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

线粒体和溶酶体对于调节神经元代谢和功能以及功能障碍都是至关重要的 这两种细胞器都与多种神经退行性疾病有关，包括帕金森病和 腓骨肌萎缩症（CMT）然而，这两种细胞器在调节 神经元稳态和驱动神经变性仍然没有很好地理解。细胞器间膜 接触形成于两个不同的细胞器之间并且是介导细胞器动力学的关键位点， 代谢物交换和信号传导，但线粒体和溶酶体是否形成类似的膜接触 调节其功能性串扰的位点以前是未知的。我最近发现了这个构造 调节细胞膜-溶酶体膜接触位点，这代表了一种新的途径， 线粒体和溶酶体的双向调节，但这些接触位点在神经元中的作用还没有 尚未被探索。重要的是，进一步阐明了神经元的作用，溶酶体接触 提供了重要的洞察耦合线粒体和溶酶体功能的神经元和潜在的 它们在多种神经退行性疾病中的耦合功能障碍的途径。在这个项目中，我建议 研究健康人和哺乳动物中溶酶体接触功能的分子机制， 在K99和R 00期使用人诱导多能神经元的长期培养物 干细胞（iPSC）衍生的神经元生长在微图案化的基底上，以促进细胞器成像， 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术。在目标1中，我将研究溶酶体如何接触 通过检查1）线粒体之间的双向关系来调节神经元的健康和稳态 轴突中的运输和突触-溶酶体接触，以及2）接触在调节钙和 脂质动力学以及神经元中线粒体和溶酶体之间的交换。此外，由于溶酶体Rab 7 由线粒体GAP驱动的GTP从GTP结合态水解为GDP结合态（GTP酶激活 蛋白）调节细胞-溶酶体接触动力学，Rab 7的破坏可能有助于 神经退行性变通过错误调节接触动力学和下游溶酶体和线粒体功能。 在目的2中，我将研究Rab 7介导的细胞-溶酶体接触失调在细胞凋亡中的作用。 两种与线粒体和溶酶体在遗传和功能上相关的疾病的神经变性 功能障碍：1）帕金森病，其中各种家族基因破坏Rab 7 GTP状态，和2）CMT， Rab 7的常染色体显性突变导致CMT 2B型。同时，建议的研究和职业生涯 该计划提供了重要的实验技术和疾病建模方面的新培训，这对我的研究至关重要。 过渡到独立，并最终实现我的长期目标，揭示细胞机制 在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.