07 Nov 2008

If a gene allele clearly raises risk for a devastating disease, it might seem a no-brainer to assume that the variant protein contributes to pathogenesis, and that correcting this rogue factor could help restore normality. But in the case of ApoE4 and Alzheimer disease, accumulating evidence—most recently from Peter Reinhart and colleagues at Wyeth Research in Princeton, New Jersey—suggests a more complex scenario. In this week’s Journal of Neuroscience, the Wyeth group reports that mice expressing the human ApoE4 allele have less total ApoE than those expressing the other two human variants. Furthermore, astrocytes seem to degrade ApoE4 more quickly than they do ApoE3, an isoform known to promote neurite outgrowth and synapse formation. “My feeling is that ApoE4 itself is not doing anything toxic,” lead author David Riddell told ARF. “Potentially the reason ApoE4 is associated with AD is that it's not as effective at repairing neuronal connections as ApoE3.” The findings raise intrigue as to whether ApoE-targeted AD treatments might fare better by replacing lost ApoE protein rather than target its biological activity.

Riddell and colleagues measured ApoE protein levels in 20-week-old mice expressing human ApoE2, E3, or E4 in place of the endogenous murine ApoE protein (Knouff et al., 1999). In these targeted replacement (TR) mice, amounts of ApoE protein in the frontal cortex and hippocampus varied in a genotype-dependent fashion. ApoE levels were highest in E2/2 animals, 12-24 percent lower in E3/3 animals, and 26-39 percent lower in the E4/4 group, consistent with the relative AD risk conferred by these ApoE alleles (E2 being linked to lowest and E4 to highest AD risk). The researchers found similar trends when assaying for ApoE protein in mouse plasma and cerebrospinal fluid (CSF) samples, and in measurements of secreted ApoE in culture medium from human astrocytoma cell lines and primary astrocytes cultured from the ApoE TR mice.

Using an ApoE4-specific immunoassay, they determined that ApoE4 represents 30-40 percent of the total ApoE pool in brain, CSF, and plasma of E3/4 heterozygote mice, which have lower levels of total ApoE than do E3/3 mice. They also showed that the per-allele amount of ApoE3 was similar in E3/4 and E3/3 mice, as was the amount of ApoE4 per allele in E3/4 and E4/4 animals. The authors took these data as evidence that reduced E4 levels account for the lower amounts of total ApoE protein in E3/4 compared with E3/3 mice. Levels of ApoE mRNA, however, did not significantly differ in cortical and hippocampal samples across E2/2, E3/3, and E4/4 mice, implicating post-translational mechanisms in the differing ApoE levels observed between the various genotypes.

To get at a possible mechanism, the researchers investigated whether differential degradation and secretion of the ApoE isoforms could account for the alterations in ApoE protein levels. Sure enough, in pulse-chase experiments with E3/3 and E4/4 primary astrocytes, ApoE degradation was higher in E4/4 cultures, with about 43 percent of E4 degraded after a 120-minute chase, compared with 22 percent for E3. The half-life of cell-associated E3 was nearly double that of E4 (96 minutes vs. 49 minutes).

“This paper and these mice are building the hypothesis that the problem with ApoE4 is not something that E4 is doing,” said Bill Rebeck of Georgetown University Medical Center in Washington, DC. “It's that E4 is being degraded, and so there's just less ApoE around.” In contrast to ApoE-based therapy built on the premise that “inheritance of ApoE4 is something bad, so I need to get rid of the ApoE4,” the new data suggest that treatments should instead focus on replenishing the lost ApoE, he said. “This really says it might be a levels question.”

But amounts may only be part of the picture. Last year, Carol Colton and Mike Vitek of Duke University Medical Center, Durham, North Carolina, also showed that ApoE protein levels in E4/4 TR mice appear to be significantly lower than in their E3/3 counterparts (Vitek et al., 2007). That study compared macrophage-mediated innate immune responses in E3/3 and E4/4 TR mice, as well as E3/0 heterozygotes that express levels of total ApoE protein equivalent to those found in E4/4 mice. The researchers found that pro-inflammatory cytokine secretion in the E3/0 heterozygotes was greater than that of E3/3 but much lower than that of E4/4 mice, suggesting that pound for pound, ApoE4 allows a stronger inflammatory response than does ApoE3. “This genetic experiment clearly shows that the effect of the APOE4 gene allele is complex and depends on both the absolute levels of ApoE protein and on an independent effect of the ApoE4 protein,” they wrote in an e-mail to ARF (see full comment below).—Esther Landhuis

