The Mucolipin TRP Ion Channels

 描述(申请人提供)：为了调节由内吞作用和自噬传递的大分子的降解，溶酶体必须经历广泛的运输，包括沿着微管轨道向携带货物的内噬小体和自噬小体迁移，以及随后的膜融合和分裂过程。溶酶体转运缺陷会导致溶酶体储存病(LSD)。这项拟议研究的长期目标是了解各种细胞信号如何调节自噬和内吞作用中的溶酶体运输。在上一个资助阶段，我们证明了溶酶体腔内钙离子的释放在调节溶酶体转运的多个步骤中起着关键的信号作用。通过直接膜片钳技术和对溶酶体内钙释放的荧光成像，我们证明了TRPML1(瞬时受体潜在粘蛋白1，ML1)是溶酶体内主要的钙释放通道。通过高通量筛选，我们已经鉴定出几种高效和选择性的ML1合成激动剂和拮抗剂。虽然人类的TRPML1突变导致了以溶酶体运输无序为特征的LSD，但使用合成激动剂对ML1进行药理激活足以诱导溶酶体的生物发生和运输。此外，合成拮抗剂的使用表明，ML1是溶酶体吞噬和膜修复过程中胞吐所必需的。我们的初步研究已经确定了两类不同的内源性分子作为ML1的激动剂。这个竞争性更新项目的中心目标是检验这样一个假设，即细胞信号通过直接激活ML1来诱导溶酶体钙释放来调节溶酶体运输和自噬。我们将利用溶酶体钙离子成像、溶酶体电生理学、小鼠基因敲除、转基因小鼠和CRISPR/Cas9体内诱变来验证这一假说，即增强ML1的活性将促进溶酶体在培养细胞中的生物合成和自噬，并缓解溶酶体储存和几种LSD小鼠模型的神经变性。目的1确定ML1在溶酶体生物发生和自噬中的作用。目的2研究ML1在钙依赖的溶酶体运动中的作用。最后，我们将研究增加ML1‘S活性对LSD相关神经退行性变的体内影响。我们这项拟议研究的最终目标是为开发与溶酶体相关的儿童和成人神经退行性疾病的新治疗策略奠定必要的基础。

 DESCRIPTION (provided by applicant): To mediate the degradation of macromolecules delivered by endocytosis and autophagy, lysosomes must undergo extensive trafficking, including migration along microtubule tracks towards cargo-carrying endosomes and autophagosomes and subsequent membrane fusion and fission processes. Defects in lysosomal trafficking lead to lysosome storage diseases (LSDs). The long term goal of the proposed research is to understand how various cellular cues regulate lysosomal trafficking in autophagy and endocytosis. During the last funding period, we demonstrated that Ca2+ release from the lysosomal lumen plays a key signaling role in regulating multiple steps of lysosomal trafficking. By directly patch-clamping lysosomal membranes and by fluorescently imaging Ca2+ release from lysosomes, we showed that TRPML1 (transient receptor potential mucolipin 1, or ML1) is the principle Ca2+ release channel in the lysosome. By performing high-throughput screening we have identified several highly potent and selective synthetic agonists and antagonists of ML1. Whereas human mutations of TRPML1 cause a LSD characterized by disordered lysosomal trafficking, pharmacological activation of ML1 using synthetic agonists is sufficient to induce lysosomal biogenesis and trafficking. Furthermore, use of synthetic antagonists shows that ML1 is required for lysosomal exocytosis during phagocytosis and membrane repair. Our preliminary studies have identified two distinct classes of endogenous molecules that act as agonists for ML1. The central goal of this competitive renewal project is to test the hypothesis that cellular cues regulate lysosome trafficking and autophagy, by directly activating ML1 to elicit lysosomal Ca2+ release. We will use lysosome Ca2+ imaging, lysosome electrophysiology, mouse knockouts, transgenic mice, and CRISPR/Cas9 in vivo mutagenesis to test the hypothesis that boosting the activity of ML1 will facilitate lysosome biogenesis and autophagy in cells in culture, and alleviate lysosome storage and neurodegeneration in mouse models of several LSDs. Aim 1 is to determine the role of ML1 in lysosome biogenesis and autophagy. Aim 2 is to investigate the roles of ML1 in Ca2+-dependent lysosomal motility. Finally, we will study the in vivo effects of increasing ML1's activity on neurodegeneration associated with LSD. Our ultimate goal of this proposed research is to lay the groundwork necessary to develop new therapeutic strategies for lysosome-related pediatric and adult neurodegenerative diseases.