Understanding the molecular functions of progranulin and granulin in FTLD

 描述（由申请人提供）：尽管最近取得了进展，但仍不清楚颗粒蛋白前体缺乏如何导致神经退行性疾病额颞叶变性（FTLD）的发展。颗粒蛋白前体被切割成多个颗粒蛋白肽，这些颗粒蛋白肽是生物活性的并且可以在功能上对抗颗粒蛋白前体全蛋白。许多人认为，颗粒蛋白前体缺乏同样耗尽颗粒蛋白前体和颗粒蛋白水平，但这从来没有被直接测量。关于颗粒蛋白前体何时、为何以及在何处裂解成颗粒蛋白，颗粒蛋白前体和颗粒蛋白发挥何种分子功能以及在人类CNS中发现了多少颗粒蛋白前体和颗粒蛋白，也存在基础知识空白。这项研究计划的长期目标是了解颗粒蛋白前体和颗粒蛋白如何促进神经退行性疾病的病理生理学。这个应用程序的总体目标是利用C。elegans和人类死后组织，以了解颗粒蛋白前体裂解成颗粒蛋白，以确定全蛋白及其裂解产物的分子功能，并表征颗粒蛋白前体突变在人类疾病中的下游效应。核心假设是，颗粒蛋白前体裂解成颗粒蛋白是在时间和空间上受到严格调控的，因为这两个物种在应激反应中发挥相互作用，颗粒蛋白前体具有保护作用，颗粒蛋白具有毒性。这项工作的基本原理是，了解颗粒蛋白前体和颗粒蛋白的功能和产生对于在FTLD和其他疾病的治疗中安全地靶向这些分子至关重要。它还将提供有关应激反应，溶酶体生物学和神经变性之间联系的基础新知识。将通过三个具体目的来检验中心假设：1）阐明在体内负责将颗粒蛋白原切割成颗粒蛋白的时间、定位和蛋白酶，2）鉴定全长颗粒蛋白原促进和切割的颗粒蛋白损害应激反应的分子机制，3）确定疾病和颗粒蛋白原突变对人CNS组织中颗粒蛋白原/颗粒蛋白水平的影响.强有力的初步数据支持这些拟议的研究，包括证明颗粒蛋白前体保护和颗粒蛋白损害应激反应，以及颗粒蛋白前体的产生和裂解随着年龄和应激刺激而增加。两组新的抗颗粒蛋白抗体（特异于C.线虫或人颗粒蛋白）用于这些研究。此外，一种新的方案，分离完整的溶酶体从C。将使用Elegans。拟议的研究是创新的，因为它试图直接涉及颗粒蛋白毒性，而不是或除了颗粒蛋白前体缺乏症，作为与颗粒蛋白前体突变相关的神经变性的驱动力。这一贡献是重要的，因为为了了解颗粒蛋白前体单倍不足和颗粒蛋白前体替代疗法的后果，必须了解颗粒蛋白前体和颗粒蛋白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.