The search for more effective AD therapies will likely involve targeting tau, given tau’s strong associations with neurodegeneration and loss of cognitive function. Pioneering research from Dr. Holtzman’s lab and others demonstrated that APOE plays a critical role in activating microglia to influence tau pathology and neurodegeneration, which suggests that APOE-targeting therapies could protect against tau-related pathologies. Further support for this idea comes from the unique case report of a woman with an Alzheimer’s-causing mutation that remained cognitively healthy decades beyond when symptoms should have appeared. Her brain autopsy showed that despite having high levels of amyloid in her brain, she had very little tau pathology. Researchers discovered that, in addition to the familial Alzheimer’s mutation, she had two copies of a rare APOE3 variant called APOE3 Christchurch (APOE3ch).
With CureAlz support, the Holtzman lab developed an APOE3ch mouse model to determine whether this variant was the key to her protection and, if so, how. The lab discovered that APOE3ch protects against amyloid-induced tau pathology, and that these protective effects are related to APOE3ch’s influence on microglia. These discoveries prompted Dr. Holtzman to hypothesize that protective APOE variants act specifically within microglia to protect against tau pathologies.
The Holtzman lab proposed three aims to test this hypothesis. In the first aim, they are testing whether microglia expressing protective APOE variants reduce the spread of pathological tau in the brains of mice with amyloid. They are studying APOE3ch and two other protective variants (APOE2 and APOE4-R251G). There is also evidence suggesting that deleting the APOE gene completely is protective against AD pathologies, so they will also knock out APOE from microglia. These experiments use an innovative approach in which all of a mouse’s normal microglia are killed and replaced with microglia grown in culture. The power of this approach is that the team can engineer cells to express protective, neutral, or risk APOE variants, or to delete APOE entirely. They are transplanting the engineered microglia into the brains of amyloid mice with APOE4 in all other resident cell types. The presence of APOE4 everywhere else should result in negative outcomes, unless Dr. Holtzman is correct, and the APOE in the microglia has the most impact. Any measurable improvement in these mice can be attributed to the addition of these microglia and their APOE variant. They will also inject toxic forms of tau into the brain to model aspects of amyloid-induced tau pathology. The lab will stain and image the brain tissue to reveal amyloid plaques and tau tangles, then compare the amount of damage across mice carrying different APOE variants. In the second aim, they are performing similar experiments in a tau mouse model (PS19; APOE4). This model develops severe atrophy, tangles, and inflammation. The team expects that mice transplanted with microglia expressing protective APOE variants will be protected against tau-related pathologies. In the third aim, they are exploring how microglial APOE impacts tau pathology. They predict that the different variants impact phagocytosis, an essential function of microglia, with APOE4 cells degrading the least amount of toxic tau, while APOE2 and APOE3ch will be the most efficient. To test this, they are measuring tau uptake and degradation by microglia in culture across the different APOE variants. Together, these results should provide insights into whether and how protective APOE variants act within microglia to protect the rest of the brain from amyloid and tau pathologies in AD.
In the first year of funding, the Holtzman team made progress in optimizing protocols, particularly for microglial transplantation into mouse models. This is a critical step that positions the team to perform the related experiments in the second year. In their cell cultures, the team observed a significant increase in tau phagocytosis by microglia lacking APOE, and by microglia with APOE2 and APOE3ch variants. However, these results were not seen when these cells expressed a mutated receptor important for the transplantation protocols. This suggests that this mutation may, on its own, sufficiently affect cell function to mask the impact of the APOE variant in microglia. The lab is studying whether this is also the case in cells extracted from mice or in the mice’s brains. If so, it will influence their approach to transplantations and provide a critical experimental finding for other labs seeking to perform similar experiments.
