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

描述（由申请方提供）：葡萄糖脑苷脂酶（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作为一种溶酶体酶在溶酶体降解途径中发挥关键作用。GBA的大多数致病突变被认为是功能缺失突变，其最终导致GBA缺乏、受损的葡萄糖基脑酰胺（GlcCer）代谢和随后的溶酶体介导的GBA底物（包括α-突触核蛋白）降解的失败。GBA的消耗导致<$-突触核蛋白积聚和神经变性，表明受损的GBA级联有助于<$-突触核蛋白病的发病机制。我们和其他人发现，有或没有GBA突变的PD患者的大脑，以及A53 T α-突触核蛋白转基因（Tg）小鼠的大脑，表现出GBA活性降低，表明该酶在疾病发病机制中的作用。有趣的是，GBA的催化活性的损失与其定量蛋白质的减少相关，这表明未鉴定的关键调节剂可能通过翻译后修饰（PTM）在GBA蛋白水平和活性中发挥重要作用。因此，我们通过使用串联亲和纯化（TAP）分析鉴定关键的GBA调节剂来研究GBA活性和蛋白水平损失的潜在机制。在初步数据中，我们发现了GIP 1（GBA相互作用蛋白1），并表明GIP 1属于一类新的E3连接酶。在这个项目中，我们建议探索GBA的GIP 1的调节和它们在调节<$-突触核蛋白在PD和LBD的发病机制中的作用。在具体目标1中，我们将确定GIP 1是否在体外和体内与GBA相互作用并泛素化GBA。在具体目标2中，我们将检查GIP 1是否靶向GBA降解，从而调节GBA活性及其底物GlcCer。由于GBA是一种可能调节α-突触核蛋白表达的溶酶体酶，我们还将探索GIP 1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，我们将利用慢GIP 1 shRNA病毒敲低GIP 1和单纯疱疹病毒GIP 1过表达GIP 1。在初步研究中，我们发现GIP 1在A53 T α-突触核蛋白Tg小鼠、PD脑和用α-突触核蛋白预形成的原纤维（PFF）处理的神经元中积累。因此，在具体目标3中，我们将进一步评估α-突触核蛋白病理学是否与GIP 1的积累相关，以及其积累是否与GBA缺乏和A53 T α-突触核蛋白Tg小鼠和PD患者以及α-突触核蛋白PFF处理的神经元中α-突触核蛋白病理学的严重程度相关。此外，初步数据表明GIP 1的消耗显著减少了α-突触核蛋白PFF诱导的神经元死亡。因此，在具体目标3中，我们还将评估GIP 1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GIP 1-GBA-alpha-synulcein神经退行性通路的全部意义，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.