relationship between ApoE and Aβ and the levels of different isomers of ApoE and their corresponding impact on Neuroinflammation and neurotoxicity, etc.

Bibliography
1) Jungsu Kim, Jacob M. Basak, & David M. Holtzman, (Aug 13th 2009), The Role of Apolipoprotein E in Alzheimer’s disease, Neuron 63.
2) Bertram, L., McQueen, M.B., Mullin, K., Blacker, D., and Tanzi, R.E. (2007) Systematic meta-analyses of Alzheimer disease genetic association studies: the AlzGene database. Nat. Genet. 39, 17–23.
3) Bertram, L., McQueen, M.B., Mullin, K., Blacker, D., and Tanzi, R.E. (2009). The AlzGene Database Alzheimer Research Forum available at, <http://www.alzgene.org.>
4) Eric M. Reiman, Kewei Chen,& Xiaofen Liu et.al (Apr 2009), Fibrillar amyloid-β burden in cognitively normal people at 3 levels of genetic risk for Alzheimer’s disease, Proc Natl Acad Sci U S A. ; 106(16): 6820–6825.
5) Bell, R.D., Sagare, A.P., Friedman, & A.E., Bedi et.al (2007). Transport pathways for clearance of human Alzheimer’s amyloid beta-peptide and apolipoproteins E and J in the mouse central nervous system. J. Cereb. Blood Flow Metab. 27, 909–918.
6) Ito, S., Ohtsuki, S., Kamiie, J., Nezu, Y., and Terasaki, T. (2007). Cerebral clearance of human amyloid-beta peptide (1-40) across the blood-brain barrier is reduced by self-aggregation and formation of low-density lipoprotein receptor-related protein-1 ligand complexes. J. Neurochem. 103, 2482–2490
Part 2
Fibrillar amyloid-β burden in cognitively normal people at 3 levels of genetic risk for Alzheimer’s disease (Reiman et.al 2009)

This research study by Reiman et.al (2009) hypothesized that fibrillar amyloid accumulation is an early development in the onset of Alzheimer’s disease. The researchers further hypothesized that the fibrillar amyloid accumulation precedes any recognizable symptoms of cognitive decline. Amyloid plaques are a distinct feature of AD as observed from post mortem studies. Given the hypothesis that fibrillar Aβ accumulation is observed even in healthy, non symptomatic adults, the researchers intended to study the effect of fibrillar Aβ burden as a predictor of future onset of AD on adults with normal cognitive function using Ante mortem brain imaging studies.

For this study, the researchers used Pittsburgh Compound B (PiB), a radio ligand that binds only with the fibrillar Aβ species. Earlier studies have also used this PIB for imaging studies of live human brain to observe the formation of fibrillar Aβ in healthy adults. This study by the authors is aimed to extend the previous studies by providing a genetic risk analysis and to understand the predisposition to AD based on an individuals APOE genotype. The APOE genotypes were identified by using blood sample analysis. The researchers used fluorodeoxyglucose PET, volumetric MRI and neuropsychological tests. Fibrillar imaging was done using 3 dimensional HR+ scanner. The subjects of this study were all cognitively normal and late middle aged people representing a mixture of APOE genotypes. This was a longitudinal study and the subjects were assessed every 2 years.
The researchers observed that cerebral glucose metabolism levels of those subjects carrying dual copy of the ApoE 4 (homozygotes) was significantly lower than that of the heterozygotes and the non carriers. With the PiB Pet images the researchers compared and correlated the fibrillar burden with the genetic risk to understand the predisposition for AD between the various isomers. The average Pittsburgh(PiB) distribution volume rations(DVR), which is a measure of the fibrillar Aβ accumulation, was on increase in all the three different groups in the 2 years period. One of the homozygote subjects developed mild amnesia and her DVR was close to that of an AD patient. Overall of all the 28 subjects (8 homozygotes, 8 heterozygotes and 12 non carriers), the ROI measurements pertaining to the different brain regions such as temporal, parietal, posterior cingulate-precuneus, basal ganglia, etc, was the highest among the homozygotes.
The researchers used statistical analysis and found that the homozygotes had considerably higher ROI values when compared to the non carriers (P < .05) while the difference between the heterozygotes and the non carriers was not significant (.05 < P < .11). When correlated the APOE4 and Fibrillar Aβ burden was found to be significant among both homozygotes and heterozygotes compared with the non carriers. The study authors successfully found that even in cognitively normal individuals there is a positive correlation between ApoE 4 and fibrillar Aβ. The results from the study suggest that the ApoE 4 is clearly associated with increased Aβ aggregation and decreased Aβ clearance. The following PET map clearly indicates the various regions of the brain that are affected by Aβ@_001

Group A represents the homozygotes while group B and C represent the heterozygotes and non carriers respectively.
Longitudinal studies are very effective in following the growth of fibrillar Aβ accumulation as they provide a clear pattern of disease progression even in the asymptomatic stage. People who are in late middle age who are at risk for AD would greatly benefit from a prophylactic intervention based on the brain mapping study and their ApoE genotype. This study clearly contributes to our understanding that the ApoE 4 is directly involved in the Aβ interaction and the formation of Amyloid Plaques. By considering live brain images for the study the authors were able to clearly confirm the association between the ApoE 4 and Aβ fibrillar density and predict the increased susceptibility of people with that genotype.
This study has large implications for the treatment of AD. By proving that Aβ fibrillar formation precedes a perceivable cognitive decline, this study recommends early interventions particularly for people with ApoE 4 genotype, as the current Aβ modifying treatments would be more effective before extensive Aβ accumulation has occurred. The researchers advocate the use of brain imaging studies combined with genotype based risk assessment as a potentially powerful tool in preventive therapy for people who are at a genetically high risk for AD. One of the drawbacks of the study is the very small study sample. A large cohort based longitudinal study would have provided more conclusive confirmation of the study results.

Bibliography
1) Eric M. Reiman, Kewei Chen,& Xiaofen Liu et.al (Apr 2009), Fibrillar amyloid-β burden in cognitively normal people at 3 levels of genetic risk for Alzheimer’s disease, Proc Natl Acad Sci U S A. ; 106(16): 6820–6825.

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