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，控制代谢，基因转录， 钙（Ca 2+）稳态和自噬。一个关键的机制，通过它溶酶体沟通， 接受指令是通过胆固醇在ER-溶酶体膜接触位点的转移。在这些接触中， Niemann Pick C1胆固醇转运蛋白（NPC 1）促进胆固醇在溶酶体形成之前流出溶酶体。 它被转移到ER以分布到其它细胞膜。因此，NPC 1是 胆固醇代谢NPC 1的功能缺失突变进一步强调了其重要性，导致 进行性神经退行性疾病NPC疾病。这种致命的疾病无法治愈，其特征是： 溶酶体腔内胆固醇的积聚和几种脑的进行性神经变性 伴随着一系列破坏性症状的地区，包括癫痫发作，精神问题， 痴呆尽管在NPC疾病中胆固醇失调具有明确的神经病理学后果， 将NPC 1功能丧失与疾病神经病理学联系起来的分子机制是未知的。最近 我们的小组已经报道了NPC 1功能的丧失导致（i）神经元过度兴奋，（ii）ER-1的重组， 溶酶体，ER-高尔基体和ER-线粒体膜接触位点，和（iii）诱导神经毒性增加， 线粒体Ca 2+。尽管有这些重要的信息，但我们在以下方面的知识仍存在重大差距：（1） NPC疾病中兴奋性增强的后果，（2）溶酶体胆固醇转运如何改变 神经元ER-质膜（ER-PM）接触部位的分子元素和编排，以及（3）如果 可以靶向质膜离子通道或ER-PM连接以降低线粒体毒性， 增加NPC疾病中神经元活力。我们的中心假设是，NPC 1功能的丧失导致 ER-PM接触处离子通道分布和功能的异常重塑， 线粒体Ca 2+导致神经变性。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、脑兴奋性的光学映射、新型小鼠模型， 和动物行为测试，以严格调查胆固醇从 溶酶体调节神经元活力。NPC胆固醇的根本重要性和普遍表达 转运蛋白意味着我们应该特别注意分子元件和信号级联， 被其活动所改变。胆固醇稳态与离子通道信号传导的关系 在NPC的ER-PM膜接触提供了一个可测试的模型，用于检查 溶酶体胆固醇和离子通道活性，并对多个领域和其他胆固醇具有广泛影响- 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.