Molecular genetic studies of progranulin regulators in FTLD and ALS

描述（由申请人提供）：颗粒蛋白前体基因（GRN）突变导致额颞叶变性（FTLD）。具有GRN突变的个体具有50%的功能性颗粒蛋白前体蛋白（PGRN）减少，并且还总是显示TDP-43病理（FTLD-TDP），表明低PGRN水平是FTLD中TDP-43功能障碍的潜在引发剂。TDP-43不仅在具有GRN突变的FTLD患者中，而且在大多数ALS患者中被鉴定为病理性蛋白，这进一步表明TDP-43在这些疾病的统一神经退行性疾病机制中的作用。因此，确定PGRN水平如何在大脑中调节可能会导致一系列神经退行性疾病的新治疗和疗法。 在过去的几年里，我们和其他人已经确定了两个PGRN调节器通过全基因组关联研究：未表征的跨膜蛋白106 B（TMEM 106 B）和多配体受体分拣蛋白（SORT 1）。TMEM 106 B被确定为FTLD-TDP的风险因素，我们实验室的后续研究表明TMEM 106 B在PGRN调节中的作用。特别令人感兴趣的是，一种特定的TMEM 106 B遗传变异（rs3173615预测会导致p.T185S）可以显着保护GRN突变携带者免受疾病的影响。我们进一步鉴定了SORT 1是人血浆中PGRN水平的主要调节因子。有趣的是，SORT 1被独立鉴定为PGRN的神经元受体。在初步数据中，我们现在提出了一个未发表的全基因组定量性状基因座分析的GRN mRNA水平在人脑中，并确定TBC 1域家族，成员1（TBC 1D 1）作为又一个新的PGRN调节。在这个项目中，我们假设TMEM 106 B、SORT 1和TBC 1D 1的遗传变异调节脑中PGRN的水平和/或功能，从而改变TDP-43蛋白病的疾病风险、发病率和表现。具体目的集中在1）使用细胞培养模型，包括小鼠Pgrn敲除和野生型海马神经元的原代培养物、生物化学和分子方法以及使用重组腺相关病毒的小鼠中的“体细胞基因转移”，来表征p.T185S保护性变体对TMEM 106 B和PGRN的作用的功能特征; 2）通过使用通过全基因组测序鉴定的变体在广泛的FTLD和ALS病例对照群体中进行遗传关联研究，系统分析TMEM 106 B、SORT 1和TBC 1D 1中的遗传变体在FTLD和ALS的发展和呈现中的作用;和3）测定新鉴定的TMEM 106 B、SORT 1和TBC 1D 1变体对体内和体外PGRN水平的影响。 拟议的研究与充分了解TMEM 106 B、SORT 1和TBC 1D 1中常见和罕见变异对FTLD和ALS发展和表现的贡献相关，并将导致对PGRN调控的更深入了解。

DESCRIPTION (provided by applicant): Mutations in the progranulin gene (GRN) cause frontotemporal lobar degeneration (FTLD). Individuals with GRN mutations have a 50% reduction in functional progranulin protein (PGRN) and also invariably display TDP-43 pathology (FTLD-TDP), indicating low PGRN levels as a potential initiator of TDP-43 dysfunction in FTLD. The identification of TDP-43 as the pathological protein, not only in patients with FTLD with mutations in GRN, but also in the majority of patients with ALS further suggests a role for TDP-43 in a unifying neurodegenerative disease mechanism underlying these disorders. Consequently, determining how PGRN levels are regulated in brain may lead to novel treatments and therapies for a range of neurodegenerative diseases. In the last few years, we and others have identified two PGRN regulators through genome-wide association studies: the uncharacterized transmembrane protein 106B (TMEM106B) and the multiligand receptor sortilin (SORT1). TMEM106B was identified as a risk factor for FTLD-TDP, with subsequent studies from our laboratory suggesting a role for TMEM106B in PGRN regulation. Of particular interest was the finding that a specific TMEM106B genetic variant (rs3173615 predicted to result in p.T185S) could significantly protect GRN mutation carriers from developing disease. We further identified SORT1 as a major regulator of PGRN levels in human plasma. Interestingly, SORT1 was independently identified as a neuronal receptor for PGRN. In preliminary data we now present an unpublished genome-wide quantitative trait locus analysis of GRN mRNA levels in human brain and identify TBC1 domain family, member 1 (TBC1D1) as yet another novel PGRN regulator. In this Project, we hypothesize that genetic variants in TMEM106B, SORT1 and TBC1D1 regulate PGRN levels and/or function in brain, thereby modifying disease risk, penetrance and presentation in TDP-43 proteinopathies. The Specific Aims are focused on 1) the functional characterization of the effect of the p.T185S protective variant on TMEM106B and PGRN using cell culture models, including primary cultures of mouse Pgrn knock-out and wild type hippocampal neurons, biochemical and molecular approaches and "somatic gene transfer" in mice using recombinant adeno-associated virus; 2) Systematic analyses of the role of genetic variants in TMEM106B, SORT1 and TBC1D1 in the development and presentation of FTLD and ALS by performing genetic association studies in extensive FTLD and ALS case- control populations using variants identified by whole-genome sequencing; and 3) determine the effect of newly identified TMEM106B, SORT1 and TBC1D1 variants on PGRN levels in vivo and in vitro. The proposed studies are relevant to fully appreciate the contribution of common and rare variants in TMEM106B, SORT1 and TBC1D1 to the development and presentation of FTLD and ALS and will lead to a greater understanding of PGRN regulation.