The E4 allele of the apolipoprotein E, a strong risk factor for many neurodegenerative diseases, most prominently late-onset Alzheimer’s disease, upsets the balance of lipids within cells. Exactly how ApoE4 does this remains unclear. In the March 3 Science Translational Medicine, researchers led by Li-Huei Tsai at MIT found that in human astrocytes and microglia, ApoE4 increased the amount of unsaturated lipids, which then accumulated in lipid droplets. Adding the phospholipid precursor choline to the cells restored their normal lipid metabolism, hinting at potential therapeutic approaches for ApoE4 carriers.

  • How exactly does ApoE4 lead to lipid imbalance in astrocytes?
  • Astrocytes and yeast expressing ApoE4 accumulated unsaturated lipids.
  • Addition of choline returned astrocytes to a homeostatic state.

“This study emphasizes the importance of balanced lipid metabolism in prevention and treatment of AD,” Hilkka Soininen from the University of Eastern Finland, Kuopio, wrote to Alzforum. “Since the discovery of ApoE4 as a risk gene for AD, I have been waiting for innovations to reverse its effect and hope to see that in the future.”

This is not the first time scientists have spotted lipid droplets in glia. In fact, Alois Alzheimer himself described lipid buildup in those cells. More recently, one prior study uncovered similar blobs in mouse microglia that were dubbed lipid droplet-associated microglia. LAMs accumulated in the mouse hippocampus with age, increasing neuroinflammation (Aug 2019 news). 

Although ApoE levels remained unchanged in LAMs, other researchers reported that human astrocytes expressing ApoE4 generate more cholesterol than their ApoE3 counterparts and don’t break it down properly, causing lipids to gather inside the cells, possibly increasing susceptibility to AD. Similarly lipid-laden astrocytes were found in AD brain tissue (Aug 2019 news). 

Less Fat. Human iPSC-derived astrocytes that express ApoE4 (bottom left) accumulate more lipid droplets (dark specks) than those that express ApoE3 (top left). Spiking the growth medium with CDP-choline (right) restored lipid metabolism to normal. [Courtesy of Sienski et al., 2021. Science Translational Medicine.]

However, the question remains: Exactly how does ApoE4 cause lipids to pile up? Tsai and colleagues focused on astrocytes because they are the main source of APOE in the brain (Boyles et al., 1985). Co-first authors Grzegorz Sienski, Priyanka Narayan, Julia Bonner and colleagues analyzed the lipid composition in ApoE3- or ApoE4-expressing isogenic human astrocytes that he had derived from induced pluripotent stem cells. Using liquid chromatography-mass spectrometry, he discovered that ApoE4 astrocytes contained more unsaturated triacylglycerides. By adding a lipophilic dye to the astrocytes, the researchers saw that ApoE4 cells accumulated lipid droplets.

How would ApoE4 alter the lipidome in the human brain? Sienski and colleagues examined gene-expression data from postmortem samples of 838 people with different genotypes, ages, and causes of death who had enrolled in the NIH-funded Genotype-Tissue Expression project. They compared the transcriptomic profiles of ApoE4 carriers with those of noncarriers across a set of 609 lipid metabolism genes pulled from the Kyoto Encyclopedia of Genes and Genomes. Genes involved in the biochemistry of neutral lipids and cholesterol were upregulated in ApoE4 carriers, whereas genes involved in the breakdown of fatty acids and neutral lipids were downregulated, suggesting a disturbance in lipid metabolism.

To further explore how ApoE4 causes lipid defects, Tsai and colleagues collaborated with researchers then at the lab of the late Susan Lindquist at MIT.  They created yeast expressing human ApoE3 or ApoE4. Similar to the human astrocytes, ApoE4 yeast had more lipid droplets and grew poorly compared to ApoE3 yeast. The authors used loss-of-function screens to look for genes that might mediate ApoE4’s effects in yeast. Their hits included OPI1, MGA2, and UBX2, all of which encode proteins that regulate lipid metabolism. Focusing on MGA2 and OPI1, they found that neither knockout altered the amount of ApoE4 protein itself, reaffirming that the growth defect seen in these yeasts stemmed from abnormal lipid metabolism.

OPI1 regulates phospholipid synthesis, hence the authors wondered whether precursors of this pathway could reverse ApoE4-related deficiencies. They tried choline and ethanolamine. Lo and behold, when the yeast grew in a medium with choline, both the growth defect and lipid imbalance disappeared. Similarly, ApoE4 astrocytes cultured in a medium supplemented with cytidine 5'-diphosphocholine (CDP-choline) contained fewer triacylglycerides, fewer unsaturated fatty acids, and fewer lipid droplets than untreated cells.

“Choline treatment ameliorated all of the lipid metabolic disruption that we observed,” Tsai told Alzforum. “It brought the yeast and astrocytes back to a homeostatic state.”

Choline Chem 101. Choline is an essential nutrient in lipid metabolism. It is acetylated to create acetylcholine, and phosphorylated to make phosphatidylcholine, a major component of cell membranes. [Courtesy of Li-Huei Tsai.]

Now, one big question is whether it may be possible to mitigate deleterious effects of ApoE4 in people by adding more choline-rich foods, such as nuts, eggs, and fish, to their diet. Tsai said she is studying ApoE4 knock-in mice that are given either a normal diet or a choline-rich one.

Choline is a component of Souvenaid, a medical food designed by Nutricia of Danone Research to enhance the formation and function of synapses. In the LipiDiDiet clinical trial, people with AD who consumed this yogurt-like drink once a day for two years had slightly less shrinkage of the hippocampus than did controls. The trial failed to meet its primary outcome, but follow-up data suggested that Souvenaid might help people who continue consuming the drink for three years (Apr 2020 conference news). 

