The Mucolipin TRP Ion Channels

 描述（由申请方提供）：为了介导通过内吞作用和自噬递送的大分子的降解，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体和自噬体迁移以及随后的膜融合和分裂过程。溶酶体运输缺陷导致溶酶体储存病（LSD）。该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。在上一个资助期间，我们证明了从溶酶体腔释放的Ca 2+在调节溶酶体运输的多个步骤中起着关键的信号作用。通过直接膜片钳溶酶体膜和通过荧光成像从溶酶体的Ca 2+释放，我们表明TRPML 1（瞬时受体电位粘脂蛋白1，或ML 1）是溶酶体中的主要Ca 2+释放通道。通过进行高通量筛选，我们已经确定了几个高度有效的和选择性的ML 1的合成激动剂和拮抗剂。尽管TRPML 1的人类突变导致以溶酶体运输紊乱为特征的LSD，但使用合成激动剂的ML 1的药理学活化足以诱导溶酶体生物发生和运输。此外，合成拮抗剂的使用表明，ML 1是在吞噬和膜修复过程中溶酶体胞吐作用所必需的。我们的初步研究已经确定了两种不同类型的内源性分子，作为激动剂的ML 1。这个竞争性更新项目的中心目标是测试细胞信号通过直接激活ML 1引起溶酶体Ca 2+释放来调节溶酶体运输和自噬的假设。我们将使用溶酶体Ca 2+成像，溶酶体电生理学，小鼠敲除，转基因小鼠和CRISPR/Cas9体内诱变来测试以下假设：增强ML 1的活性将促进培养细胞中的溶酶体生物发生和自噬，并减轻几种LSD小鼠模型中的溶酶体储存和神经变性。目的1是确定ML 1在溶酶体生物合成和自噬中的作用。目的二是研究ML 1在钙依赖性溶酶体运动中的作用。最后，我们将研究增加ML 1活性对LSD相关神经退行性变的体内影响。我们这项研究的最终目标是为开发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.