Alzheimer’s disease (AD) affects people of both sexes but is more prevalent in women. This may be explained in part by women’s longer lifespans, but sex-based differences in genes, hormones, and other biological processes are also likely contributors. Notably, emerging evidence shows that male and female immune systems respond differently to the same insults and pathologies. Given the key role of brain immune cells, such as microglia, and inflammation in the onset and progression of AD, identifying the signaling molecules involved in these responses in both sexes may clarify why the disease affects men and women differently. These insights may also reveal novel therapeutic targets tailored by sex.
Dr. Gan and Dr. Sinha are investigating a sex-specific immune signaling pathway that plays an important role in demyelination. Demyelination occurs when the fatty protective sheath (myelin) that wraps around neuronal axons is lost or damaged. This myelin acts like insulation on electrical wires—it allows neurons to transmit signals quickly and efficiently. Demyelination is a common feature of aging and multiple neurodegenerative conditions, including AD. Loss of this protective coating contributes to motor deficits and cognitive decline. However, the cells and molecular pathways that drive myelin damage and clearance across various diseases remain largely unknown, and recent research suggests these mechanisms differ between males and females.
Dr. Gan’s team found that in mice, demyelination induced by the chemical CPZ caused microglia in female brains to shift into an activated state. Levels of TLR7, an immune-related gene expressed in microglia and located on the X chromosome, were decreased in female— but not male— mice. To determine the consequence of this change, the team tested CPZ in mice lacking TLR7. Strikingly, these mice were protected against myelin loss and motor deficits. Activating TLR7 increased pro-inflammatory signaling in male microglia. Together, these findings strongly support the hypothesis that inhibiting TLR7 in microglia will protect against demyelination in both sexes, with potentially stronger benefits in males. Their long-term goal is to develop compounds to block TLR7 as treatments for AD.
The Gan and Sinha labs are pursuing this hypothesis across two aims. In the first aim, the team is examining how TLR7 in human microglia affects other brain cell types during demyelination and in the presence of tau pathology. They are using a novel mouse model in which the mouse’s microglia are replaced with human microglia derived from cultured cells (replaced with stem cells). To assess sex-related differences, they are transplanting male human microglia, with and without TLR7, into mice of both sexes and treating them with CPZ to induce demyelination. Motor behavior, myelin integrity, and gene expression changes will be measured across all brain cell types using single-cell transcriptomics. To determine the relevance of this pathway in AD, these experiments will be repeated in a tau mouse model (PS19). In these studies, the impact of TLR7 on multiple tau-related pathologies, including demyelination and the spread of tau aggregates, will be measured. In the second aim, the team is focusing on identifying and developing compounds that cross the blood-brain barrier and block TLR7. They are using computer modeling to predict which compound will have the greatest chance of blocking TLR7, then creating the most promising ones in the lab and testing them in human cell cultures. They are also evaluating the leading compounds in several cell-based models to assess their effects on demyelination and immune responses. Finally, they are testing whether each promising compound can survive in the body long enough to work, reach the brain from the bloodstream, and remain active at the right concentrations.
At the end of the first year of funding, the team has made great progress. They successfully generated humanized microglial chimera models using male human microglia with or without TLR7 and engrafted them into male and female mice. This system allowed them to begin defining how TLR7 and biological sex shape microglial responses during demyelination. They also initiated CPZ-induced demyelination studies, completed single-nucleus RNA sequencing across all groups, and built the analytical framework to map TLR7-dependent and sex-dependent cellular changes. In parallel, they advanced their therapeutic pipeline by developing new computer models, establishing functional screening assays, and synthesizing several selective, brain-permeable TLR7 inhibitor candidates. In the coming year, the team is analyzing the full CPZ dataset to pinpoint how TLR7 loss alters microglia and oligodendrocyte signaling and myelin repair in males and females. They are expanding this work into a tauopathy model by generating human-microglia chimeras in tau mice to test how TLR7 influences tau aggregation and tau-related demyelination. At the same time, they are refining and screening next-generation TLR7 inhibitors and evaluating promising compounds in human cell-based models, alongside ongoing pharmacokinetic studies to identify the most viable drug candidates.
