Molecular genetic studies of progranulin regulators in FTLD and ALS

描述(申请人提供)：原颗粒基因(GRN)突变导致额颞叶变性(FTLD)。GRN突变个体的功能性原颗粒蛋白(PGRN)减少了50%，并且总是表现出TDP-43病理(FTLD-TDP)，这表明低水平的PGRN是FTLD中TDP-43功能障碍的潜在起始者。TDP-43作为病理蛋白的鉴定，不仅在GRN突变的FTLD患者中，而且在大多数ALS患者中也进一步表明TDP-43在这些疾病背后的统一的神经退行性疾病机制中发挥作用。因此，确定PGRN在大脑中的水平是如何调节的，可能会导致一系列神经退行性疾病的新疗法和疗法。在过去的几年里，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：未鉴定的跨膜蛋白106B(TMEM106B)和多配体受体索迪林(SORT1)。TMEM106B被确定为FTLD-TDP的危险因素，我们实验室的后续研究表明TMEM106B在PGRN调控中发挥作用。特别令人感兴趣的是，发现一个特定的TMEM106B基因变异(rs3173615，预测会导致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是人血浆中PGRN水平的主要调节因子。有趣的是，SORT1被独立地确定为PGRN的神经元受体。在初步数据中，我们现在提供了一个尚未发表的对人脑中GRN mRNA水平的全基因组数量性状基因座分析，并确定TBC1结构域家族成员1(TBC1D1)是另一个新的PGRN调节因子。在这个项目中，我们假设TMEM106B、SORT1和TBC1D1的基因变异调节大脑中PGRN的水平和/或功能，从而改变TDP-43蛋白病变的疾病风险、外显性和呈现。其具体目标集中在1)利用细胞培养模型，包括小鼠PGRN基因敲除和野生型海马神经元的原代培养，生化和分子方法，以及利用重组腺相关病毒进行“体细胞基因转移”，来表征p.T185S保护性变异体对TMEM106B和PGRN的影响；2)通过对广泛的FTLD和ALS病例对照人群进行遗传关联研究，利用全基因组测序确定的变异体，系统地分析TMEM106B、SORT1和TBC1D1的遗传变异体在FTLD和ALS的发生和呈现中的作用；3)确定新发现的TMEM106B、SORT1和TBC1D1突变体在体内和体外对PGRN水平的影响。建议的研究有助于充分认识TMEM106B、SORT1和TBC1D1的常见和罕见变异在FTLD和ALS的发生和表现中的作用，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.