Modulation of lysosomal pH by soluble Adenylyl Cyclase (sAC)

描述（由申请人提供）：在与年龄相关的神经变性过程中，受损的蛋白质和细胞器在神经元内积聚。这些蛋白和细胞器通常在溶酶体中被组织蛋白酶降解，组织蛋白酶在酸性pH下最具活性。溶酶体pH的升高阻碍了溶酶体内的正常降解，损害了自噬，导致与衰老、年龄相关疾病和溶酶体储存障碍相关的神经元功能障碍。调节溶酶体pH值的分子过程尚不清楚。在这个应用中，我证明了sAC，一种pH敏感的cAMP来源，是溶酶体pH的调节剂。遗传或药物消融sAC会升高溶酶体pH，导致体内和体外蛋白质和自噬体的积累。因此，sAC是溶酶体pH的pH敏感调节剂，是唯一已知的溶酶体pH传感器。本建议的目的是阐明sAC调节溶酶体ph的机制。我建议测试sAC通过调节v - atp酶的运输和/或活性和/或负责溶酶体酸化所需的反离子电流的任何通道和转运体（如ClC-7）来调节溶酶体功能的假设。我将追求两个具体目标：第一个目标将阐明sAC调节细胞系统中溶酶体pH值的机制。第二个目的是通过检查sAC KO小鼠是否表现出与年龄相关的溶酶体病理进展来研究体内sAC丢失的分子后果。本应用程序中提出的实验将更好地了解溶酶体pH调节的机制，从而增强我们对衰老，年龄相关的神经变性和溶酶体储存疾病的理解。如果这一假设得到证实，将为未来的研究奠定基础，研究由sAC破坏引起的特定神经元缺陷，并可能将sAC确定为年龄相关和遗传性神经退行性疾病的新治疗策略的潜在靶点。

DESCRIPTION (provided by applicant): During age-related neurodegeneration, damaged proteins and organelles accumulate within neurons. These proteins and organelles are normally degraded in the lysosomes by cathepsins, which are optimally active at acidic pH. Elevation of lysosomal pH hinders normal degradation within lysosomes and impairs autophagy leading to neuronal dysfunction associated with aging, age-related diseases and lysosomal storage disorders. The molecular processes that regulate lysosomal pH are not clearly understood. In this application, I demonstrate that sAC, a pH-sensitive source of cAMP, is a modulator of lysosomal pH. Genetic or pharmacologic ablation of sAC elevates lysosomal pH leading to accumulation of proteins and autophagosomes both in vitro and in vivo. Thus, sAC is a pH-sensitive regulator of lysosomal pH, defining it as the only known lysosomal pH sensor. The goal of this proposal is to elucidate the mechanism by which sAC regulates lysosomal pH. I propose to test the hypothesis that sAC regulates lysosomal function by regulating trafficking and/or activity of V-ATPase and/or any of the channels and transporters (such as ClC-7) responsible for the counter ion current necessary for lysosomal acidification. I will pursue two Specific Aims: the first aim will elucidate the mechanism by which sAC regulates lysosomal pH in cellular systems. The second aim will investigate the molecular consequences of loss of sAC in vivo by examining whether sAC KO mice exhibit age related progression of lysosomal pathology. The experiments proposed in this application will better our understanding of the mechanism by which lysosomal pH is regulated, which in turn will enhance our understanding of aging, and age-related neurodegeneration and lysosomal storage disorders. If validated this hypothesis will lay the groundwork for future studies examining the particular neuronal deficits caused by sAC disruption, and may identify sAC as a potential target for novel treatment strategies for age-related and genetic neurodegenerative diseases.