ApoE4 predisposes people to Alzheimer’s disease by modulating astrocytes and microglia, suggest researchers led by Julia TCW and Alison Goate at the Icahn School of Medicine at Mount Sinai, New York. In a preprint on bioRχiv, the researchers describe transcriptional differences between iPSC-derived human astrocytes and microglia that express ApoE4/4 or ApoE3/3. The ApoE4/4 glia generated more cholesterol than their E3/3 counterparts. They exported and degraded it poorly, causing lipid to build up inside them. The E4/4 glia also pumped out greater amounts of proinflammatory cytokines and extracellular matrix proteins than E3/3s.

  • ApoE4 astrocytes accumulate cholesterol because production and export are unbalanced
  • E4 astrocytes and microglia make more extracellular matrix and inflammatory proteins than do E3 cells
  • This glial phenotype is also seen in AD brain, regardless of ApoE status

Does this have anything to do with Alzheimer’s? Lo and behold, in AD brains, astrocytes and microglia behaved quite similarly to these ApoE4/4 glia. They accumulated lipid and ratcheted up inflammation. Importantly, they did so regardless of their ApoE genotype. To TCW, the data imply that ApoE4 may nudge microglia and astrocytes toward an Alzheimer's-like state. Perhaps faulty lipid metabolism is one of the earliest changes on the path to Alzheimer’s. If so, restoring glial lipid regulation could be a therapeutic approach, she suggested. The paper is under review at Cell (Sneak Peek).

Production Up, Disposal Down. Genes for making cholesterol are expressed more in cultured human ApoE4 than ApoE3 microglia (red, orange), while genes for exporting and breaking cholesterol are mostly lower (blue, green). [Courtesy of Julia TCW.]

Other scientists agreed that the data shed light on how ApoE4 may push the brain toward disease. “This unbiased work demonstrates that in CNS-relevant glial cells, cholesterol metabolism likely represents a major ApoE4-related pathobiology in Alzheimer’s and aging brains,” Guojun Bu at the Mayo Clinic in Jacksonville, Florida, wrote to Alzforum (full comment below). Shane Liddelow at New York University was impressed by how comprehensive the analyses were. “This is a great paper. It opens up a whole new avenue of hypotheses for us to investigate,” he said.

TCW and others previously reported that ApoE4 prods microglia and astrocytes to crank up inflammation in response to external stressors (Apr 2017 conference news). At recent meetings, TCW laid out problems with lipid metabolism in ApoE4 glia (Jul 2018 conference news; Apr 2019 conference news).

In the new paper, the scientists elaborate. They generated 13 iPSC lines: six from people homozygous for ApoE4/4, the remainder from people with the ApoE3/3 genotype. They differentiated each line into pure cultures of microglia or astrocytes, and analyzed gene expression. ApoE4/4 cells, regardless of type, more highly expressed genes involved in the synthesis of cholesterol and steroids than did ApoE3/3 cells. At the same time, ApoE4/4 microglia expressed fewer Liver X and retinoid-X receptors and ligands. LXRs and RXRs are transcription factors that bind lipids and switch on expression of lipid exporters such as ApoE.

Because LXRs and RXRs are lipid sensors, the finding suggested that E4/4 glia might have trouble sensing and regulating their lipid levels. In keeping with this, E4/4 astrocytes made less ApoE and ABCA1 than did ApoE3/3 cells. These proteins help ferry cholesterol from the cell. Meanwhile, ApoE4/4 microglia expressed less LAMP1, a lysosomal protein that binds cholesterol and facilitates its breakdown. Overall, the findings depicted an imbalance between cholesterol production and disposal in E4/4 glia (see image above).

If true, then cholesterol levels should rise in these cells. The authors confirmed this using gas chromatography and mass spectrometry in ApoE4/4 and ApoE3/3 isogenic lines. (To make sure whatever effect was observed was due to ApoE genotype and nothing else, the scientists picked two E4/4 lines and used CRISPR to edit the genotype to E3/3.) Astrocytes from the E4/4 parent lines contained 20 percent more cholesterol than the isogenic E3/3 lines did. This was in the form of free cholesterol, rather than cholesterol esters, and primarily accumulated in lysosomes. The data support the idea that E4/4 glia fail to properly dispose of cholesterol, and gradually become stuffed with lipid.

How, if at all, does lipid overload affect interactions with other cell types? To find out, the authors analyzed gene expression in mixed cortical cultures of neurons and astrocytes generated from human iPSCs. Compared with ApoE3/3 astrocytes, ApoE4/4 astrocytes not only turned up expression of lipid synthesis proteins, but also highly overexpressed extracellular matrix proteins. ECM proteins, collectively known as the matrisome, include structural proteins such as collagens and proteoglycans, as well as signaling proteins, including cytokines and growth factors. The highly expressed matrisome proteins in these E4/4 cultures would heighten lipid synthesis, cell migration, and the inflammatory response, the authors predicted, tying lipid dysregulation to inflammation.

