Investigating contributions of late endosomal and lysosomal chloride/proton antiporter dysfunction to neuronal storage

项目总结/摘要 进行性神经退行性疾病如阿尔茨海默氏症和帕金森氏症的特征在于： 神经元内蛋白质物质的不适当积累，并伴有一系列 症状，包括运动逐渐退化和痴呆。溶酶体中物质的积累， 降解废弃蛋白质的细胞内区室，也是溶酶体贮积症的特征 （LSD）。CLCN 6和CLCN 7基因编码CLC-6和CLC-7。CLC-6和CLC-7与 辅助亚基OSTM 1（CLC-7/OSTM 1）是具有跨膜转运氯离子和质子的功能的蛋白质。 膜限定连续的细胞内室-晚期内体和溶酶体。 导致CLC-6和CLC-7功能丧失（LoF）的变异体与这些细胞内物质的积累有关。 LSD的特征。与CLC-6和CLC-7/OSTM 1 LoF相关的LSD具有不同的 在发病和疾病严重程度上的差异。有趣的是，产生CLC-6和CLC-7/OSTM 1的变体 功能获得（GoF）与不同深度神经退行性疾病相关。目前，知识 关于它们的功能机制，指出了一般的相似之处，以及微妙的监管差异。 对野生型转运蛋白的精确转运机制以及LoF和 GoF疾病相关变异对于理解疾病病因很重要，但也需要额外的研究。 更深入的功能分析。为了获得这一需要的洞察力，目标1结合了生物物理学，电生理学， 和计算建模方法来提取和比较详细的生物物理参数调节 CLC-6和CLC-7的野生型和疾病变体的功能。目标2最初提出产生人类iPSC- 衍生的CLCN 6基因编辑的分化神经元系，以研究房室稳态的早期变化 假设为加速疾病，目的是监测进行性功能障碍。时间允许的话，类似 可以进行CLCN 7基因编辑系的开发。总之，测量、分析和详细 CLC-6和CLC-7/OSTM 1转运功能的建模提供了对 疾病相关的转运蛋白变异体。此外，所提出的工作提供了独特的神经元细胞模型， 以精确的分辨率监测疾病进展。即将到来的基础知识为战略提供信息 治疗过多的破坏性神经退行性疾病，从而产生持续的影响。

Project Summary/Abstract Progressive neurodegenerative disorders such as Alzheimer’s and Parkinson’s are characterized by inappropriate accumulation of proteinaceous matter within neurons, and are accompanied by a constellation of symptoms, including gradual degeneration of movement and dementia. Accumulation of matter in lysosomes, an intracellular compartment that degrades obsolete protein, also characterizes lysosomal storage disorders (LSDs). The CLCN6 and CLCN7 genes encode CLC-6 and CLC-7. CLC-6, and CLC-7 in combination with an accessory subunit, OSTM1 (CLC-7/OSTM1), are proteins that function to transport chloride and protons across membranes defining contiguous intracellular compartments—late endosomes and lysosomes, respectively. Variants causing CLC-6 and CLC-7 loss of function (LoF) associate with accumulation of matter within these compartments, characteristic of LSDs. The LSDs associated with CLC-6 and CLC-7/OSTM1 LoF have distinct differences, in onset as well as disease severity. Interestingly, variants producing CLC-6 and CLC-7/OSTM1 gain of function (GoF) associate with distinct profound neurodegenerative disease. Presently, knowledge regarding their mechanism of function points to general similarities, as well as subtle regulatory differences. Greater understanding about the precise transport mechanism of wild type transporters, as well as both LoF and GoF disease-associated variants, is important for understanding disease etiologies, but also requires additional and deeper functional analysis. To gain this needed insight, Aim 1 combines biophysical, electrophysiological, and computational modeling approaches to extract and compare the detailed biophysical parameters regulating function of wild-type and disease variants of CLC-6 and CLC-7. Aim 2 proposes initially to generate human iPSC- derived CLCN6 gene-edited, differentiated neuronal lines to study early changes in compartmental homeostasis hypothesized to precipitate disease, and aims to monitor progressive dysfunction. Time-permitting, similar development of CLCN7 gene-edited lines can proceed. In summary, measurement, analysis, and detailed modeling of CLC-6 and CLC-7/OSTM1 transport function offer needed insight into functional disruption in disease-associated, transporter variants. Moreover, the proposed work provides unique neuronal cell models for monitoring disease progression at refined resolution. Forthcoming foundational knowledge informs strategies toward treatment of a plethora of devastating neurodegenerative diseases, thereby exerting sustained impact.