Carriers of the detrimental ApoE4 genotype have long been hypothesized to have slower Aβ catabolism, leading to earlier onset and more amyloid burden than non-ApoE4 carriers. Critical questions involve defining the mechanisms by which ApoE4 leads to sluggish Aβ turnover so that therapeutic methods to correct this can be developed. Brad Hyman and colleagues add to the evidence that ApoE is a critical modulator of Aβ metabolism.

Using AD brains of various ApoE genotypes that were carefully matched for amyloid burden, this group characterized the soluble fraction and observed significantly more higher-molecular-weight Aβ by direct visualization on SDS gels and after separation by size exclusion chromatography (SEC). Therefore, soluble Aβ is independent of amyloid burden, and the proportion of soluble oligomeric Aβ species is higher in ApoE4 carriers. Remarkably, ApoE co-eluted with Aβ during SEC and immunoprecipitated with Aβ, and it is possible that the ApoE-Aβ association makes specific Aβ epitopes under native conditions.

In a key advance, Hyman and colleagues...  Read more

Carriers of the detrimental ApoE4 genotype have long been hypothesized to have slower Aβ catabolism, leading to earlier onset and more amyloid burden than non-ApoE4 carriers. Critical questions involve defining the mechanisms by which ApoE4 leads to sluggish Aβ turnover so that therapeutic methods to correct this can be developed. Brad Hyman and colleagues add to the evidence that ApoE is a critical modulator of Aβ metabolism.

Using AD brains of various ApoE genotypes that were carefully matched for amyloid burden, this group characterized the soluble fraction and observed significantly more higher-molecular-weight Aβ by direct visualization on SDS gels and after separation by size exclusion chromatography (SEC). Therefore, soluble Aβ is independent of amyloid burden, and the proportion of soluble oligomeric Aβ species is higher in ApoE4 carriers. Remarkably, ApoE co-eluted with Aβ during SEC and immunoprecipitated with Aβ, and it is possible that the ApoE-Aβ association makes specific Aβ epitopes under native conditions.

In a key advance, Hyman and colleagues demonstrate that physiologically lipidated ApoE4 increases Aβ oligomerization in vitro. This appears to be a specific structural property of extracellular ApoE4. They first purified secreted ApoE particles from immortalized murine astrocytes expressing human ApoE2, ApoE3, or ApoE4, mixed these with synthetic Aβ, and observed that oligomer levels were significantly greater in the presence of ApoE4, which seems to stabilize the oligomers compared with ApoE2 and ApoE3. That Aβ oligomerization follows an ApoE4->ApoE3->ApoE2 relationship was confirmed using a split-luciferase assay in which Aβ oligomerization is required to observe luminescence. Furthermore, Aβ purified from the soluble fraction of AD patient brains forms high-molecular-weight Aβ species when incubated with ApoE. Importantly, ApoA-II, which is another apolipoprotein found on brain HDL particles, did not increase Aβ oligomerization using the split-luciferase assay. No effects on Aβ oligomerization were observed in cell lysates, suggesting that ApoE specifically affects Aβ in the extracellular milieu. Finally, reducing a salt bridge in ApoE4 by converting the arginine at amino acid 61 to a threonine reduced the level of Aβ oligomerization back to the level of ApoE3. This finding provides further support that the structural differences caused by arginine 61 specifically in human ApoE4 may be at the root of ApoE4’s dysfunction.

Further experiments explored the selectivity of the effect on Aβ oligomerization by other lipoproteins. Both ApoA-I and ApoJ led to significant increases in Aβ oligomers in the split-luciferase assay, albeit far more modestly than ApoE4. Hyman’s group then demonstrated that the lipid binding C-terminal domain of ApoE is necessary and sufficient for Aβ oligomerization.

Finally, in a series of elegant experiments, the Hyman group demonstrated that human ApoE purified from brain tissue affects Aβ oligomerization in an isoform-dependent manner, with ApoE4->ApoE3. Immunodepletion of ApoE from these samples significantly reduced Aβ oligomerization.

What is critically important about these experiments is that the researchers used sources of ApoE that are physiologically lipidated, unlike innumerable previous studies that used either unlipidated ApoE or ApoE reconstituted with non-physiological lipids. However, the ApoE-containing HDL particles used in these experiments can be considered “baseline” with respect to their lipid content. This is an important point, as modifying the lipidation status of ApoE has been under considerable study as a therapeutic approach for AD. For example, agonists of Liver-X-Receptor (LXR) and Retinoid-X-receptor (RXR) nuclear receptors, increase the expression of genes such as ABCA1 and ABCG1 that physiologically add lipids to apolipoprotein acceptors including ApoE. These agonists consistently improve cognitive behavior in APP-expressing mice. The studies vary in terms of the efficacy of these agonists to reduce amyloid or Aβ levels, or to shift Aβ from the insoluble to the soluble fraction. Furthermore, ABCA1, which is the rate-limiting step in apolipoprotein lipidation, is a key player in Aβ metabolism in vivo, as deletion of ABCA1 slows Aβ turnover, whereas selective overexpression of ABCA1 in the brain nearly eliminates amyloid deposits. What is not yet known is the relationship between ApoE lipidation and ApoE structure, particularly with respect to the argining 61 salt bridge. Whether LXR/RXR agonists or genetic manipulation of ABCA1 activity affect ApoE structure, thereby affecting Aβ oligomer formation, stability, or degradation, is not fully understood.

An important caveat to the studies using LXR agonists and genetically modified ABCA1 murine lines is that they have thus far been conducted on murine ApoE. Whether these pathways rescue the deficits in human ApoE4 with respect to Aβ metabolism, or whether they enhance the detrimental effects of ApoE4, is now a crucial question to address.

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