Does this happen in brain? The authors analyzed RNAseq data from healthy ApoE4/4 and ApoE3/3 brains in the Mount Sinai Brain Bank as well as from the Religious Orders Study and Memory and Aging Project (ROSMAP). In multiple regions of the temporal and frontal cortex that the authors examined, ApoE4/4 astrocytes and microglia indeed expressed more ECM proteins than E3/3s did. As in culture, E4/4 astrocytes and microglia made more cholesterol than their E3/3 counterparts did, but poorly exported and degraded it.

What about Alzheimer’s disease? Astrocytes and microglia in AD brains expressed much higher levels of matrisome proteins and inflammatory cytokines than did glial cells from healthy controls. Curiously, this did not depend on the person’s ApoE genotype. In essence, glia in ApoE3/3 AD brains had a similar phenotype to glial cells in healthy ApoE4/4 brains. To the authors, this implies that ApoE4/4 brains may already be on the path to AD even while the person is outwardly healthy.

The involvement of the matrisome in Alzheimer’s is a new finding, TCW noted. While it has been extensively studied in cancer and acute injury models, the matrisome has not drawn much attention in neurodegenerative disease.

In future work, TCW wants to determine how all these glial expression changes interact, and which come first. She believes the buildup of lipids in glia may trigger both inflammatory response and matrisome upregulation. To test this idea, she will stress cultured glia with cholesterol, cytokines, or lipid debris, and see what changes downstream.

She noted that in the brain, failure to export lipids from astrocytes could starve neurons and oligodendrocytes of the raw materials needed for proper myelin maintenance (Camargo et al., 2017). If ApoE4 carriers have thinner myelin sheaths, that might leave them more vulnerable to neurodegeneration late in life, TCW speculated. Intriguingly, some evidence suggests that women who carry an ApoE4 allele lose more myelin at menopause than do ApoE3 carriers (Aug 2018 conference news).

If lipid dysregulation is the root of the problem, would restoring normal lipid metabolism help ward off Alzheimer’s? Direct activators of LXR/RXR, such as the cancer drug bexarotene, restore cholesterol homeostasis and have been tested for atherosclerosis, but their liver toxicity makes them problematic for chronic use (for review, see Fessler, 2018). Pharmaceutical companies are developing indirect modulators of LXR/RXR that appear safer (Fan et al., 2018). In collaboration with Eli Lilly and Company, TCW is testing whether indirect modulators improve lipid regulation in cultured glia.

Bexarotene lowers amyloid plaques in mouse models, but a small Phase 2 trial gave conflicting results, reporting a slight benefit only in people who did not carry the ApoE4 allele (Feb 2012 news; Feb 2016 news). No follow-up bexarotene trial has been announced. A trial of bexarotene kinetics in cerebrospinal fluid found that very little of the drug enters the central nervous system. With low nanomolar exposure in CSF, bexarotene boosted ApoE CSF levels by one-quarter, but did not alter Aβ peptides (Ghosal et al., 2016). TCW noted that her findings suggest bexarotene and other LXR/RXR activators could be given at an early disease stage before fibrillar amyloid has formed. The goal would be to restore normal glial lipid metabolism rather than solubilizing plaque.

Meanwhile, Liddelow was particularly intrigued by potential interactions between astrocytes and microglia. Microglia need exogenous cholesterol to survive, and they get most of it from astrocytes (Jun 2017 news). Without it, microglia enter an inflammatory state, Liddelow said. Because ApoE4 astrocytes poorly export cholesterol, they may contribute to microglial inflammation, he suggested. “It’s exciting, because this is a description of astrocyte dysfunction that could be driving microglial dysfunction,” Liddelow told Alzforum.

Importantly, all findings reported in this manuscript were specific to human cells. The authors purified astrocytes and microglia from transgenic mice carrying human ApoE3 and ApoE4, and analyzed their gene expression. In stark contrast to the human iPSC-derived glia, E4 and E3 mouse glia had similar lipid metabolism. However, E4 mouse cells did recapitulate the spike in ECM and inflammatory pathways seen in human cultures and brain samples. This highlights the importance of using human iPSC models to study human disease, TCW noted.—Madolyn Bowman Rogers

Comments

  1. This is an exciting study demonstrating that APOE4 is associated with dysregulation of cholesterol homeostasis in human but not mouse astrocytes and microglia. Although such an association was implicated in previous studies, including those addressing APOE genotype effects in peripheral cells such as macrophages, this unbiased work demonstrates that in CNS-relevant glial cells, cholesterol metabolism likely represents a major APOE4-related pathobiology in Alzheimer’s and aging brains.

