By developing a laboratory-designed model of the human blood-brain barrier (BBB), neuroscientists at the Picower Institute for Learning and Memory at the Massachusetts Institute of Technology revealed how the most common risk genes for Alzheimer’s disease cause amyloid plaques, thereby disrupting the brain’s vasculature, while also demonstrating that they can prevent these injuries through drugs that have been approved for human use.
About 25% of people carry the APOE4, variant of the APOE gene, which greatly increases their risk of developing Alzheimer’s disease. Almost every Alzheimer’s disease patient, even some non-Alzheimer’s disease patients, has cerebral amyloid angiopathy (CAA). In this case, amyloid deposits on the walls of blood vessels, impairing the ability of the blood-brain barrier to transport nutrients normally, remove waste, and prevent the invasion of pathogens and harmful substances. The researchers determined that the APOE4 gene mutation promotes CAA pathology through specific vascular cell types (pericytes) and molecular pathways (calcineurin/NFAT).
Studies have shown that in people with APOE4 mutants, the pericytes in the blood vessels produce too much APOE protein. APOE causes amyloid to aggregate, and amyloid is more abundant in Alzheimer’s disease. Meanwhile, the increased activation of the calcineurin/NFAT molecular pathway in the diseased pericytes seems to promote the increase in APOE expression.
There are already drugs that can inhibit this pathway. Currently, they are used to suppress the immune system after transplantation. When the researchers used some drugs including cyclosporine A and FK506 for the laboratory-cultivated blood-brain barrier with APOE4 variants, they accumulate much less amyloid protein than they did without treatment.
In order to study the connection between Alzheimer’s disease, APOE4 mutation and CAA, Blanchard, Tsai and co-authors induced human induced pluripotent stem cells, making them three types of cells that constitute the blood-brain barrier: brain endothelial cells, astrocytes and pericytes. Pericytes are simulated by mural cells, and they have extensively tested mural cells to ensure that they exhibit characteristics and gene expression similar to pericytes.
After growing for two weeks in the three-dimensional hydrogel scaffold, the blood-brain barrier model cells assemble into blood vessels, showing the natural characteristics of the blood-brain barrier, including low permeability to molecules, and the same key genes, proteins and molecular pumps as the natural blood-brain barrier. When immersed in a high amyloid culture medium to simulate the brain condition of Alzheimer’s disease, the blood-brain barrier model cultured in the laboratory showed the same accumulation of amyloid as human disease.
After establishing the BBB model, scientists tried to test the differences in APOE4. Through several measurements, they found that the blood-brain barrier model carrying APOE4 accumulated more amyloid from the culture medium than the blood-brain barrier model carrying APOE3, and APOE3 is a more typical and healthy genetic variant. When these models were exposed to amyloid-rich media, only the model of apolipoprotein APOE 4 pericyte-like mural cells showed excessive accumulation of amyloid. Replacing APOE4-carrying mural cells with APOE3 mural cells can reduce amyloid deposition. These results directly attribute the CAA-like pathology to pericytes.
To further verify the clinical significance of these findings, the scientists also observed the expression of APOE in the vascular system samples of the prefrontal cortex and hippocampus in the human brain, which have important effects on Alzheimer’s disease. Consistent with the laboratory blood-brain barrier model, in the vascular system, especially pericytes, people carrying APOE4 showed higher levels of gene expression than people carrying APOE3.
The next step is to determine how APOE4 is overexpressed by pericytes. Therefore, the researchers identified hundreds of transcription factors—proteins that determine how genes are expressed—that are regulated differently in APOE3 and APOE4-pericyte-like mural cells. Then they browsed the list to see which factors affected APOE’s expression. A group of factors that are up-regulated in APOE4 cells stand out, which are part of the calcineurin/NFAT pathway.
As part of an investigation into whether increased signaling activity of this pathway resulted in amyloid deposition and increased CAA, they tested cyclosporin A and FK506 because they reduced the activity of this pathway. They found that the drug reduced the expression of APOE in pericyte-like mural cells, and therefore reduced APOE4-mediated amyloid deposition in the blood-brain barrier model. They also tested the drug on mice carrying APOE4 and found that the drug reduced APOE expression and amyloid accumulation.
The researchers also clearly pointed out that these drugs may have significant side effects, so their results may not recommend the use of these drugs to treat patients with CAA. It points to the value of understanding its mechanism to find more effective drugs and less off-target effects.