The authors also noticed that ApoE4 alters lipid droplets in microglia, and are exploring this finding in animal models. “Microglia show tremendous disruption of lipid metabolism,” said Tsai. How this relates to the lipid droplets in LAMs remains to be seen.—Helen Santoro

Comments

  1. The work by Sienski and colleagues, demonstrating the perturbation of lipid homeostasis by APOE4, is of much interest because it suggests another pathway by which APOE polymorphisms may increase susceptibility to Alzheimer’s disease.

    In particular, the authors showed an increase in unsaturated tricylglycerides and a higher phosphatidylcholine requirement in cultured human astrocytes with the APOE4 genotype, a defect reversed by choline supplementation. The focus on lipids is important since the brain is highly enriched for them, and several lipid changes have been demonstrated in AD brains and AD plasma (Wong et al., 2017). 

    Lipids change with age, and aging is the strongest risk factor for AD. Lipids can also be manipulated by alterations to the diet and gut microbiome, suggesting several therapeutic approaches. The work therefore needs to be taken further. Since APOE2 is known to offer some protection against AD, it would be interesting to study the impact of this genotype on lipids as well. 

    The authors focused on tricylglycerides and phospholipids and did not report other lipid species such as sphingolipids and ceramides, which are also of interest. The reversal with choline supplementation is interesting. There have been several trials of choline in mice models of AD, with reports of a protective effect (Velazquez et al., 2019). Several mechanisms for this have been suggested: an increase in levels of acetylcholine, reduced production of amyloid-β plaques, reduction in homocysteine levels, reduced activation of microglia, and even epigenetic alterations. Sienski et al. now add another potential mechanism to this list.

    However, initial human trials of choline in AD were generally negative (Amenta et al., 2001). More recent multinutrient interventions have been more promising (Baumel et al., 2020), but better understanding of the mechanisms is needed so that appropriate intervention strategies can be developed.

    References:

    . Dysregulation of lipids in Alzheimer's disease and their role as potential biomarkers. Alzheimers Dement. 2017 Jul;13(7):810-827. Epub 2017 Feb 24 PubMed.

    . Maternal choline supplementation ameliorates Alzheimer's disease pathology by reducing brain homocysteine levels across multiple generations. Mol Psychiatry. 2019 Jan 8; PubMed.

    . Treatment of cognitive dysfunction associated with Alzheimer's disease with cholinergic precursors. Ineffective treatments or inappropriate approaches?. Mech Ageing Dev. 2001 Nov;122(16):2025-40. PubMed.

    . Potential Neuroregenerative and Neuroprotective Effects of Uridine/Choline-Enriched Multinutrient Dietary Intervention for Mild Cognitive Impairment: A Narrative Review. Neurol Ther. 2020 Dec 26; PubMed.

  2. This study sheds more light on the role of ApoE in the brain. It is known that ApoE activates a range of growth-factor like receptors and plays a role in lipid metabolism (Liu et al., 2015Zhao et al., 2017). APOE4 appears to be less able to activate these receptors. Therefore, the buildup of lipids in the microglia indicates a problem in processing lipids for energy production. This can explain why APOE4 carriers are more prone to develop AD, as we know that brain energy metabolism is increasingly challenged in AD, and that insulin signalling and glucose utilization is decreased (Talbot et al., 2012; Arnold et al., 2018). In APOE2 carriers, it might be possible to compensate for loss of glucose utilization, while in APOE4 carriers, this is not as effective.

    References:

    . Neuronal LRP1 regulates glucose metabolism and insulin signaling in the brain. J Neurosci. 2015 Apr 8;35(14):5851-9. PubMed.

    . Apolipoprotein E4 Impairs Neuronal Insulin Signaling by Trapping Insulin Receptor in the Endosomes. Neuron. 2017 Sep 27;96(1):115-129.e5. PubMed.

    . Demonstrated brain insulin resistance in Alzheimer's disease patients is associated with IGF-1 resistance, IRS-1 dysregulation, and cognitive decline. J Clin Invest. 2012 Apr;122(4):1316-38. PubMed.

    . Brain insulin resistance in type 2 diabetes and Alzheimer disease: concepts and conundrums. Nat Rev Neurol. 2018 Mar;14(3):168-181. Epub 2018 Jan 29 PubMed.

  3. It would be very helpful if the authors, or other ApoE experts, could weigh in on the mechanism(s) by which ApoE secreted from yeast might interact with cellular processes. Does yeast have any surface receptors capable of binding ApoE? Or do we suppose that the effects are all due to mass-action effects by which ApoE, soluble in the growth medium, influences the availability of free lipids for uptake by other mechanisms?

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References

News Citations

  1. Newly Identified Microglia Contain Lipid Droplets, Harm Brain
  2. ApoE4 Glia Bungle Lipid Processing, Mess with the Matrisome
  3. Non-Aβ, Non-Tau Drugs Tweak Markers, Cognition in Alzheimer’s, Huntington’s

Therapeutics Citations

  1. Souvenaid

Paper Citations

  1. . Apolipoprotein E associated with astrocytic glia of the central nervous system and with nonmyelinating glia of the peripheral nervous system. J Clin Invest. 1985 Oct;76(4):1501-13. PubMed.

External Citations

  1. Genotype-Tissue Expression
  2. Kyoto Encyclopedia of Genes and Genomes

Further Reading

No Available Further Reading

Primary Papers

  1. . APOE4 disrupts intracellular lipid homeostasis in human iPSC-derived glia. Sci Transl Med. 2021 Mar 3;13(583) PubMed.