Comments

1. • Michael Vitek
     Duke University Medical Center and Cognosci
   • Posted: 07 Nov 2008

   Compelling and widely accepted genetic evidence shows that the presence of one or more APOE4 genes is associated with more severe disease in Alzheimer’s, stroke, traumatic brain injury, multiple sclerosis, and many others. The question is, How does APOE4/ApoE4 protein contribute to disease or conversely, how does the absence of APOE3/ApoE3 protein contribute to disease? The likely explanations are quantity and quality of ApoE differs between APOE4 carriers and non-APOE4 carriers (i.e., APOE3 carriers). Riddell and colleagues confirm, as we had previously reported (Vitek et al., 2007), that the amount of ApoE protein in APOE4/4 targeted replacement (TR) mice appears to be significantly less than in their APOE3/3 counterparts. Thus, like many diseases where some critical factor is reduced in the disease, a critical therapeutic path becomes supplementation of that missing critical factor with either the authentic protein or a mimetic of that protein.

   But in the case of ApoE, amount is not the whole story. We (Colton et al., 2002; Vitek et al., 2007) have shown that the presence of the APOE4 gene is associated with a significantly greater pro-inflammatory innate immune response. This is observed despite the lower ApoE4 mRNA and ApoE4 protein levels in brain. We asked the question if the increased pro-inflammatory immune response observed in macrophages from the APOE4/4 TR compared to APOE3/3 TR mice was due to the lower level of ApoE protein. Thus, we generated a targeted replacement mouse that expressed levels of total ApoE protein equivalent to those found in APOE4/4 TR mice by crossing APOE knockout mice (APOE0/0) to APOE3/3 TR mice to generate heterozygous APOE3/0 TR mice. The response of macrophages in the APOE3/0 mice showed increased levels of pro-inflammatory cytokines compared to macrophages from APOE3/3 TR mice, cytokine levels that were significantly lower than those seen in APOE4/4 TR cells. This genetic experiment clearly shows that the effect of the APOE4 gene allele is complex and depends on both the absolute levels of ApoE protein and on an independent effect of the ApoE4 protein. Although understanding the mechanisms underlying how the unique APOE4 gene affects inflammation is clearly important, replacement of lost ApoE protein using ApoE mimetics may help to reduce the pathophysiological outcomes associated with APOE4 gene expression in many neurological disease states.

   M. P. Vitek is also a Prinicipal in Cognosci, Inc.

   References:

   Colton CA, Brown CM, Cook D, Needham LK, Xu Q, Czapiga M, Saunders AM, Schmechel DE, Rasheed K, Vitek MP. APOE and the regulation of microglial nitric oxide production: a link between genetic risk and oxidative stress. Neurobiol Aging. 2002 Sep-Oct;23(5):777-85. PubMed.

   Vitek MP, Brown CM, Colton CA. APOE genotype-specific differences in the innate immune response. Neurobiol Aging. 2009 Sep;30(9):1350-60. PubMed.

   Beffert U, Cohn JS, Petit-Turcotte C, Tremblay M, Aumont N, Ramassamy C, Davignon J, Poirier J. Apolipoprotein E and beta-amyloid levels in the hippocampus and frontal cortex of Alzheimer's disease subjects are disease-related and apolipoprotein E genotype dependent. Brain Res. 1999 Oct 2;843(1-2):87-94. PubMed.

   View all comments by Michael Vitek

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References

Paper Citations

1. Knouff C, Hinsdale ME, Mezdour H, Altenburg MK, Watanabe M, Quarfordt SH, Sullivan PM, Maeda N. Apo E structure determines VLDL clearance and atherosclerosis risk in mice. J Clin Invest. 1999 Jun;103(11):1579-86. PubMed.
2. Vitek MP, Brown CM, Colton CA. APOE genotype-specific differences in the innate immune response. Neurobiol Aging. 2009 Sep;30(9):1350-60. PubMed.

Further Reading

Papers

1. Vitek MP, Brown CM, Colton CA. APOE genotype-specific differences in the innate immune response. Neurobiol Aging. 2009 Sep;30(9):1350-60. PubMed.

News

1. ApoE4 and Aβ: Partners in Crime? 5 May 2008
2. ApoE’s Secret Revealed? Protein Promotes Aβ Degradation 15 Jun 2008