Membrane contact sites regulate cellular excitability

该提案的长期目标是为治疗人类疾病制定治疗策略 以细胞胆固醇代谢的病理变化为特征的C型尼曼-匹克病（Niemann-Pick type C，NPC）。 NPC疾病是一种致命的神经退行性疾病，由于关键看门人的突变而发生 胆固醇转运蛋白：溶酶体尼曼-匹克C1型。NPC患者的神经元 由于减少胆固醇从溶酶体到溶酶体的转运， 内质网神经学上，溶酶体胆固醇在NPC患者神经元中的积累， 导致运动功能受损、精神问题、癫痫发作、痴呆，通常在 成年尽管NPC疾病中胆固醇失调有明确的神经病理学后果， 很少有研究关注于了解改变的离子通道功能、神经元 兴奋性和钙处理，有助于NPC疾病的神经病理学。鉴于 磷酸肌醇控制广泛的细胞过程，包括调节质膜离子 通道活动，因此，这些脂质的膜池中的任何相互依赖性都可能具有关键性 对这种疾病的重要影响。我们的中心假设是， 溶酶体通过NPC 1与ER的连接是磷酸肌醇的变阻器，因此是神经元兴奋性的变阻器。到 为了验证这一假设，我们采用了多尺度方法来严格研究 胆固醇可以调节磷酸肌醇的丰度和分布，以改变磷酸肌醇- 依赖离子通道功能。具体目标1将检验NPC 1功能的改变 修饰质膜磷酸肌醇，改变离子通道活性，从而改变神经元 膜的兴奋性和功能。在具体目标2中，我们检验了兴奋性和 膜胆固醇控制神经元储存和释放钙的能力。第三，具体目标 我们将确定在NPC中表达谱改变的特定蛋白质， 磷酸肌醇依赖性离子通道功能。拟议的研究在以下领域具有具体意义： 神经科学，细胞生物学和生物物理学，但磷酸肌醇和 胆固醇对过多的细胞事件，意味着它将有广泛的医学意义。

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.