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Functional Characterization of Alzheimer's Disease APOE4 Variant Shows How it Changes Brain Cells

NEW YORK (GenomeWeb) – The Alzheimer's disease risk variant APOE4 leads to a number of changes within brain cells, including differences in gene expression, according to a new study.

People with the apolipoprotein E4 variant have a higher risk of developing late-onset Alzheimer's disease — the variant is three times more common among people with the condition than the general public. But while APOE4 has clearly been linked to higher levels of the amyloid protein that forms plaques seen in patients' brains, it's been unclear how this increase takes place.

In a new study appearing today in Neuron, a Massachusetts Institute of Technology-led team used an induced pluripotent stem cells, CRISPR-Cas9 gene editing, and transcriptomic analysis to study the effects of APOE4 on various brain cell types by engineering healthy cells with the more common APOE3 to instead express APOE4. They reported that hundreds of genes were differentially expressed in different types of brain cells when APOE4 was expressed instead of APOE3, and that this led to changes in cell behavior — including the dysregulation of cholesterol metabolism — that were linked to the disease phenotype.

"From this gene expression profiling, we can narrow down to certain signaling pathways that are dysregulated by APOE4," senior author Li­Huei Tsai from MIT said in a statement says. "I think that this definitely can reveal potential targets for therapeutic intervention." She and her colleagues also reported that tweaking APOE4 to APOE3 in brain cells via gene editing could reverse many of these disease-related effects.

To examine the effect of the APOE4 variant, Tsai and her colleagues used CRISPR to generate APOE4 iPSCs from parental APOE3 cells, which were obtained from an unaffected individual. Using these APOE4 iPSC and APOE3 iPSC lines, the researchers generated neurons, astrocytes, and microglia-like cells, all with the same genetic background.

When the researchers performed RNA sequencing on their cells, they noted that the transcriptomic signatures of the different cell types they generated clustered with those of their corresponding cell types, validating the cellular identity of the derived cells.

But, they also reported that 857 genes have expression patterns similar to that of APOE, many of which are involved in lipid metabolism or immune response, processes that both have been implicated in Alzheimer's disease.

In neurons, 250 genes were downregulated in cells expressing APOE4 and 190 were upregulated. In particular, they noted that cell proliferation-related transcripts were downregulated by APOE4 in neurons, while differentiation-associated genes were upregulated. Additionally, 1,131 genes were downregulated and 329 upregulated in microglia expressing APOE4.

The behavior of the cell types also varied by APOE status. APOE4-expressing neurons had increased synaptic density and early endosomes, as compared to APOE3-expression neurons, and also secreted higher levels of the amyloid protein.

Meanwhile, APOE4-expressing astrocyteshad reduced levels of APOE protein, but much higher levels of cholesterol than their APOE3 counterparts, suggesting dysregulation of cholesterol metabolism. They also took up and cleared Aβ more slowly. APOE4 microglia-like cells likewise had reduced Aβ uptake and exhibited morphological changes.

The researchers also developed APOE4-expressing brain organoids, through which they found that the APOE4 variant alone was sufficient to cause amyloid protein aggregates, a hallmark of the disease.

In subsequent experiments using iPSC cells generated from an Alzheimer's disease patient, the researchers edited the cells' APOE4 variant using CRISPR to become APOE3. When they then characterized brain cells derived from these edited iPSCs, they noted that much of the disease-related phenotype was reversed.

This suggested to the researchers that not only could targeting certain signaling pathways treat Alzheimer's disease, but that gene editing itself could potentially be harnessed as a treatment mechanism. "If you can convert the gene from E4 to E3, a lot of the Alzheimer's associated characteristics can be diminished," Tsai noted.