Molecular genetic studies of progranulin regulators in FTLD and ALS

描述（由申请人提供）：前颗粒蛋白基因（GRN）突变导致额颞叶变性（FTLD）。GRN突变个体的功能性前颗粒蛋白（PGRN）减少50%，并且总是表现出TDP-43病理（FTLD- tdp），这表明低PGRN水平是FTLD中TDP-43功能障碍的潜在引发因素。不仅在GRN突变的FTLD患者中，而且在大多数ALS患者中，TDP-43被鉴定为病理蛋白，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发挥作用。因此，确定大脑中PGRN水平的调节方式可能会导致一系列神经退行性疾病的新治疗和疗法。在过去的几年中，我们和其他人通过全基因组关联研究确定了两种PGRN调节因子：未表征的跨膜蛋白106B （TMEM106B）和多配体受体SORT1 （SORT1）。TMEM106B被确定为FTLD-TDP的一个危险因素，我们实验室的后续研究表明TMEM106B在PGRN调节中起作用。特别令人感兴趣的是，一种特定的TMEM106B遗传变异（rs3173615预计会导致p.T185S）可以显著保护GRN突变携带者免受疾病的侵袭。我们进一步确定SORT1是人类血浆中PGRN水平的主要调节因子。有趣的是，SORT1被独立鉴定为PGRN的神经元受体。在初步数据中，我们现在提出了一项未发表的人类大脑GRN mRNA水平的全基因组数量性状位点分析，并确定了TBC1结构域家族成员1 （TBC1D1）是另一种新的PGRN调节因子。在本项目中，我们假设TMEM106B、SORT1和TBC1D1的遗传变异调节PGRN在大脑中的水平和/或功能，从而改变TDP-43蛋白病变的疾病风险、外显率和表现。具体目标是：1)利用细胞培养模型，包括小鼠PGRN敲除和野生型海马神经元的原代培养、生化和分子方法以及重组腺相关病毒在小鼠体内的“体细胞基因转移”，研究p.T185S保护变异体对TMEM106B和PGRN的功能特性；2)系统分析TMEM106B、SORT1和TBC1D1基因变异在FTLD和ALS发展和表现中的作用，利用全基因组测序鉴定的变异在广泛的FTLD和ALS病例对照人群中进行遗传关联研究；3)确定新发现的TMEM106B、SORT1和TBC1D1变异对体内和体外PGRN水平的影响。这些建议的研究与充分认识TMEM106B、SORT1和TBC1D1中常见和罕见变异对FTLD和ALS的发展和表现的贡献有关，并将有助于更好地理解PGRN的调控。

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.