ApoE4 Promotes Aβ Oligomerization

How does apolipoprotein E (ApoE) boost risk for late-onset Alzheimer’s disease (LOAD)? While prior work suggests it accelerates amyloid-β deposition, a study in the October 24 Journal of Neuroscience examines how ApoE, the top LOAD risk gene, influences the formation of oligomers, widely seen as the most toxic forms of Aβ. Researchers led by Brad Hyman at Massachusetts General Hospital, Boston, show that ApoE purified from AD patient brains enhances Aβ oligomerization in an isoform-dependent manner, with E4 having the strongest effect. The data “add to the evidence that ApoE is a critical modulator of Aβ metabolism,” noted Cheryl Wellington of the University of British Columbia, Vancouver, Canada, who was not involved in the study.

Research has long indicated that ApoE4 drives up Aβ fibrillization. AD model mice that overexpress amyloid precursor protein (APP) develop fewer plaques if they lack endogenous ApoE (Holtzman et al., 2000; Fagan et al., 2002) or express just one copy (ARF related news story on Kim et al., 2011). Among late-onset AD patients, people with two copies of the E4 isoform rack up more brain Aβ than E3 homozygotes do (Rebeck et al., 1993; Gomez-Isla et al., 1996), and clear the peptide more slowly (Castellano et al., 2011). Furthermore, ApoE binds Aβ in vitro (Strittmatter et al., 1993; LaDu et al., 1994), as well as in human brain (Näslund et al., 1995) and cerebrospinal fluid (Strittmatter et al., 1993). Since research has fingered soluble multimeric forms of Aβ as being most dangerous to neurons (see, e.g., ARF related news story), scientists have wondered if and how ApoE might directly influence oligomerization of the peptide.

In the current study, first author Tadafumi Hashimoto and colleagues measured Aβ oligomers in postmortem brain extracts of AD patients who had various ApoE genotypes. They separated peptides in soluble fractions by size-exclusion chromatography and probed them with Aβ antibodies on immunoblots. Even when matched for amyloid burden, E4/E4 samples had nearly three times more oligomers than E3 homozygote brains did, and about seven times more than samples from E2 carriers.

To look at oligomer formation, the researchers purified ApoE lipid particles from immortalized mouse astrocytes expressing human ApoE2, ApoE3, or ApoE4, then mixed them with synthetic Aβ. Again, immunoblots showed oligomers forming most robustly in the ApoE4 samples and least in the presence of ApoE2. The team confirmed the E4->E3->E2 effect on oligomers formed from luciferase-tagged Aβ. In this quantitative assay, HEK293 cells were transfected with Aβ genes that had been fused with a gene for either the N- or C-terminal ends of luciferase. Formation of Aβ dimers or larger oligomers enabled cells to express functional luciferase and glow (Hashimoto et al., 2011, and ARF related news story). When introduced into these cells, lipidated ApoE from human brain also promoted Aβ oligomerization in an isoform-dependent manner.

Hashimoto and colleagues went on to determine that the ApoE’s C-terminal lipid-binding domain is necessary and sufficient for enhancing oligomerization. Plus, a mutation (R61T) that makes ApoE4 structurally more similar to ApoE3 brought Aβ oligomer levels down in the HEK293 split-luciferase assay, suggesting that differences in protein conformation may underlie ApoE’s effects.

A key strength of the study was its use of physiologically lipidated ApoE, “unlike innumerable previous studies that used either unlipidated ApoE or ApoE reconstituted with non-physiological lipids,” Wellington suggested (see full comment below). On the flipside, Joachim Herz of the University of Texas, Southwestern, Dallas, noted that HEK293-secreted ApoE particles are poorly lipidated (LaDu et al., 2006). In his view, the present data suggest that lipidation status matters little in ApoE’s enhancement of Aβ oligomerization, since similar effects were seen using lipidated and HEK293-derived forms of ApoE.

