The role of gasdermins in microglial activation and neurodegeneration in ALS/FTD

7.项目概要/摘要 这项研究的目的是阐明小胶质细胞gasdermin-D在严重的脑梗死中的作用。 在肌萎缩性侧索硬化（ALS）和额颞叶痴呆（FTD）模型中的神经变性。 尽管有广泛的证据表明小胶质细胞在几种神经退行性疾病中活化，但精确的 控制这些小胶质细胞反应的机制尚不清楚。Gasdermin-D（GSDMD）是一种多孔的 在外周巨噬细胞中表达的一种蛋白质，它启动称为 焦亡我有证据表明，小胶质细胞在中枢神经系统中高度表达GSDMD，并且在体外 当暴露于炎性小体触发物时，初级小胶质细胞类似地形成GSDMD孔。利用这口井- 描述了ALS的SOD 1G 93 A模型，我发现小胶质细胞Gsdmd转录和激活显著增加， 在脊髓中的蛋白质水平。在C9 ORF 72相关FTD的单独模型中， 我发现，在神经元过度表达二肽重复序列扩增后， (DRE)蛋白虽然Gsdmd表达在两种模型中类似地增加，但使用Gsdmd缺失动物， 任一种疾病背景对动物存活产生相反的影响，其中GSDMD在动物中驱动保护作用。 ALS疾病模型，但FTD的C9 ORF 72模型中的疾病进展。小胶质细胞的转录组学研究 在发育和神经退行性疾病中，小胶质细胞具有不同的免疫功能， 它们在大脑中的驻留位置，动物年龄，以及健康与疾病的关系。我的目标是进一步了解 通过解决以下问题，研究神经变性中CNS中小胶质细胞反应的异质性： 1)Gsdmd表达式如何在ALS的SOD 1G 93 A模型中驱动保护（目标1），但反过来驱动 C9 ORF 72 FTD模型中的疾病进展（目的2）？2)小胶质细胞转录谱如何改变 ALS和FTD疾病模型中脊髓和脑中Gsdmd表达缺失（目的1和2）？我 假设小胶质细胞GSDMD由于疾病背景而对生存结果产生相反影响- 小胶质细胞免疫谱的依赖性变化。为了验证这一假设，我计划使用流式细胞术和单克隆抗体。 细胞RNAseq是询问小胶质细胞功能的强大方法，我将 获得新的科学技能。我还建立了强有力的合作，以模拟FTD与AAV策略， 以及一个强大的指导团队，包括我的导师Isaac Chiu博士以及Judy Lieberman博士，Beth博士 Stevens和伦纳德·彼得鲁切利博士是神经免疫学、gasdermin生物学、小胶质细胞和 神经退行性变。我们一起来研究小胶质细胞Gsdmd的激活 ALS和FTD神经退行性变在完成本提案中的实验过程中，我将收到 我接受了各种免疫学和神经生物学方法的培训，这将为我的未来奠定基础。 作为一个独立的首席研究员在学术机构的职业生涯，专注于解剖功能， 神经回路中的小胶质细胞

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 will provide a foundation for my future career as an independent principal investigator at an academic institution focused on dissecting functions for microglia within neural circuits.