In the brain, both astrocytes and microglia produce and secrete large amounts of the APOE protein, which can influence not only the cell where it was made but other cells in the vicinity. This complicates determining which cell type mediates the positive or negative effects of APOE variants on Alzheimer’s pathologies. Recent CureAlz-supported studies from the labs of Dr. Bu and Dr. Butovsky have shown that APOE4 prevents microglia from appropriately responding to and clearing amyloid pathology. Dr. Haney also recently identified a unique population of microglia in APOE4 carriers with AD that are overloaded with lipids. Several studies have shown that the accumulation of lipids in microglia can dramatically influence their function. Using human microglial cells in culture, the team demonstrated that APOE4 microglia accumulate more lipids than APOE3 microglia and also secrete factors that drive tau pathology and neuronal death. These results support the conclusion that APOE4 in microglia drives a damaging microglial state. However, we don’t know whether protective APOE variants, like APOE3ch, are acting in and through microglia to reduce amyloid pathology. Dr. Haneyhypothesizes that both damaging and protective effects of APOE variants are mediated by their expression in microglia.
The Haney lab proposed three experimental aims. In the first aim, they are determining whether expressing protective APOE variants in microglia alone will prevent or reduce amyloid pathology. They are using the same method described above for the Holtzman lab experiments, in which mouse microglia are killed and replaced with engineered microglia. The only difference is that the engineered cells will be mouse microglia rather than human. An amyloid mouse model (5xFAD) with either human APOE3 or APOE4 in the other brain cell types (astrocytes, neurons) will be used. They will measure several outcomes, including which genes are turned on or off in microglia (to determine whether microglia are shifting toward a helpful or harmful state), lipid levels in the brain, and other signs of amyloid-related pathology. In the second aim, the team is evaluating the effects of protective APOE variants on human microglia functions in cultured cells by adding toxic forms of amyloid and measuring phagocytosis, lipid levels, and changes in microglial gene expression. In their earlier work, the Haney lab found that APOE4 microglia secreted factors that triggered tau pathology in neurons. Now they will perform similar experiments to assess whether APOE2, APOE3ch, or other protective variant microglia secrete factors that promote survival and reduce pathology. In the third aim, Dr. Haney is identifying novel protective APOE variants. He is using an innovative screen based on CRISPR methods to identify variants that modify lipid accumulation in microglia after amyloid exposure in mice.
During the first year of funding, Dr. Haney’s team completed the preparation and optimization for all of his proposed aims. They have obtained the necessary cell lines and completed breeding of the mouse models. The second year will see the team complete the transplantation experiments, as well as screen for novel APOE variants. Their work thus far has helped inform the Holtzman lab and others in defining the protocols most appropriate for these experiments and in identifying the hurdles to their implementation.
