Lysosomal control of plasma membrane -endoplasmic reticulum membrane contacts regulates neuronal excitability

项目摘要 溶酶体是复杂和动态的细胞信号中心，控制新陈代谢、基因转录、 钙(Ca~(2+))动态平衡和自噬。一种关键的机制，溶酶体通过它进行沟通和 接受指令是通过在内质网-溶酶体膜接触部位转移胆固醇。在这些联系人中， Niemann Pick C1胆固醇转运体(NPC1)促进胆固醇从溶酶体内流出 它被转移到内质网，然后分配到其他细胞膜。因此，NPC1是 胆固醇代谢。进一步强调其重要性的是，NPC1功能突变的丢失导致 进行性神经退行性疾病，鼻咽癌疾病。这种致命的疾病无法治愈，其特点是 胆固醇在溶酶体腔内的积聚与几种脑组织的进行性神经变性 伴随着一系列破坏性症状的区域，包括癫痫，精神问题和 痴呆症。尽管鼻咽癌疾病中胆固醇调节失调的神经病理后果很明显， NPC1功能缺失与疾病神经病理之间的分子机制(S)尚不清楚。最近 我们的研究小组已经报道，NPC1功能的丧失导致(I)神经元过度兴奋，(Ii)内质网重组。 溶酶体、ER-高尔基体和ER-线粒体膜接触部位，以及(Iii)诱导神经毒性增加。 线粒体钙离子。尽管有这些至关重要的信息，但我们在以下方面的知识存在严重差距： 增强的兴奋性在鼻咽癌疾病中的后果，(2)溶酶体胆固醇转运如何改变 神经元内质网-质膜(ER-PM)接触部位的分子成分和编排，以及(3)IF 质膜离子通道或ER-PM连接可以靶向降低线粒体毒性和 提高鼻咽癌疾病中神经元的活性。我们的中心假设是NPC1功能的丧失会导致 内质网-质膜接触处离子通道分布和功能的异常重塑导致细胞毒作用增加 线粒体钙离子导致神经退行性变。为了验证这一假设，我们实施了多尺度方法， 包括超分辨率成像，电生理学，大脑兴奋性的光学标测，新的小鼠模型， 和动物行为测试，以严格调查胆固醇流出的机制 溶酶体调节神经元的活性。鼻咽癌胆固醇的基本重要性和普遍表达 转运体意味着我们应该特别注意分子元件和信号级联，这些分子和信号级联是 被它的活动改变的。胆固醇稳态与离子通道信号转导关系的研究 在鼻咽癌的ER-PM膜接触提供了一个可测试的模型来检验相互依赖 溶酶体胆固醇和离子通道活性，对几个领域和其他胆固醇- 阿尔茨海默氏症和帕金森氏症等相关疾病。

Project Summary Lysosomes are sophisticated and dynamic cellular signaling centers that control metabolism, gene transcription, calcium (Ca2+) homeostasis, and autophagy. A key mechanism through which lysosomes communicate and receive instruction is via transfer of cholesterol at ER–lysosome membrane contact sites. At these contacts the Niemann Pick C1 cholesterol transporter (NPC1) facilitates the efflux of cholesterol out of the lysosome before it is transferred to the ER for distribution to other cellular membranes. Thus, NPC1 is a key gatekeeper in cholesterol metabolism. Further underscoring its importance, loss of function mutations in NPC1 lead to the progressive neurodegenerative disorder, NPC disease. This fatal condition has no cure and is characterized by the accumulation of cholesterol within lysosome lumen and the progressive neurodegeneration of several brain regions that are accompanied by a host of devastating symptoms including seizures, psychiatric problems, and dementia. Notwithstanding clear neuropathological consequences for cholesterol dysregulation in NPC disease, the molecular mechanism(s) linking loss of NPC1 function to disease neuropathology are unknown. Recently our group has reported that loss of NPC1 function results in (i) neuron hyperexcitability, (ii) reorganization of ER– Lysosome, ER–Golgi, and ER–mitochondrial membrane contact sites, and (iii) induces neurotoxic increases in mitochondrial Ca2+. Despite this crucial information there are critical gaps in our knowledge regarding (1) the consequences of enhanced excitability in NPC disease, (2) how lysosomal cholesterol transport alters the molecular elements and choreography at neuronal ER–plasma membrane (ER–PM) contact sites, and (3) if plasma membrane ion channels or ER–PM junctions can be targeted to reduce mitochondrial toxicity and increase neuron viability in NPC disease. Our central hypothesis is that loss of NPC1 function results in aberrant remodeling of ion channel distribution and function at ER–PM contacts to drive cytotoxic increases in mitochondrial Ca2+ leading to neurodegeneration. To test this hypothesis, we implement a multi-scale approach, including super-resolution imaging, electrophysiology, optical mapping of brain excitability, novel murine models, and animal behavior testing to rigorously investigate the mechanisms by which cholesterol efflux from the lysosome tunes neuron viability. The fundamental importance and ubiquitous expression of the NPC cholesterol transporter means we should pay particular attention to molecular elements and signaling cascades that are modified by its activity. Investigating the relationship between cholesterol homeostasis and ion channel signaling at ER–PM membrane contacts in NPC provides a testable model for examining the interdependence of lysosomal cholesterol and ion channel activity and has broad implications for several fields and other cholesterol- linked diseases such as Alzheimer’s and Parkinson’s.