Regulation of Degradative Pathways in Tauopathies

项目摘要 额颞叶变性（FTLD）包括一组神经退行性疾病 以认知和行为障碍为特征。颗粒蛋白前体（PGRN）中的杂合突变导致 PGRN表达降低，占家族性FTLD的约25%。相反，纯合子PGRN 突变导致PGRN的完全丧失并导致神经元蜡样质脂褐质沉积症（NCL），一组 神经退行性溶酶体贮积症因此，PGRN突变似乎会导致不同的疾病， (FTLD相对于NCL）以剂量依赖性方式，表明杂合PGRN突变可能导致 通过溶酶体功能部分丧失导致FTLD。我的博士论文项目旨在确定减少PGRN是否 由于FTLD连锁的PGRN杂合突变，表达导致溶酶体功能障碍， FTLD的神经退行性变。使用源自FTLD患者的iPSC衍生的人皮质神经元 与同基因对照相比，携带PGRN突变，我们已经证明PGRN突变神经元 已经严重损害了溶酶体的蛋白水解。为了确定这种受损的溶酶体 蛋白水解，我们研究了PGRN和溶酶体酶组织蛋白酶D之间的关系。突变 在PGRN和CTSD中均导致类似形式的NCL，并且组织蛋白酶D主要在 大脑负责降解长寿蛋白质。我们发现组织蛋白酶D活性， 而PGRN突变体神经元中其表达不显著降低。此外，我们证明， PGRN与组织蛋白酶D相互作用，并且PGRN的裂解产物颗粒蛋白显著增加了PGRN的表达。 体外组织蛋白酶D活性。基于这些初步结果，我们提出了一个新的作用，PGRN在调节 溶酶体组织蛋白酶D活性，其被FTLD连锁杂合子中PGRN表达的丧失所破坏。 突变的神经元，导致FTLD中溶酶体功能缺陷。为了进一步研究这些结果，我们将 使用重组颗粒蛋白进行体外剂量依赖性组织蛋白酶D活性曲线，以确定 从PGRN裂解的单个颗粒蛋白特异性调节组织蛋白酶D活性。此外，我们将 确定PGRN是否与除组织蛋白酶D之外的其他溶酶体酶相互作用或改变其活性。 最后，我们将使用FTLD患者来源的PGRN突变iPSC皮质神经元的长期培养物， 确定组织蛋白酶D活性降低导致的溶酶体功能障碍是否有助于致病性 FTLD标志物如泛素和TDP-43阳性包涵体形成。本博士论文项目将 为PGRN在调节溶酶体功能和细胞凋亡中的正常作用提供了重要的见解。 PGRN突变导致人类神经元FTLD的机制。作为一名博士后，我将 通过研究神经胶质细胞如何促进神经退行性表型来扩展我的博士培训。 最终，我计划通过成为一名学术研究人员， 研究神经元-神经胶质相互作用在神经变性发病机制中的作用。

Project Summary Frontotemporal lobar degeneration (FTLD) encompasses a group of neurodegenerative disorders characterized by cognitive and behavioral impairments. Heterozygous mutations in progranulin (PGRN) result in decreased PGRN expression and account for ~25% of familial FTLD. In contrast, homozygous PGRN mutations result in complete loss of PGRN and lead to neuronal ceroid lipofuscinosis (NCL), a group of neurodegenerative lysosomal storage disorders. Thus, PGRN mutations appear to cause different diseases (FTLD vs NCL) in a dose-dependent manner, suggesting that heterozygous PGRN mutations might cause FTLD via partial loss of lysosomal function. My PhD Dissertation Project aims to determine if reduced PGRN expression, due to FTLD-linked PGRN heterozygous mutations, causes lysosomal dysfunction and contributes to neurodegeneration in FTLD. Using iPSC-derived human cortical neurons derived from FTLD patients harboring PGRN mutations compared to isogenic controls, we have demonstrated that PGRN mutant neurons have significantly impaired lysosomal proteolysis. To determine the mechanism of this impaired lysosomal proteolysis, we examined the relationship between PGRN and the lysosomal enzyme cathepsin D. Mutations in PGRN and CTSD both lead to similar forms of NCL, and cathepsin D is predominantly expressed in the brain where it is responsible for the degradation of long-lived proteins. We found that cathepsin D activity, but not its expression was significantly decreased in PGRN mutant neurons. Furthermore, we demonstrated that PGRN interacts with cathepsin D, and that granulins, cleavage products of PGRN, significantly increase cathepsin D activity in vitro. Based upon these initial results, we propose a novel role for PGRN in regulating lysosomal cathepsin D activity, which is disrupted by loss of PGRN expression in FTLD-linked heterozygous mutant neurons, leading to defective lysosomal function in FTLD. To further investigate these results, we will perform in vitro dose-dependent cathepsin D activity curves using recombinant granulins to determine if individual granulins cleaved from PGRN specifically regulate cathepsin D activity. Furthermore, we will determine if PGRN interacts with or alters the activity of other lysosomal enzymes in addition to cathepsin D. Finally, we will use long-term cultures of FTLD patient-derived PGRN mutant iPSC cortical neurons to determine if lysosomal dysfunction resulting from decreased cathepsin D activity contributes to pathogenic FTLD hallmarks such as ubiquitin and TDP-43 positive inclusion formation. This PhD Dissertation Project will provide important insight into both the normal role of PGRN in regulating lysosomal function and the cellular mechanisms by which PGRN mutations cause FTLD in human neurons. As a Postdoctoral trainee, I will expand upon my PhD training by studying how glial cells contribute to neurodegenerative phenotypes. Ultimately, I plan to merge my PhD and postdoctoral training by becoming an academic researcher investigating the role of neuronal-glial interactions in the pathogenesis of neurodegeneration.