Black gold staining in postmortem human brain samples from three individuals reveals significantly less myelination in people carrying a copy of the APOE4 Alzheimer's disease risk gene. [Tsai Laboratory/MIT Picower Institute]

APOE4 is the strongest genetic risk factor for Alzheimer’s disease. Carrying one copy of the APOE4 gene variant increases one’s risk for Alzheimer’s disease threefold (and two copies about tenfold.) However, the biological mechanisms underlying the reasons for the increased risk are unclear.

Now, a new study used single-cell transcriptomics to profile post-mortem human brains from APOE4 carriers compared with noncarriers. The findings provide comprehensive insights into the impact of APOE4 on the human brain. The research provides a single-cell atlas describing the transcriptional effects of APOE4 on the aging human brain. In doing so, the researchers have established a functional link between APOE4, cholesterol, myelination, and memory, offering therapeutic opportunities for Alzheimer’s disease.

The study is published in Nature, in the article, “APOE4 impairs myelination via cholesterol dysregulation in oligodendrocytes.

“This paper shows very clearly from the snRNAseq of postmortem human brains in a genotype-specific manner that APOE4 influences different brain cell types very distinctly,” said Li-Huei Tsai, professor at the Picower and a member of MIT’s Brain and Cognitive Sciences faculty. “We see convergence of lipid metabolism being disrupted, but when you really look into further detail at the kind of lipid pathways being disturbed in different brain cell types, they are all different.”

“I feel that lipid dysregulation could be this very fundamental biology underlying a lot of the pathology we observe,” she continued.

The study combines evidence from histological and lipidomic analysis of the post-mortem human brain, induced pluripotent stem-cell-derived cells, and targeted-replacement mice, and showed that when people have one or two copies of APOE4 (rather than the more common and risk-neutral APOE3 version) oligodendrocytes mismanage cholesterol, failing to transport it to wrap axons. More specifically, they showed that cholesterol is aberrantly deposited in oligodendrocytes—myelinating cells that are responsible for insulating and promoting the electrical activity of neurons.

Deficiency of myelin may be a significant contributor to the pathology and symptoms of Alzheimer’s disease because, without proper myelination, neuronal communication is degraded.

Not only does the new study suggest how APOE4 disrupts myelination, it also provides the first systematic analysis across major brain cell types using single nucleus RNA sequencing (snRNAseq) to compare how gene expression differs in people with APOE4 compared to APOE3.

The team’s snRNAseq results, a dataset that Djuna Von Maydell, a PhD student in the Tsai lab has made freely available, encompasses more than 160,000 individual cells of 11 different types from the prefrontal cortex of 32 people—12 with two APOE3 copies, 12 with one copy of each APOE3 and APOE4, and eight with two APOE4 copies.

The APOE3/3 and APOE3/4 samples were balanced by Alzheimer’s diagnosis, gender, and age. All APOE4/4 carriers had Alzheimer’s and 5 of 8 were female.

Some results reflected known Alzheimer’s pathology, but other patterns were novel. One, in particular, showed that APOE4-carrying oligodendrocytes exhibited greater expression of cholesterol synthesis genes and disruptions to cholesterol transport. The more APOE4 copies people had, the greater the effect. This was especially interesting given the results from a prior analysis by the labs of Tsai and Manolis Kellis, PhD, professor of computer science at MIT, in 2019 that linked Alzheimer’s disease to reduced expression of myelination genes among oligodendrocytes.

Using a variety of techniques to look directly at the tissue, the team saw that in APOE4 brains, aberrant amounts of cholesterol accumulated within cell bodies, especially of oligodendrocytes, but was relatively lacking around neural axons.

To understand why, the team used patient-derived induced pluripotent stem cells to create lab cell cultures of oligodendrocytes engineered to differ only in APOE4 or APOE3. Again, APOE4 cells showed major lipid disruptions. In particular, the afflicted oligodendrocytes hoarded extra cholesterol within their bodies, showed signs that the extra internal fats were stressing the endoplasmic reticulum, and indeed transported less cholesterol out to their membranes. Later when they were co-cultured with neurons, the APOE4 oligodendrocytes failed to myelinate the neurons as well as APO3 cells did, regardless of whether the neurons carried APOE4 or APOE3.

The team also observed that in post-mortem brains there was less myelination in APOE4 carriers than in APOE3 carriers. For instance, the sheaths around axons running through the corpus callosum (the structure that connects brain hemispheres) were notably thinner in APOE4 brains. The same was true in mice engineered to harbor human APOE4 versus those engineered to have APOE3.

The team focused on drugs that affect cholesterol including statins (which suppress synthesis) and cyclodextrin, which aids cholesterol transport. The statins did not have an effect, but applying cyclodextrin to APOE4 oligodendrocyte cultured in a dish reduced accumulation of cholesterol within the cells and improved myelination in co-cultures with neurons. It showed similar effects in APOE4 mice.

Finally, the team treated some APOE4 mice with cyclodextrin, left others untreated, and subjected them all to two different memory tests.  The cyclodextrin-treated mice performed both tests significantly better, suggesting an association between improved myelination and improved cognition.

Tsai said a clear picture is emerging in which intervening to correct specific lipid dysregulations by cell type could potentially help counteract APOE4’s contributions to Alzheimer’s pathology.

“It’s encouraging that we’ve seen a way to rescue oligodendrocyte function and myelination in lab and mouse models,” Tsai said. “But in addition to oligodendrocytes, we may also need to find clinically effective ways to take care of microglia, astrocytes, and vasculature to really combat the disease.”

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