Microglia, the resident innate immune cells of the brain, are clearly involved in Alzheimer’s disease (AD). These cells typically monitor the brain’s environment and respond as needed to protect and clean the brain. They can switch between several states, including inflammatory or phagocytic (engulfing and clearing amyloid or damaged synaptic connections). The ability to dynamically transition between these states and then return to a resting state is essential for a healthy brain. While the field has made significant progress in understanding the importance of microglia functions in AD pathology, many questions remain about the specific mechanisms that control microglia states and which molecules carry out critical operations such as phagocytosis of amyloid plaques. Identifying these molecules could lead to new drug targets for therapies that promote amyloid clearance without causing excessive neuroinflammation.
Dr. Piao identified a potential lead molecule based on CureAlz RLG member Dr. Li-Huei Tsai’s research. Dr. Tsai and her team recently reported that a microglial gene called ADGRG1 was one of only five genes with higher expression levels in older AD patients who had less brain pathology than expected for their age at autopsy. ADGRG1 is a receptor protein located on the surface of microglia. Dr. Piao suggests that people with increased ADGRG1 expression in microglia are more likely to survive to older ages with only mild AD pathology. The mechanism by which ADGRG1 limits amyloid pathology is not clear. To explore this observation, the Piao lab created amyloid mouse models (5xFAD) lacking ADGRG1 in microglia. Their preliminary results show that deleting ADGRG1 worsens amyloid-related pathologies: mice have more plaques, greater neuron loss, more swollen axons, and worse memory. They also observed notable differences in other microglia genes and signaling pathways, including lower levels of genes involved in phagocytosis and in regulating the actin cytoskeleton, a network of structural filaments closely associated with the cell membrane where ADGRG1 resides. The team also found that microglia’s ability to engulf and clear amyloid is impaired when ADGRG1 is deleted in both mouse brains and human microglia cells in culture. Building on these results, Dr. Piao specifically hypothesized that microglial ADGRG1 limits the severity of AD pathology.
The team proposed two experimental aims to test this hypothesis. In the first aim, they are studying how ADGRG1 is involved in the overall microglia response to amyloid in the brain. They are measuring the levels of all microglia genes using advanced sequencing approaches (snRNA-seq) in control and 5xFAD mice, with and without ADGRG1, at three ages before and after the onset of amyloid deposition. The team is also analyzing these data to identify signaling pathways and sets of genes affected by the loss of ADGRG1 in the context of amyloid pathology. Additionally, they are validating whether their mouse findings translate to human AD by analyzing human data for signaling pathways and genes that correlate with ADGRG1 expression. In the second aim, they are conducting two different experiments to determine how ADGRG1 regulates microglia phagocytosis. First, Dr. Piao hypothesizes that ADGRG1 causes changes to the actin cytoskeleton that could impact phagocytosis. Since phagocytosis can only occur if microglia temporarily change their shape to engulf amyloid, they are treating brain sections from amyloid mice lacking ADGRG1 with a drug (CCB-E) that effectively mobilizes actin, allowing the cell to remodel as needed for engulfment. They expect this drug to increase microglia phagocytosis of amyloid in 5xFAD brains, even when ADGRG1 is deleted. Second, they will collaborate with CureAlz-funded investigator Dr. Martin Kampmann to perform an unbiased CRISPR-based screen to identify genes that modulate ADGRG1-dependent phagocytosis. These experiments will be performed in human microglia-like cells in culture. The top candidates will be validated and pursued as potential drug targets in future studies.
During the first funding period, Dr. Piao’s team made significant progress in identifying the signaling pathways affected by ADGRG1. They demonstrated that ADGRG1 is essential for amyloid plaques to trigger the upregulation of a protein (RhoA) needed for the initiation of phagocytosis. The team also showed that the downstream effects on phagocytosis resulted from changes in the actin cytoskeleton, as they hypothesized. When they knocked out the ADGRG1 gene in microglia, phagocytosis dropped considerably, but treatment with CCB-E restored phagocytic levels. Initial analyses of human data support their findings in mice. In the second year, they will focus on identifying druggable targets within the newly characterized signaling pathway.
This project investigates the role of ADGRG1 in microglia and how it affects the structural flexibility needed for microglia to engulf and clear amyloid plaques. By identifying new pathways that link the structural dynamics of microglia to AD pathogenesis, this project creates new possibilities for therapeutic interventions.
