Investigating the regulation of lipid homeostasis and lysosomal function in neurodegenerative disease by prosaposin and progranulin in C. elegans

神经退行性疾病是现代健康面临的最大威胁之一，而针对这些复杂且进行性疾病的治疗方法很少。该提案研究了颗粒体蛋白前体 (PGRN) 的基础生物学，PGRN 是一种溶酶体蛋白，其单倍体不足会导致阿尔茨海默病相关痴呆 (ADRD) 额颞叶痴呆 (FTD) [1]。虽然 PGRN 缺乏是 FTD 的主要原因之一，但其功能尚不完全清楚。为了进一步了解 FTD 的病因和病理生理学，有必要确定 PGRN 在溶酶体中的内源功能。 PGRN 与另一种溶酶体前蛋白 proaposin (PSAP) 有着千丝万缕的联系，PSAP 也与颗粒体蛋白前体-FTD 有关 [2]。 PSAP 被裂解为鞘脂酶激活蛋白（称为 saposin），其催化溶酶体中的鞘脂代谢。皂苷的损失会扰乱溶酶体脂肪酶的活性，导致鞘脂的积累和溶酶体功能障碍。有趣的是，鞘脂代谢紊乱最近已成为 ADRD（包括 FTD）发生和进展的关键危险因素[3-11]。此外，PGRN 与 ADRD 相关的脂质稳态失调有关 [12, 13]，但其潜在的分子机制尚不清楚。总之，这表明 FTD 风险因子 PGRN 通过 PSAP 调节脂质稳态，进而促进 ADRD 病理生理学。尽管皂苷在鞘脂代谢中具有明确的作用，但皂苷从 PSAP 中释放并因此可用于调节鞘脂酶、鞘脂稳态和下游溶酶体功能的机制尚不清楚。我假设 PSAP 裂解成皂苷受到溶酶体酶、FTD 危险因素 PGRN 和年龄的调节，并且 PSAP 裂解的调节在脂质稳态和溶酶体功能中起着至关重要的作用。为了检验这一假设，我提出了几项创新性实验，其中包括体外重组人类蛋白、人类细胞系以及秀丽隐杆线虫体内行为和成像技术。我将利用体外蛋白酶功能和线虫表型的成熟方案以及新开发的技术来测量体内溶酶体 pH 值和脂质含量。我将确定 PGRN 如何调节 PSAP 裂解为皂苷，PGRN 是否通过其对 PSAP 的调节影响脂质稳态，以及改变的脂质稳态如何影响溶酶体功能。该提案的成功完成将填补关于溶酶体中 FTD 风险因子 PGRN 和 PSAP 的关系和功能的基础生物学知识的空白，并通过 PSAP 的溶酶体功能阐明 PGRN 与 ADRD 中脂质稳态改变之间的关键联系。该提案的成功完成将通过产生关于内溶酶体系统在蛋白质和脂质稳态中的内源性功能的基础知识来扩大我们对 FTD 的理解，这可能是产生 FTD、AD 和其他 ADRD 的新型诊断技术、预防策略和治疗的关键。 关键词: FTD, 颗粒体蛋白前体, 神经变性, 衰老, 溶酶体, 鞘脂, 前塞塞辛, 线虫

Neurodegenerative diseases are one of the greatest threats to modern health, and there are few treatments available for these complex and progressive diseases. This proposal investigates the basic biology underlying progranulin (PGRN), a lysosomal protein whose haploinsufficiency causes the Alzheimer’s Disease-Related Dementia (ADRD) frontotemporal dementia (FTD) [1]. While PGRN deficiency is one of the leading genetic causes of FTD, its function is not completely understood. To further our understanding of the causes and pathophysiology of FTD, it’s imperative to determine the endogenous functions of PGRN in the lysosome. PGRN is inextricably linked with another lysosomal pro-protein, prosaposin (PSAP), which is also implicated in progranulin-FTD [2]. PSAP is cleaved into sphingolipidase activator proteins called saposins, which catalyze sphingolipid metabolism in the lysosome. Loss of saposins perturbs lysosomal lipase activity, resulting in accumulation of sphingolipids and lysosomal dysfunction. Interestingly, perturbed sphingolipid metabolism has recently emerged as a critical risk factor for the onset and progression of ADRDs, including FTD [3-11]. Moreover, PGRN has been implicated in ADRD-related dysregulation in lipid homeostasis [12, 13], however, the underlying molecular mechanisms are unknown. Together, this suggests a pathway where the FTD-risk factor PGRN regulates lipid homeostasis via PSAP, which in turn contributes to ADRD pathophysiology. Despite the clear role for saposins in sphingolipid metabolism, the mechanism by which saposins are released from PSAP, and thus available to modulate sphingolipidases, sphingolipid homeostasis, and downstream lysosomal function, is not well understood. I hypothesize that PSAP cleavage into saposins is subject to regulation by lysosomal enzymes, the FTD-risk factor PGRN, and age, and that regulation of PSAP cleavage plays a crucial role in lipid homeostasis and lysosome function. To test this hypothesis, I propose a several innovative experiments with recombinant human proteins in vitro, human cell lines, and in in vivo behavioral and imaging techniques in Caenorhabditis elegans. I will utilize well-established protocols for both in vitro protease function and C. elegans phenotypes alongside newly developed techniques to measure lysosomal pH and lipid content in vivo. I will determine how PGRN regulates the cleavage of PSAP into saposins, if PGRN influences lipid homeostasis via its regulation of PSAP, and how altered lipid homeostasis impacts lysosomal function. Successful completion of this proposal will fill a gap in knowledge about the basic biology underlying the relationship and function of the FTD-risk factors PGRN and PSAP in the lysosome and elucidate a critical link between PGRN and altered lipid homeostasis in ADRD via the lysosomal function of PSAP. Successful completion of this proposal will expand our understanding of FTD by generating fundamental knowledge on the endogenous function of the endolysosomal system in protein and lipid homeostasis, which could be the key to the generation of novel diagnostic techniques, prevention strategies, and treatments for FTD, AD, and other ADRDs. Key Words: FTD, progranulin, neurodegeneration, aging, lysosome, sphingolipid, prosaposin, C. elegans