Molecular mechanisms underlying mitochondria-lysosome membrane contact sites in neuronal function and neurodegeneration

线粒体和溶酶体都是调节神经元新陈代谢和功能以及功能障碍的关键。 都与多种神经退行性疾病有关，包括帕金森氏症和 Charcot-Marie-Tooth(CMT)病。然而，这两个细胞器之间的相互作用在调节 神经元的动态平衡和驱动神经退行性变仍然没有被很好地理解。细胞器间膜 接触形成在两个不同的细胞器之间，是调节细胞器动力学的关键部位， 代谢物交换和信号传递，但线粒体和溶酶体是否形成类似的膜接触 此前，人们还不知道有哪些网站可以调节它们的功能性串音。我最近确定了它的队形 线粒体-溶酶体膜接触部位的调节代表了一条新的途径 线粒体和溶酶体的双向调节，但这些接触部位在神经元中的作用还没有 还没有被探索过。重要的是，进一步阐明线粒体-溶酶体接触的神经元作用 对神经元中线粒体和溶酶体的耦合功能和电位提供了重要的见解 在多种神经退行性疾病中其偶联功能障碍的途径。在这个项目中，我建议 探讨正常人和正常人线粒体-溶酶体接触功能的分子机制 长期培养人诱导多能细胞在K99和R00期的病变神经元 干细胞(IPSC)来源的神经元生长在微图案化的衬底上，通过 先进的显微技术，包括超分辨率成像、电子显微镜和高空间和 时间分辨率活细胞显微镜。在目标1中，我将研究线粒体-溶酶体是如何联系的 通过检查1)线粒体之间的双向关系调节神经元的健康和动态平衡 轴突内的运输和线粒体-溶酶体接触，以及2)接触在调节钙和 神经元中线粒体和溶酶体之间的脂质动力学和交换。此外，作为溶酶体Rab7 线粒体间隙(GTP酶激活)驱动GTP从GTP结合态到GDP结合态的GTP水解 蛋白)调节线粒体-溶酶体接触动力学，破坏Rab7可能有助于 通过错误调节接触动力学和下游溶酶体和线粒体功能而导致的神经退化。 在目标2中，我将研究Rab7介导的线粒体-溶酶体接触错误调控在 与线粒体和溶酶体基因和功能相关的两种疾病的神经变性 功能障碍：1)各种家族基因扰乱Rab7GTP状态的帕金森病，以及2)CMT AS Rab7常染色体显性突变导致CMT 2B型。总之，拟议的研究和职业生涯 Plans提供了重要的实验技术和疾病建模方面的新培训，这对我的 过渡到独立，并最终实现我揭开细胞机制的长期目标 细胞器间接触和神经变性的交叉点的潜在疾病发病机制。

Both mitochondria and lysosomes are critical for regulating neuronal metabolism and function, and dysfunction of both organelles has been implicated in multiple neurodegenerative diseases including Parkinson’s and Charcot-Marie-Tooth (CMT) disease. However, the interplay between these two organelles in regulating neuronal homeostasis and driving neurodegeneration are still not well understood. Inter-organelle membrane contacts form between two different organelles and are critical sites for mediating organelle dynamics, metabolite exchange and signaling, but whether mitochondria and lysosomes form similar membrane contact sites to regulate their functional crosstalk was previously unknown. I recently identified the formation and regulation of mitochondria-lysosome membrane contact sites which represent a new pathway for the bidirectional regulation of mitochondria and lysosomes, but the role of these contact sites in neurons has not yet been explored. Importantly, further elucidating the neuronal role of mitochondria-lysosome contacts provides important insight into coupled mitochondrial and lysosomal function in neurons and potential pathways for their coupled dysfunction in multiple neurodegenerative diseases. In this project, I propose to investigate the molecular mechanisms underlying mitochondria-lysosome contact function in healthy and diseased neurons during both the K99 and R00 phases using long-term cultures of human induced pluripotent stem cell (iPSC)-derived neurons grown on micropatterned substrates to facilitate organelle imaging via advanced microscopy techniques including super-resolution imaging, electron microscopy and high spatial and temporal resolution live cell microscopy. In Aim 1, I will investigate how mitochondria-lysosome contacts regulate neuronal health and homeostasis by examining 1) the bidirectional relationship between mitochondrial trafficking and mitochondria-lysosome contacts in axons, and 2) the role of contacts in regulating calcium and lipid dynamics and exchange between mitochondria and lysosomes in neurons. Moreover, as lysosomal Rab7 GTP hydrolysis from GTP-bound state to GDP-bound state driven by a mitochondrial GAP (GTPase activating protein) regulates mitochondria-lysosome contact dynamics, disruption of Rab7 may contribute to neurodegeneration by misregulating contact dynamics and downstream lysosomal and mitochondrial function. In Aim 2, I will investigate the role of Rab7-mediated mitochondria-lysosome contact misregulation in the neurodegeneration of two diseases genetically and functionally linked to both mitochondrial and lysosomal dysfunction: 1) Parkinson’s disease in which various familial genes disrupt Rab7 GTP state, and 2) CMT as autosomal dominant mutations in Rab7 result in CMT Type 2B. Together, the proposed research and career plan offers important new training in experimental techniques and disease modeling, which are essential for my transition to independence and for ultimately achieving my long-term goal of uncovering cellular mechanisms underlying disease pathogenesis at the intersection of inter-organelle contacts andneurodegeneration.