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退行性变期间，受损的蛋白质和细胞器在神经元内积聚。这些蛋白质和细胞器通常在溶酶体中被组织蛋白酶降解，所述组织蛋白酶在酸性pH下具有最佳活性。溶酶体pH的升高阻碍溶酶体内的正常降解并损害自噬，导致与衰老、年龄相关疾病和溶酶体贮积症相关的神经元功能障碍。调节溶酶体pH的分子过程还不清楚。在这个应用程序中，我证明，sAC，cAMP的pH值敏感的来源，是一个调节剂的溶酶体pH值。遗传或药理学消融sAC升高溶酶体pH值，导致积累的蛋白质和自噬体在体外和体内。因此，sAC是溶酶体pH的pH敏感性调节剂，将其定义为唯一已知的溶酶体pH传感器。这个建议的目标是阐明的机制，其中sAC调节溶酶体pH值。我建议测试的假设，sAC调节溶酶体功能，通过调节贩运和/或活性的V-ATP酶和/或任何负责的通道和转运蛋白（如ClC-7）的反离子电流溶酶体酸化。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的机制。第二个目的将通过检查sAC KO小鼠是否表现出年龄相关的溶酶体病理学进展来研究sAC丧失的体内分子后果。本申请中提出的实验将使我们更好地理解溶酶体pH调节的机制，这反过来将增强我们对衰老、年龄相关性神经变性和溶酶体贮积症的理解。如果验证这一假设将奠定基础，为未来的研究检查特定的神经元缺陷所造成的sAC中断，并可能确定sAC作为一个潜在的目标，为新的治疗策略，与年龄相关的遗传性神经退行性疾病。

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.