    It is also interesting that the defective cholesterol metabolism is unique to human cells. However, this observation should be interpreted with caution. In particular, it is known that primary cultured microglial cells behave very differently from when present in vivo. Other factors that could also impact the results, such as aging and sex, are difficult to be modeled and factored into consideration using cell culture systems.

    Future studies using, for example, single cell/single nucleus RNA sequencing in animal and human brains across different APOE genotype, age and sex with or without Alzheimer-related pathologies, might offer more definitive answers in terms of relevance to humans and in-vivo environments.

  2. This is an interesting and comprehensive whole-transcriptome study of APOE4 and Alzheimer’s disease (AD), using human iPSC-derived brain cells, mouse brain cells, and cell-type deconvoluted transcriptomic data from postmortem AD brains. An important finding is that APOE4 is associated with dysregulation of cholesterol homeostasis in human but not mouse astrocytes and microglia, suggesting a species-dependent effect of APOE4. In contrast, elevated matrisome signaling associated with chemotaxis, glial activation, and lipid biosynthesis is observed in APOE4 mixed human neuron/astrocyte culture, mouse APOE4 glial culture, and cell-type deconvoluted transcriptomic data of APOE4 glia from AD brains, suggesting that the effect of APOE4 on matrisome signaling is not species dependent.

    The human-specific transcriptional effect of APOE4 stresses the importance of studying APOE4 genotype-dependent effects in human model systems. In this regard, we have shown that APOE4 is associated with increased A production in human iPSC-derived neurons, but not in mouse neurons (Wang et al., 2018), again suggesting a species-dependent effect of APOE4. With increasing evidence of species differences in various aspects of AD modeling, it seems important to test drug candidates using human iPSC-derived brain cells before moving them into clinical trials in the future.

    One important question raised by this study is whether the human-specific effect of APOE4 on cholesterol homeostasis is a loss-of-function or a gain-of-detrimental-function in glial cells. It would be worth experimentally dissecting these opposite possibilities, since answering this question is crucial for potential therapeutic development based on this study.

    References:

    . Gain of toxic apolipoprotein E4 effects in human iPSC-derived neurons is ameliorated by a small-molecule structure corrector. Nat Med. 2018 May;24(5):647-657. Epub 2018 Apr 9 PubMed.

  3. This great and very extensive study by Julia TCW et al. demonstrates that the involvement of APOE4 in AD pathology likely falls back to the key role of ApoE in lipid metabolism. Additionally, they discovered a novel gene set/pathway called “matrisome” in APOE4 and AD cases. These findings are of tremendous importance to the field, since they make us researchers think about why APOE isoforms contribute to coronary artery disease, myocardial infarction, and AD in the same isoform-specific stepwise pattern: APOE4>APOE3>APOE2. Whereas this work demonstrates potential drawbacks of using animal models for complex human diseases, it suggests that hiPSC-derived brain cell cultures provide a translatable in vitro model to study APOE4-dysregulated pathways in AD.

    Please also check out the preLight article I wrote for this preprint: Cholesterol and matrisome pathways dysregulated in human APOE ε4 glia 

    I am excited to see it in the Sneak Peek with some additional data!

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References

News Citations

  1. ApoE and Tau: Unholy Alliance Spawns Neurodegeneration
  2. ApoE Has Hand in Alzheimer’s Beyond Aβ, Beyond the Brain
  3. Could Greasing the Wheels of Lipid Processing Treat Alzheimer’s?
  4. Do Brain Changes at Menopause Make Women More Prone to Alzheimer’s?
  5. Upping Brain ApoE, Drug Treats Alzheimer's Mice
  6. Bexarotene—First Clinical Results Highlight Contradictions
  7. What Makes a Microglia? Tales from the Transcriptome

Therapeutics Citations

  1. Bexarotene

Paper Citations

  1. . Oligodendroglial myelination requires astrocyte-derived lipids. PLoS Biol. 2017 May;15(5):e1002605. Epub 2017 May 26 PubMed.
  2. . The challenges and promise of targeting the Liver X Receptors for treatment of inflammatory disease. Pharmacol Ther. 2018 Jan;181:1-12. Epub 2017 Jul 16 PubMed.
  3. . Small molecule inducers of ABCA1 and apoE that act through indirect activation of the LXR pathway. J Lipid Res. 2018 May;59(5):830-842. Epub 2018 Mar 21 PubMed.
  4. . A randomized controlled study to evaluate the effect of bexarotene on amyloid-β and apolipoprotein E metabolism in healthy subjects. Alzheimers Dement (N Y). 2016 Jun;2(2):110-120. Epub 2016 Jun 17 PubMed.

External Citations

  1. Sneak Peek

Further Reading

Primary Papers

  1. . Cholesterol and matrisome pathways dysregulated in human APOE ε4 glia. bioRχiv, July 25, 2019