Understanding the molecular functions of progranulin and granulin in FTLD

 描述(申请人提供)：尽管最近取得了进展，但原颗粒缺乏如何导致神经退行性疾病额颞叶变性(FTLD)的发展仍不清楚。原颗粒蛋白被裂解成具有生物活性的多个颗粒蛋白多肽，并可能在功能上对抗原颗粒蛋白全蛋白。许多人认为，原颗粒蛋白缺乏同样会消耗原颗粒蛋白和颗粒蛋白水平，但这一点从未被直接测量过。关于原颗粒何时、为什么和在哪里裂解成颗粒，原颗粒和颗粒在什么分子功能中发挥作用，以及在人类中枢神经系统中发现多少原颗粒和颗粒，也存在基本的知识空白。这项研究计划的长期目标是了解原颗粒和颗粒如何在神经退行性疾病的病理生理学中起作用。这项应用的总体目标是利用线虫和人类死后组织来了解原颗粒蛋白裂解成颗粒蛋白，确定完整蛋白及其裂解产物的分子功能，并表征原颗粒蛋白突变在人类疾病中的下游效应。核心假设是原颗粒蛋白的裂解在时间和空间上受到严格的调控，因为这两个物种在应激反应中扮演着相互作用的角色，原颗粒蛋白是保护性的，而颗粒蛋白是有毒的。这项工作的基本原理是，了解原颗粒和颗粒的功能和产生对于在FTLD和其他疾病的治疗中安全地针对这些分子至关重要。它还将提供关于应激反应、溶酶体生物学和神经退化之间联系的基本新知识。这一中心假设将通过三个具体目标得到验证：1)阐明在体内将原颗粒裂解成颗粒的时间、位置和酶；2)确定全长原颗粒促进和裂解颗粒抑制应激反应的分子机制；3)确定疾病和原颗粒突变对人中枢神经系统组织中原颗粒/颗粒水平的影响。强有力的初步数据支持这些拟议的研究，包括证明原颗粒对应激反应有保护作用，颗粒对应激反应有损害，原颗粒的产生和裂解随着年龄和应激刺激的增加而增加。已经为这些研究产生了两组新的抗颗粒蛋白抗体(针对线虫或人类颗粒蛋白)。此外，还将利用一种新的方法从线虫中分离完整的溶酶体。拟议的研究具有创新性，因为它寻求直接涉及颗粒毒性，而不是或除了原颗粒缺乏之外，作为与原颗粒突变相关的神经退行性变的驱动力。这一贡献意义重大，因为为了了解原颗粒单倍体功能不全和原颗粒替代疗法的后果，必须了解原颗粒和颗粒的正常生物学功能和相对水平。

 DESCRIPTION (provided by applicant): Despite recent progress, it remains unclear how progranulin deficiency leads to development of the neurodegenerative disease frontotemporal lobar degeneration (FTLD). Progranulin is cleaved into multiple granulin peptides that are bioactive and may functionally oppose the progranulin holoprotein. Many believe that progranulin deficiency equally depletes progranulin and granulin levels yet this has never been directly measured. Fundamental knowledge gaps also exist regarding when, why and where progranulin is cleaved into granulins, what molecular functions progranulin and granulin play and how much progranulin and granulin are found in human CNS. The long-term goal of this research program is to understand how progranulin and granulins contribute to the pathophysiology of neurodegenerative disease. The overall objective of this application is to utilize C. elegans and human post-mortem tissue to understand progranulin cleavage into granulins, to determine the molecular function of the holoprotein and its cleavage products and to characterize the down- stream effects of progranulin mutations in human disease. The central hypothesis is that the cleavage of progranulin into granulins is tightly regulated in a temporal and spatial manner because the two species play reciprocal roles in stress response with progranulin being protective and granulins being toxic. The rationale for this work is that understanding the function and production of progranulin and granulins is critical to safely targeting these molecules in the treatment of FTLD and other disease. It will also provide fundamental new knowledge regarding the links between stress response, lysosome biology and neurodegeneration. The central hypothesis will be tested through three specific aims: 1) elucidate the timing, localization and proteases responsible for cleavage of progranulin into granulins in vivo, 2) identify the molecular mechanisms by which full- length progranulin promotes and cleaved granulins impair stress response, 3) determine the effects of disease and progranulin mutations on progranulin/granulin levels in human CNS tissue. Strong preliminary data sup- ports these proposed studies, including demonstration that progranulin protects against and granulins impair stress response and that progranulin production and cleavage increase with age and stressful stimuli. Two sets of novel anti-granulin antibodies (specific to either C. elegans or human granulins) have been generated for these studies. Additionally, a novel protocol for isolating intact lysosomes from C. elegans will be utilized. The proposed research is innovative because it seeks to directly implicate granulin toxicity, rather than or in addition to progranulin deficiency, as the driving force in neurodegeneration related to progranulin mutations. This contribution is significant because in order to understand progranulin haploinsufficiency and the consequences of progranulin replacement therapy, one must understand the normal biological functions and relative levels of both progranulin and granulins.