Glucocerebrosidase Biology and It's Role in Parkinson's Disease

描述(由申请人提供)：葡萄糖脑苷酶(GBA)的突变和激活在阿尔法-突触核病、PD和LBD的发病机制中起着重要作用。GBA作为溶酶体的一种酶，在溶酶体的降解途径中起着关键作用。大多数GBA的致病突变被认为是功能突变的丧失，最终导致GBA缺乏，葡萄糖礼仪(GlcCer)代谢受损，随后溶酶体介导的GBA底物包括α-突触核蛋白的降解失败。GBA耗竭导致突触核蛋白积聚和神经变性，提示GBA级联反应受损参与了突触核病的发病机制。我们和其他人发现，无论是否存在GBA突变，帕金森病患者的大脑以及A53Tα-突触核蛋白转基因(TG)小鼠的大脑都显示出GBA活性降低，这表明该酶在疾病的发病机制中发挥了作用。有趣的是，GBA的催化活性的丧失与其定量的蛋白质还原有关，这表明未知的关键调节因子可能通过翻译后修饰(PTM)在GBA的蛋白质水平和活性中发挥重要作用。因此，我们通过串联亲和纯化(TAP)分析确定了关键的GBA调节因子，从而研究了GBA活性和蛋白质水平丧失的机制。在初步的数据中，我们发现了GIP1(GBA相互作用蛋白1)，并证明GIP1属于一类新的E3连接酶。本研究旨在探讨GIP1对GBA的调节及其在PD和LBD发病机制中的作用。在特定的目标1中，我们将确定GIP1在体外和体内是否与GBA相互作用并泛化GBA。在具体目标2中，我们将研究GIP1是否针对GBA进行降解，从而调节GBA的活性及其底物GlcCer。由于GBA是一种潜在调节α-突触核蛋白表达的溶酶体酶，我们还将探讨GIP1介导的GBA降解是否最终调节α-突触核蛋白的表达和聚集。为了达到这些特定的目的，我们将利用Lenti-GIP1 shRNA病毒敲除GIP1，并利用单纯疱疹病毒GIP1过表达GIP1。在初步研究中，我们发现GIP1在A53Tα-突触核蛋白TG小鼠、帕金森病脑和经α-突触核蛋白预形成纤维(PFF)处理的神经元中积聚。因此，在特定的目标3中，我们将进一步评估α-突触核蛋白病理是否与GIP1的积聚有关，以及它的积聚是否与A53Tα-突触核蛋白TG小鼠和PD患者以及突触核蛋白pff处理的神经元中GBA缺乏和α-突触核蛋白病理的严重程度相关。此外，初步数据表明，GIP1的缺失显著减少了α-突触核蛋白pff诱导的神经元死亡。因此，在特定的目标3中，我们还将评估GIP1在α-突触核蛋白pff诱导的神经元死亡中的作用。最终，该项目将确定GIP1-GBA-α-突触蛋白神经退变途径的全部含义，并将确定PD和LBD治疗干预的新靶点。

DESCRIPTION (provided by applicant): Mutations and activation of glucocerebrosidase (GBA) play a prominent role in the pathogenesis of the alpha- synucleinopathies, PD and LBD. GBA plays a pivotal role in the lysosomal degradation pathway by functioning as a lysosomal enzyme. Most disease causing mutations of GBA are thought to be loss of function mutations that ultimately lead to the GBA deficiency, compromised glucosylceremide (GlcCer) metabolism and the subsequent failure of lysosomal mediated degradation of GBA substrates including alpha-synuclein. Depletion of GBA results in ¿-synuclein accumulation and neurodegeneration suggesting compromised GBA cascade contributes to the pathogenesis of ¿-synucleinopathies. We and others have found that brains of PD patients with or without mutations in GBA, as well as brains of A53T alpha-synuclein transgenic (Tg) mice, exhibit reduced GBA activity suggesting a role of the enzyme in the pathogenesis of the disease. Interestingly, the loss of catalytic activity of GBA correlated with its quantitative protein reduction, suggesting that unidentified key modulators might play an important role in GBA protein levels and activity though posttranslational modifications (PTMs). Accordingly, we investigated the mechanisms underlying the loss of GBA activity and protein levels by identifying key GBA modulators using tandem affinity purification (TAP) analysis. In preliminary data, we discovered GIP1 (GBA interacting protein 1) and show that GIP1 belongs to a new class of E3 ligases. In this project, we propose to explore the regulation of the GBA by GIP1 and their roles in regulating the pathogenesis of ¿-synuclein in PD and LBD. In specific aim 1 we will determine whether GIP1 interacts with and ubiquitinates GBA in vitro and in vivo. In specific aim 2 we will examine whether GIP1 targets GBA for degradation, thus regulating GBA activity and its substrates, GlcCer. Since GBA is a lysosomal enzyme that potentially regulates the expression of alpha synuclein, we will also explore whether GIP1 mediated GBA degradation ultimately regulates alpha-synuclein expression and aggregation. To accomplish these specific aims we will utilize lenti-GIP1 shRNA virus to knockdown GIP1 and a herpes simplex-GIP1 virus to overexpress GIP1. In preliminary studies we discovered that GIP1 accumulates in A53T alpha-synuclein Tg mice, PD brains and neurons treated with alpha-synuclein preformed fibrils (PFFs). Thus, in specific aim 3 we will further evaluate whether alpha-synuclein pathology is associated with accumulation of GIP1 and if its accumulation correlates with GBA deficiency and the severity of the alpha-synuclein pathology in A53T alpha-synuclein Tg mice and PD patients, as well as ¿-synuclein PFFs treated neurons. Moreover, preliminary data indicates that depletion of GIP1 significant reduces alpha-synuclein PFFs induced neuronal death. Thus, in specific aim 3, we will also evaluate the role of GIP1 in alpha-synuclein PFFs induced neuronal death. Ultimately this project will determine the full implications of the GIP1-GBA-alpha-synulcein neurodegenerative pathway and will identify new targets for therapeutic intervention in PD and LBD.