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α -突触核蛋白病、PD和LBD的发病机制中起着重要作用。GBA作为溶酶体酶在溶酶体降解途径中起关键作用。大多数引起GBA疾病的突变被认为是功能突变的丧失，最终导致GBA缺陷，葡萄糖醛酸盐（glcer）代谢受损，以及随后溶酶体介导的GBA底物（包括α -突触核蛋白）降解失败。GBA耗竭导致-突触核蛋白积累和神经退行性变，表明GBA级联受损有助于-突触核蛋白病的发病机制。我们和其他人发现，GBA突变或不突变的PD患者的大脑，以及A53T α -突触核蛋白转基因（Tg）小鼠的大脑，都表现出GBA活性降低，这表明该酶在疾病发病机制中的作用。有趣的是，GBA催化活性的丧失与其定量蛋白减少相关，这表明未知的关键调节剂可能通过翻译后修饰（PTMs）在GBA蛋白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）分析鉴定关键的GBA调节剂，研究了GBA活性和蛋白质水平丧失的机制。在初步数据中，我们发现了GIP1 (GBA相互作用蛋白1)，并表明GIP1属于一类新的E3连接酶。本项目拟探讨GIP1对GBA的调控及其在PD和LBD中¿-synuclein发病机制中的作用。在具体的目标1中，我们将确定GIP1是否在体外和体内与GBA相互作用并泛素化。在具体目标2中，我们将研究GIP1是否靶向GBA降解，从而调节GBA活性及其底物glcer。由于GBA是一种溶酶体酶，可能调节α -突触核蛋白的表达，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。为了实现这些特定的目标，我们将利用lentii -GIP1 shRNA病毒来敲低GIP1，并利用单纯疱疹-GIP1病毒来过表达GIP1。在初步研究中，我们发现GIP1在A53T α -突触核蛋白Tg小鼠、PD脑和经α -突触核蛋白预形成原纤维（PFFs）处理的神经元中积累。因此，在具体的目的3中，我们将进一步评估α -synuclein病理是否与GIP1的积累有关，以及它的积累是否与A53T α -synuclein Tg小鼠和PD患者以及¿-synuclein pff治疗的神经元的GBA缺乏和α -synuclein病理的严重程度有关。此外，初步数据表明，GIP1的缺失可显著减少α -突触核蛋白pff诱导的神经元死亡。因此，在特定的目的3中，我们也将评估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 -突触蛋白神经退行性通路的全部含义，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.