The situation seems more complex in vivo, though. In a recent study, AD mice accumulated less soluble brain Aβ and fared better cognitively if fed a cancer drug (bexarotene) that drives up ApoE production (ARF related news story on Cramer et al., 2012). The drug also increased ApoE lipidation, which apparently confers benefits that outweigh ApoE’s enhancement of Aβ deposition, Herz said. Compounds that raise ApoE lipidation have been reported to curb plaque formation in APP-overexpressing mice (ARF related news story on Jiang et al., 2008). Bexarotene had no effect on plaques. Taken together, the evidence to date suggests that ApoE4 drives up Aβ oligomerization, but whether it needs to be lipidated to achieve that remains unclear.—Esther Landhuis

Comments

  1. 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.

    View all comments by Cheryl Wellington

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References

News Citations

  1. Lowering ApoE Brings Down Amyloid in Mice
  2. Bad Guys—Aβ Oligomers Live Up to Reputation in Human Studies
  3. San Diego: ApoE, Aβ, and AD—Strengthening the Synaptic Connection
  4. Upping Brain ApoE, Drug Treats Alzheimer's Mice
  5. ApoE’s Secret Revealed? Protein Promotes Aβ Degradation

Paper Citations

  1. Holtzman DM, Bales KR, Tenkova T, Fagan AM, Parsadanian M, Sartorius LJ, Mackey B, Olney J, McKeel D, Wozniak D, Paul SM. Apolipoprotein E isoform-dependent amyloid deposition and neuritic degeneration in a mouse model of Alzheimer's disease. Proc Natl Acad Sci U S A. 2000 Mar 14;97(6):2892-7. PubMed.
  2. Fagan AM, Watson M, Parsadanian M, Bales KR, Paul SM, Holtzman DM. Human and murine ApoE markedly alters A beta metabolism before and after plaque formation in a mouse model of Alzheimer's disease. Neurobiol Dis. 2002 Apr;9(3):305-18. PubMed.
  3. Kim J, Jiang H, Park S, Eltorai AE, Stewart FR, Yoon H, Basak JM, Finn MB, Holtzman DM. Haploinsufficiency of human APOE reduces amyloid deposition in a mouse model of amyloid-β amyloidosis. J Neurosci. 2011 Dec 7;31(49):18007-12. PubMed.
  4. Rebeck GW, Reiter JS, Strickland DK, Hyman BT. Apolipoprotein E in sporadic Alzheimer's disease: allelic variation and receptor interactions. Neuron. 1993 Oct;11(4):575-80. PubMed.
  5. Gomez-Isla T, West HL, Rebeck GW, Harr SD, Growdon JH, Locascio JJ, Perls TT, Lipsitz LA, Hyman BT. Clinical and pathological correlates of apolipoprotein E epsilon 4 in Alzheimer's disease. Ann Neurol. 1996 Jan;39(1):62-70. PubMed.
  6. Castellano JM, Kim J, Stewart FR, Jiang H, DeMattos RB, Patterson BW, Fagan AM, Morris JC, Mawuenyega KG, Cruchaga C, Goate AM, Bales KR, Paul SM, Bateman RJ, Holtzman DM. Human apoE isoforms differentially regulate brain amyloid-β peptide clearance. Sci Transl Med. 2011 Jun 29;3(89):89ra57. PubMed.
  7. Strittmatter WJ, Weisgraber KH, Huang DY, Dong LM, Salvesen GS, Pericak-Vance M, Schmechel D, Saunders AM, Goldgaber D, Roses AD. Binding of human apolipoprotein E to synthetic amyloid beta peptide: isoform-specific effects and implications for late-onset Alzheimer disease. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8098-102. PubMed.
  8. LaDu MJ, Falduto MT, Manelli AM, Reardon CA, Getz GS, Frail DE. Isoform-specific binding of apolipoprotein E to beta-amyloid. J Biol Chem. 1994 Sep 23;269(38):23403-6. PubMed.
  9. Näslund J, Thyberg J, Tjernberg LO, Wernstedt C, Karlström AR, Bogdanovic N, Gandy SE, Lannfelt L, Terenius L, Nordstedt C. Characterization of stable complexes involving apolipoprotein E and the amyloid beta peptide in Alzheimer's disease brain. Neuron. 1995 Jul;15(1):219-28. PubMed.
  10. Strittmatter WJ, Saunders AM, Schmechel D, Pericak-Vance M, Enghild J, Salvesen GS, Roses AD. Apolipoprotein E: high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1977-81. PubMed.
  11. Hashimoto T, Adams KW, Fan Z, McLean PJ, Hyman BT. Characterization of oligomer formation of amyloid-beta peptide using a split-luciferase complementation assay. J Biol Chem. 2011 Aug 5;286(31):27081-91. PubMed.
  12. LaDu MJ, Stine WB Jr, Narita M, Getz GS, Reardon CA, Bu G. Self-assembly of HEK cell-secreted ApoE particles resembles ApoE enrichment of lipoproteins as a ligand for the LDL receptor-related protein. Biochemistry. 2006 Jan 17;45(2):381-90. PubMed.
  13. Cramer PE, Cirrito JR, Wesson DW, Lee CY, Karlo JC, Zinn AE, Casali BT, Restivo JL, Goebel WD, James MJ, Brunden KR, Wilson DA, Landreth GE. ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models. Science. 2012 Mar 23;335(6075):1503-6. Epub 2012 Feb 9 PubMed.
  14. Jiang Q, Lee CY, Mandrekar S, Wilkinson B, Cramer P, Zelcer N, Mann K, Lamb B, Willson TM, Collins JL, Richardson JC, Smith JD, Comery TA, Riddell D, Holtzman DM, Tontonoz P, Landreth GE. ApoE promotes the proteolytic degradation of Abeta. Neuron. 2008 Jun 12;58(5):681-93. PubMed.

