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的。原颗粒蛋白(PGRN)结果中的杂合性突变 在家族性FTLD中，PGRN表达降低，约占家族性FTLD的25%。相比之下，纯合子PGRN 突变导致PGRN完全丧失并导致神经元蜡样脂褐素沉着症(NCL)，一组 神经退行性溶酶体储存障碍。因此，PGRN突变似乎会导致不同的疾病 (FTLD与NCL)呈剂量依赖关系，提示PGRN杂合突变可能导致 部分溶酶体功能丧失所致的FTLD。我的博士论文项目旨在确定PGRN是否减少 由于FTLD连锁的PGRN杂合突变导致的表达，会导致溶酶体功能障碍并导致 到FTLD的神经变性。使用来自FTLD患者的IPSC来源的人皮质神经元 与同基因对照相比，我们已经证明了PGRN突变的神经元 显著损害了溶酶体的蛋白分解。为了确定这种溶酶体受损的机制 蛋白分解，我们研究了PGRN和溶酶体酶组织蛋白酶D突变的关系。 在PGRN和CTSD中，都会导致类似形式的NCL，组织蛋白酶D主要在 负责降解长寿蛋白质的大脑。我们发现组织蛋白酶D具有活性，但是 在PGRN突变型神经元中，NOT的表达显著降低。此外，我们还展示了 PGRN与组织蛋白酶D相互作用，其裂解产物颗粒蛋白显著增加 组织蛋白酶D的体外活性。基于这些初步结果，我们提出了PGRN在调节中的新作用 FTLD连锁杂合子中PGRN表达缺失对溶酶体组织蛋白酶D活性的影响 突变型神经元，导致FTLD溶酶体功能缺陷。为了进一步调查这些结果，我们将 使用重组颗粒绘制体外剂量依赖的组织蛋白酶D活性曲线以确定 从PGRN中分离出来的单个颗粒专门调节组织蛋白酶D的活性。此外，我们还将 确定PGRN是否与组织蛋白酶D以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突变的IPSC皮质神经元的长期培养来 确定组织蛋白酶D活性降低引起的溶酶体功能障碍是否与致病因素有关 FTLD标志如泛素和TDP-43阳性包涵体形成。这个博士论文项目将 对PGRN在调节溶酶体功能和细胞中的正常作用提供重要的见解 PGRN突变导致人类神经元FTLD的机制。作为博士后实习生，我会 通过研究神经胶质细胞对神经退行性变表型的贡献，扩展我的博士训练。 最终，我计划把我的博士和博士后培训结合起来，成为一名学术研究员 探讨神经元-神经胶质细胞相互作用在神经退行性变发病机制中的作用。

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.