The Role of Lysosomal Glucocerebrosidase in Synucleinopathies

总结 溶酶体功能障碍越来越多地与神经退行性疾病的发展有关 包括帕金森病（PD）。编码β-葡萄糖脑苷脂酶（GCase）的GBA 1突变导致 戈谢病（GD）是最常见的溶酶体贮积症，是一种重要的遗传风险 突触核蛋白病的因子，包括PD和路易体痴呆（DLB）。显著减少 在散发性PD患者的脑中也有GCase活性的报道，这表明GCase的减少 活性可能代表PD发病机制的共同特征。与PD和DLB的遗传连锁也已被发现。 证明了溶酶体膜蛋白LIMP-2，GCase和GD的溶酶体运输受体 修饰语SCARB 2编码LIMP-2本身的突变是一种罕见的进行性乳腺癌的致病原因。 肾衰竭相关性肌阵挛性癫痫（AMRF）。我们之前的数据显示， LIMP-2导致GCase的误分泌和溶酶体消耗，并且还与PD样细胞凋亡相关。 小鼠病理学。我们还证明，通过提高LIMP-2水平， 活性增强，α-syn水平降低，表明这两种蛋白质在功能上是联系在一起的。 调节溶酶体功能和α-syn代谢。因此，我们假设， 重要的是在开发GCase激活剂时考虑LIMP-2-GCase相互作用 用于PD和相关突触核蛋白病的治疗剂。为了验证这一假设，我们建议进一步研究 LIMP-2在突触核蛋白病发病机制中的作用， LIMP-2/GCase复合物的形成和运输，及其在正常和患病的人类神经元中的作用。 目的1将通过以下方法检查野生型LIMP-2/GCase复合物在活细胞中的组装和化学计量： 使用光活化氨基酸、脉冲追踪和免疫沉淀实验。这些研究报告将 扩展到GCase和LIMP-2患者相关突变，以确定它们对肿瘤形成的影响。 LIMP-2/GCase复合体。目的2将检查LIMP-2和GCase缺陷型中的时间依赖性表型。 人类中脑神经元。我们将从LIMP-2患者成纤维细胞产生iPSC衍生的中脑神经元， 检查GCase和GCase/LIMP-2复合物的亚细胞定位，溶酶体蛋白水解和 形态学、脂质底物和α-突触核蛋白以时间依赖性方式蓄积。在目标3中， 对表达LIMP-2的小鼠模型进行神经病理学表征，所述LIMP-2缺乏与 GCase。为了直接检查我们先前在LIMP-2基因敲除小鼠中描述的神经学表型是否 由于溶酶体GCase缺失，我们建立了表达LIMP-2-Y163 D突变体的小鼠模型 无法绑定GCase。将分析这些小鼠的α-syn和脂质蓄积、溶酶体功能障碍， 炎症和神经毒性。这些实验还将确定是否由神经病理学介导的 LIMP-2缺陷可能至少部分独立于GCase。

SUMMARY Lysosomal dysfunction has been increasingly implicated in the development of neurodegenerative diseases including Parkinson's disease (PD). Mutations in GBA1 encoding β-glucocerebrosidase (GCase) cause Gaucher disease (GD), the most prevalent lysosomal storage disorder and represent an important genetic risk factor for synucleinopathies including PD and dementia with Lewy Bodies (DLB). A significant reduction in GCase activity has been also reported in brains of sporadic PD patients, suggesting that reduced GCase activity may represent a common feature of PD pathogenesis. Genetic linkage with PD and DLB has been also demonstrated for lysosomal membrane protein LIMP-2, the lysosomal trafficking receptor for GCase and GD modifier. Mutations in SCARB2 encoding LIMP-2 itself are disease-causing for a rare form of progressive myoclonic epilepsy associated with renal failure (AMRF). Our previous data showed that loss of function of LIMP-2 results in mistrafficking and lysosomal depletion of GCase and is also associated with a PD-like pathology in mice. We have also demonstrated that by elevating the levels of LIMP-2, lysosomal GCase activity was enhanced and α-syn levels reduced, suggesting that both proteins are functionally linked in the regulation of lysosomal function and α-syn metabolism. Therefore, we hypothesize that it will be critically important to consider LIMP-2-GCase interaction in the development of activators of GCase as potential therapeutics for PD and related synucleinopathies. To test this hypothesis, we propose to further examine the contribution of LIMP-2 in the pathogenesis of synucleinopathies by providing molecular insights into the formation and trafficking of the LIMP-2/GCase complex, and its role in normal and diseased human neurons. Aim 1 will examine the assembly and stoichiometry of the wild type LIMP-2/GCase complex in live cells by using photoactivatable amino acids, pulse chase and immunoprecipitations experiments. These studies will be extended to GCase and LIMP-2 patient-linked mutations to determine their impact on the formation of the LIMP-2/GCase complex. Aim 2 will examine time-dependent phenotypes in LIMP-2- and GCase-deficient human midbrain neurons. We will generate iPSC-derived midbrain neurons from LIMP-2 patient fibroblasts to examine subcellular localization of GCase and GCase/LIMP-2 complex, lysosomal proteolysis and morphology, accumulation of lipid substrates and α-synuclein in a time-dependent manner. In Aim 3 we will perform neuropathological characterization of a mouse model expressing LIMP-2 that is deficient in binding to GCase. To directly examine if the neurological phenotypes we previously described in LIMP-2 knockout mice result from depletion of lysosomal GCase, we generated a mouse model expressing LIMP-2-Y163D mutant that cannot bind GCase. These mice will be analyzed for α-syn and lipid accumulation, lysosomal dysfunction, inflammation and neurotoxicity. These experiments will also establish whether the neuropathology mediated by LIMP-2 deficiency may be at least in part independent of GCase.