Membrane contact sites regulate cellular excitability

这项提议的长期目标是开发治疗人类疾病的治疗策略。 以细胞胆固醇代谢的病理变化为特征的尼曼-皮克C型(NPC)病。 鼻咽癌疾病是一种致命的神经退行性疾病，由关键看门人内的突变引起。 胆固醇转运中的蛋白质：溶酶体Niemann-Pick C1型。鼻咽癌患者的神经元 由于减少了胆固醇从溶酶体到体内的运输，显著改变了胆固醇的稳态 内质网。神经学上，鼻咽癌患者神经元中溶酶体胆固醇的积聚给 出现运动功能受损、精神问题、癫痫、痴呆症，通常在 成人期。尽管鼻咽癌疾病的胆固醇失调有明确的神经病理后果，但 很少有研究关注于了解离子通道功能、神经元 兴奋性和钙的处理，有助于鼻咽癌疾病的神经病理。考虑到 肌醇磷脂控制着广泛的细胞过程，包括对质膜离子的调节 因此，这些脂类的膜池中的任何相互依赖都可能对 对这种疾病的重要影响。我们的中心假设是胆固醇的调节外流 溶酶体通过NPC1进入内质网，是磷脂酰肌醇和神经元兴奋性的变阻器。至 为了验证这一假设，我们实施了一种多尺度的方法来严格研究 胆固醇可以调节肌醇磷脂的丰度和分布，从而改变肌醇磷脂- 依赖离子通道功能。特定目标1将检验NPC1功能改变的假设 修饰质膜磷脂酰肌醇，改变离子通道活性，从而改变神经元 膜的兴奋性和功能。在特定的目标2中，我们检验了兴奋性和兴奋性的改变 膜胆固醇控制神经元储存和释放钙的能力。最后，在具体目标3 我们将确定在鼻咽癌中表达谱改变的特定蛋白质以进行修饰 肌醇磷脂依赖的离子通道功能。拟议的研究在以下领域具有具体的相关性 神经科学、细胞生物学和生物物理学，但肌醇磷脂和 胆固醇对于过多的细胞事件来说，意味着它将对医学产生广泛的影响。

The long-term goal of this proposal is to develop therapeutic strategies for the treatment of a human disease characterized by pathological changes in cellular cholesterol metabolism, Niemann-Pick type C (NPC) disease. NPC disease is a fatal neurodegenerative disorder that occurs due to mutations within a key gatekeeper protein in cholesterol transport: lysosomal Niemann-Pick type C1. Neurons from NPC patients have dramatically altered cholesterol homeostasis due to reduce transport of cholesterol out of lysosomes to the endoplasmic reticulum. Neurologically, accumulation of lysosomal cholesterol in NPC patient neurons, gives rise to impaired motor functions, psychiatric problems, seizures, dementia, and typically death prior to adulthood. Despite clear neuropathological consequences for cholesterol dysregulation in NPC disease, there has been little research attention invested in understanding whether altered ion channel function, neuronal excitability, and calcium handling, contribute to the neuropathology of NPC disease. Given that phosphoinositides control a wide range of cellular processes including the regulation of plasma membrane ion channel activity, it follows that any interdependence in the membrane pools of these lipids could have critically important implications for this disease. Our central hypothesis is that regulated efflux of cholesterol from lysosomes to the ER, via NPC1, is a rheostat for phosphoinositides and consequently neuronal excitability. To test this hypothesis, we implement a multi-scale approach to rigorously investigate the mechanisms by which cholesterol can regulate the abundance and distribution of phosphoinositides to alter phosphoinositide- dependent ion channel function. Specific Aim 1 will test the hypothesis that alterations in NPC1 function modifies plasma membrane phosphoinositides, to alter ion channel activity, and consequently neuron membrane excitability and function. In Specific Aim 2 we test the hypothesis that alterations in excitability and membrane cholesterol controls the ability of neurons to store and release calcium. Finally, in Specific Aim 3 we will determine specific proteins that have their expression profiles altered in NPC to modify phosphoinositide-dependent ion channel function. The proposed studies have specific relevance in the fields of neuroscience, cell biology and biophysics, but the fundamental importance of phosphoinositides and cholesterol for a plethora of cellular events, mean it will have broad implications for medicine.