7. Project Summary/Abstract The goal of this proposal is to elucidate a role for microglial gasdermin-D in the severity of neurodegeneration in models of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Despite widespread evidence for microglial activation in several neurodegenerative diseases, the precise mechanisms governing these microglial responses are not well understood. Gasdermin-D (GSDMD) is a pore- forming protein expressed in peripheral macrophages that initiates a proinflammatory form of cell death termed pyroptosis. I have evidence that microglia highly express GSDMD in the central nervous system, and that in vitro primary microglia similarly form GSDMD pores when exposed to inflammasome triggers. Using the well- described SOD1G93A model of ALS, I found a significant increase in microglial Gsdmd transcription and activation at the protein level in the spinal cord as paralysis progresses. In a separate model of C9ORF72-associated FTD, I found Gsdmd expression increases in the brain after neuronal overexpression of a dipeptide repeat expansion (DRE) protein. While Gsdmd expression is similarly increased in both models, using a Gsdmd null animal in either disease context results in an opposite effect on animal survival where GSDMD drives protection in the ALS disease model but disease progression in the C9ORF72 model of FTD. Transcriptomic work on microglia across development and in neurodegeneration suggests microglia have differing immune functions based on their resident location in the brain, animal age, and in health versus disease. I aim to further understand the heterogeneity of microglial responses in the CNS in neurodegeneration by addressing the following questions: 1) How does Gsdmd expression drive protection in the SOD1G93A model of ALS (Aim 1) but conversely drive disease progression in a model of C9ORF72 FTD (Aim 2)? 2) How do microglial transcriptional profiles change with loss of Gsdmd expression in the spinal cord and brain in ALS and FTD disease models (Aims 1 and 2)? I hypothesize microglial GSDMD results in opposing effects on survival outcome due to disease context- dependent changes in microglial immune profiles. To test this hypothesis, I plan to use flow cytometry and single cell RNAseq which are powerful approaches to interrogating microglial function and techniques for which I will acquire new scientific skills. I also have developed strong collaborations to model FTD with AAV strategies, as well as a strong mentoring team including my mentor Dr. Isaac Chiu as well as Dr. Judy Lieberman, Dr. Beth Stevens, and Dr. Leonard Petrucelli who are experts in neuroimmunology, gasdermin biology, microglia, and neurodegeneration, respectively. Together, I am in a strong position to interrogate microglial Gsdmd activation in ALS and FTD neurodegeneration. In the process of completing the experiments in this proposal, I will receive training in a variety of immunology and neurobiology approaches that...