How Alzheimer’s Risk Gene APOE4 Disrupts Myelination

One of the main ways that the APOE4 gene variant—the biggest genetic risk factor for late onset Alzheimer’s disease—undermines brain circuit function is by disrupting myelination, the process of insulating neurons with a fatty sheath that optimizes their electrical transmission. Studies headed by neuroscientists at The Picower Institute for Learning and Memory at MIT have now provided several lines of evidence to implicate a specific molecular pathway in that disruption.

Headed by Picower Professor Li-Huei Tsai, PhD, and Leyla Akay, PhD, a former graduate student in Tsai’s lab, the researchers showed that APOE4 leads to a suppression of Wnt signaling in the oligodendrocyte cells that provide neurons with fatty myelin insulation. The results indicated that when Wnt signaling is deficient the cells accumulate droplets of lipids rather than deploying fats to properly myelinate the neurons’ axon branches.

The team discovered a key role for Wnt using multiple methods, including a screen in which they used CRISPR gene editing to systematically knock out each protein-coding gene in the genome, one by one, in cultures of human APOE4-carrying oligodendrocytes. The screen showed that when genes promoting Wnt activity in oligodendrocytes were lacking, lipid droplets accumulated, and that when genes suppressing Wnt were lacking, lipid droplets were, too.

Experiments in both human cell cultures and in mice modeling Alzheimer’s disease showed that when Wnt signaling in APOE4-carrying oligodendrocytes was restored, myelination rebounded. “This proof of principle of doing CRISPR screens for genetic modifiers of APOE4-associated lipid accumulation in a central nervous system cell type is very exciting,” said Tsai, who also directs MIT’s Aging Brain Initiative. “I think this type of screen not only provides new insights into cellular pathways contributing to lipid metabolism defects, but really also has the potential to provide new targets for drug development.”

Tsai is co-senior author, and Akay is first author of the researchers’ published paper in Cell Stem Cell, titled “Genome-wide CRISPR screen reveals Wnt signaling as a potential therapeutic target for lipid accumulation and impaired myelination in APOE4 oligodendrocytes.”

In 2022, Tsai’s Lab led by Akay, together with co-author Joel Blanchard, PhD, and colleagues, showed that APOE4 caused lipid droplet accumulation in oligodendrocytes and upset myelination. But, Akay said, that droplet accumulation could happen for many reasons. In their newly reported study, which sought to dig deeper into the underlying molecular mechanisms, the team noted, “We previously found that APOE4 expression causes lipid droplet accumulation in oligodendrocytes, reducing myelination, but the underlying mechanisms were unknown.”

For the study now reported in Cell Stem Cell, the team performed a genome-wide CRISPR knockout screen in induced pluripotent stem cell (iPSC)-derived oligodendrocytes. “We used a well-characterized human iPSC line, homozygous for the APOE4 allele, to generate oligodendrocytes, as previously described,” they explained. “This CRISPR screen allowed us an unbiased way to test every single possibility,” Akay said. “What kind of lipid enzymes are involved in this process? And what are upstream pathways that could be targeted?”

In parallel with the CRISPR screen, the team also analyzed the chromatin accessibility of the DNA in human oligodendrocyte cell cultures and in postmortem human brain samples, in each case comparing APOE4-carrying samples to ones carrying the neutral APOE3 variant. “To further understand how APOE4 perturbs the epigenome of oligodendrocytes and to identify putative master regulators of APOE4’s effects on lipid accumulation, we next used assay for transposase-accessible chromatin using sequencing (ATAC-seq) to compare  transposase-accessible regions of the genome between iPSC-derived oligodendrocytes, isogenic except for APOE genotype,” they continued.

This analysis of ATAC-seq data allowed the investigators to assess which genes were most accessible for expression, versus which ones were locked down from transcription. The chromatin analysis showed that genes promoting Wnt signaling were less accessible in APOE4 carriers. “There was a striking overlap between ATAC-seq and CRISPR screen results,” Tsai said. “Both pointed to a dysregulation of the Wnt signaling pathway.”

Further analysis, including direct observations in cells at the lab bench, revealed how Wnt signaling became depressed in cultured APOE4 oligodendrocytes. Two proteins, beta-catenin and GSK3b, each compete to regulate Wnt. Beta-catenin promotes Wnt and GSK3b suppresses it. The team found that in APOE4 oligodendrocytes compared to APOE3 oligodendrocytes, there was reduced beta-catenin expression and excessive GSK3b expression.

The team then tested interventions that manipulate GSK3b activity. In human APOE3 oligodendrocyte cultures they showed that increasing GSK3b with a chemical called Dif3 increased lipid droplets. In APOE4 oligodendrocytes, they showed that inhibiting GSK3b with a chemical called CHIR99021 reduced droplets. In more complex “organoid” cultures combining APOE4 oligodendrocytes with neurons, they found that inhibiting GSK3b increased myelination and promoted the growth of neural axons.

The tests suggested that GSK3b inhibition could be a proof-of-concept therapeutic strategy for restoring Wnt signaling and myelination. As a final test, they treated mice carrying APOE4 and genetically modeling Alzheimer’s tau pathology with doses of CHIR99021. The treated mice exhibited a reduction in lipid droplets in the hippocampus of the brain and showed increased myelination, when compared to untreated controls.

“Our results suggest that inhibition of GSK3B could reduce lipid droplet burden in APOE4 oligodendrocytes through increasing mitochondrial us age of stored lipids, or potentially by upregulating lipophagic mechanisms,” the team wrote in summary.

In addition to highlighting suppression of Wnt signaling, the CRISPR screen and chromatin analysis provided key clues about the molecular mechanisms by which loss of Wnt alters how oligodendrocytes metabolize lipids. Akay and Tsai said they are eager to follow up on those clues. They are also interested in investigating GSK3b’s linkage to tau phosphorylation, a major feature of Alzheimer’s pathology. “Given GSK3B’s established role as a major tau phosphorylating kinase, our work draws an interesting potential parallel between oligodendrocyte lipid accumulation and neuronal tau pathology,” the authors noted in their report.

Raw data from the study is available on the Open Science Framework. Akay and Tsai have applied for a patent on the CRISPR screen results.

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