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

7.项目摘要/摘要 这项建议的目的是阐明小胶质细胞Gasdermin-D在高血压严重程度中的作用。 肌萎缩侧索硬化症(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激活，但准确的 管理这些小胶质细胞反应的机制还不是很清楚。Gasdermin-D(GSDMD)是一种孔道- 在外周巨噬细胞中表达的形成蛋白，启动一种称为 上睑下垂。我有证据表明，小胶质细胞在中枢神经系统高表达GSDMD，而且在体外 当暴露在炎性小体触发下时，初级小胶质细胞类似地形成GSDMD孔。利用水井- 描述了SOD1G93A ALS模型，我发现小胶质细胞Gsdmd转录和激活显著增加 在脊髓的蛋白质水平上，随着瘫痪的进展。在与C9ORF72相关的FTD的单独模型中， 我发现在神经元过度表达二肽重复扩增后，Gsdmd在大脑中的表达增加。 (DRE)蛋白质。虽然Gsdmd在两个模型中的表达类似地增加，但使用Gsdmd缺失的动物 任何一种疾病背景都会对动物生存产生相反的影响，其中GSDMD推动对 ALS疾病模型，但在FTD的C9ORF72模型中疾病进展。小胶质细胞的转录工作 在发育和神经退行性变过程中，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，动物的年龄，以及在健康与疾病之间的关系。我的目标是进一步了解 神经变性中中枢神经系统小胶质细胞反应的异质性通过回答以下问题： 1)Gsdmd如何在ALS的SOD1G93A模型(目标1)中表达驱动保护，但反过来又如何驱动 C9ORF72 FTD模型中的疾病进展(目标2)？2)小胶质细胞转录谱如何变化 在ALS和FTD疾病模型(目标1和2)中，Gsdmd在脊髓和脑中的表达缺失？我 假设小胶质细胞GSDMD因疾病背景而对生存结局产生相反的影响- 小胶质细胞免疫图谱的依赖性变化。为了验证这一假设，我计划使用流式细胞术和单细胞 细胞RNAseq是询问小胶质细胞功能的强大方法和技术，我将 掌握新的科学技能。我还建立了强大的合作伙伴关系，将FTD与AAV战略相结合，如 以及一个强大的指导团队，包括我的导师Isaac Chiu博士和Judy Lieberman博士，Beth博士 Stevens和Leonard Petrucelli博士，他们是神经免疫学、Gasdermin生物学、小胶质细胞和 神经退行性变。总而言之，我在审问小胶质细胞Gsdmd激活方面处于有利地位 在ALS和FTD神经退行性变中。在完成本提案中的实验的过程中，我将收到 接受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 作为一家学术机构的独立首席调查员，专注于解剖 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.