Mechanism and importance of innate immune activation in a Drosophila GBA mutant model of Parkinson's disease

摘要： 葡萄糖神经酰胺酶β(GBA)基因突变导致溶酶体脂肪储存障碍 是帕金森氏病和路易体痴呆症最常见的基因关联。 GBA编码葡萄糖脑苷酶，这是一种催化鞘糖脂分解的溶酶体酶 将葡萄糖神经酰胺转化为神经酰胺和葡萄糖。探讨GBA基因突变易感的机制 针对这些疾病，我们建立了一种果蝇葡萄糖脑苷酶缺乏症模型，通过灭活 果蝇Gba同源基因，Gba1b。Gba1b突变体概括了这些疾病的许多特征， 包括寿命缩短、运动障碍、葡萄糖神经酰胺堆积、蛋白质聚集 在大脑和其他组织中，以及神经退化。在最近发表的工作中，我们报告了一项 果蝇Gba1b突变体的蛋白质组学研究表明，Gba1b的丰度和 胞外囊泡(EV)蛋白的周转。我们的实验还证明了这些蛋白质组 研究结果反映了电动汽车组成的实际变化，以及针对因素的基因扰动 参与EVS的生产抑制了Gba1b突变表型。在最近未出版的著作中， 我们使用RNA-Seq来比较Gba1b突变体和对照中的转录丰度。这项研究揭示了 深刻诱导gba1b突变体的先天免疫反应途径。这一归纳是特定于 Gba1b突变体，并在我们的蛋白质组数据中得到进一步证实，以及RNAi介导的 天然免疫途径成分部分抑制Gba1b脑蛋白聚集表型 变种人。根据这些和其他发现，我们假设葡萄糖神经酰胺的产生富含 Gba1b突变体的EV会触发一种先天免疫反应，因为这些EV类似于 病原体在感染过程中释放的富含葡萄糖神经酰胺的肠道病毒。我们进一步假设这一点 Gba1b突变体的表型与先天免疫反应有关。我们提出了两个目标来解决 这些假说；第一个将研究免疫激活的机制，第二个将 探讨免疫激活在Gba1b突变发病机制中的重要性。因此，其主要目标是 我们的研究建议是通过问两个最多的问题来为进一步的机械化工作提供基础 我们的初步发现提出了一些基本问题：Gba1b是如何发生天然免疫激活的 突变体，这对他们的表型重要吗？鉴于越来越多的证据表明 神经退行性疾病，包括那些由GBA突变引起的疾病，我们预计我们的工作将有 具有广泛的医学意义。

ABSTRACT: Mutations in the glucosylceramidase beta (GBA) gene cause the lysosomal lipid storage disorder Gaucher’s disease and are the most frequent genetic association with Parkinson’s disease and Lewy body dementia. GBA encodes glucocerebrosidase, a lysosomal enzyme that catalyzes the breakdown of the sphingolipid glucosylceramide to ceramide and glucose. To explore the mechanism by which mutations in GBA predispose to these diseases, we created a Drosophila model of glucocerebrosidase deficiency by inactivating the Drosophila GBA ortholog, Gba1b. Gba1b mutants recapitulate many of the features of these diseases, including shortened lifespan, locomotor impairment, accumulation of glucosylceramide, protein aggregation in brain and other tissues, and neurodegeneration. In recently published work, we reported the results of a proteomic study of Drosophila Gba1b mutants that revealed dramatic alterations in the abundance and turnover of extracellular vesicle (EV) proteins. Our experiments also demonstrated that these proteomic findings reflected actual changes in the composition of EVs, and that genetic perturbations targeting factors involved in the production of EVs suppressed a Gba1b mutant phenotype. In more recent unpublished work, we used RNA-Seq to compare transcript abundance in Gba1b mutants and controls. This study revealed a profound induction of the innate immune response pathway in Gba1b mutants. This induction was specific to Gba1b mutants and was further corroborated in our proteomic data, and RNAi-mediated knockdown of an innate immune pathway component partially suppressed the brain protein aggregation phenotype of Gba1b mutants. From these and other findings, we hypothesize that the production of glucosylceramide-enriched EVs by Gba1b mutants triggers an innate immune response because these EVs resemble the glucosylceramide-enriched EVs released by pathogens during infection. We further hypothesize that this innate immune response accounts for the phenotypes of Gba1b mutants. We propose two aims to address these hypotheses; the first will investigate the mechanism of immune activation, and the second will investigate the importance of immune activation to Gba1b mutant pathogenesis. Thus, the primary goal of our research proposal is to provide a foundation for further mechanistic work by asking the two most fundamental questions raised by our preliminary findings: how does innate immune activation occur in Gba1b mutants, and is it important to their phenotypes? Given the increasing evidence for neuroinflammation in neurodegenerative disorders, including those caused by GBA mutations, we anticipate that our work will have broad medical significance.