Further Reading

Papers

  1. Kim J, Jiang H, Park S, Eltorai AE, Stewart FR, Yoon H, Basak JM, Finn MB, Holtzman DM. Haploinsufficiency of human APOE reduces amyloid deposition in a mouse model of amyloid-β amyloidosis. J Neurosci. 2011 Dec 7;31(49):18007-12. PubMed.
  2. Holtzman DM, Bales KR, Tenkova T, Fagan AM, Parsadanian M, Sartorius LJ, Mackey B, Olney J, McKeel D, Wozniak D, Paul SM. Apolipoprotein E isoform-dependent amyloid deposition and neuritic degeneration in a mouse model of Alzheimer's disease. Proc Natl Acad Sci U S A. 2000 Mar 14;97(6):2892-7. PubMed.
  3. Cramer PE, Cirrito JR, Wesson DW, Lee CY, Karlo JC, Zinn AE, Casali BT, Restivo JL, Goebel WD, James MJ, Brunden KR, Wilson DA, Landreth GE. ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models. Science. 2012 Mar 23;335(6075):1503-6. Epub 2012 Feb 9 PubMed.
  4. Koffie RM, Hashimoto T, Tai HC, Kay KR, Serrano-Pozo A, Joyner D, Hou S, Kopeikina KJ, Frosch MP, Lee VM, Holtzman DM, Hyman BT, Spires-Jones TL. Apolipoprotein E4 effects in Alzheimer's disease are mediated by synaptotoxic oligomeric amyloid-β. Brain. 2012 Jul;135(Pt 7):2155-68. PubMed.
  5. Fagan AM, Watson M, Parsadanian M, Bales KR, Paul SM, Holtzman DM. Human and murine ApoE markedly alters A beta metabolism before and after plaque formation in a mouse model of Alzheimer's disease. Neurobiol Dis. 2002 Apr;9(3):305-18. PubMed.

Primary Papers

  1. Hashimoto T, Serrano-Pozo A, Hori Y, Adams KW, Takeda S, Banerji AO, Mitani A, Joyner D, Thyssen DH, Bacskai BJ, Frosch MP, Spires-Jones TL, Finn MB, Holtzman DM, Hyman BT. Apolipoprotein E, especially apolipoprotein E4, increases the oligomerization of amyloid β peptide. J Neurosci. 2012 Oct 24;32(43):15181-92. PubMed.

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