Mechanisms of non-cell-autonomous regulation of brain protein aggregation in Drosophila

大脑蛋白质聚集物的积累是阿尔茨海默病和许多其他神经退行性疾病的标志。大脑蛋白质聚集最初被认为是阿尔茨海默病中的一种细胞自主现象，但越来越多的证据表明，非细胞自主过程也通过促进有毒聚集倾向蛋白的传播发挥重要作用。尽管在阿尔茨海默病患者体内积累的大脑蛋白质聚集体的主要成分已在细胞外小泡(EVS)中被检测到，但阿尔茨海默病蛋白质聚集体扩散的机制尚不清楚。我们实验室最近的工作表明，GBA基因在这一过程中发挥着重要作用。GBA编码溶酶体酶葡萄糖脑苷酶，该酶催化鞘脂葡萄糖神经酰胺转化为葡萄糖和神经酰胺。GBA的突变会导致几种神经退行性疾病，其特征是蛋白质聚集体在大脑中积累。为了研究GBA突变致病的机制，我们通过删除果蝇GBA同源基因GBA1b建立了果蝇葡萄糖脑苷酶缺乏症模型。GBA1b突变体积累泛素化的蛋白质聚集体，表现出与年龄相关的神经变性，EV蛋白的周转和丰度发生变化，EV丰度增加6倍。此外，GBA1b在肌肉或肠道等外周组织中的异位表达拯救了大脑中的蛋白质聚集。这些发现导致我们假设，GBA1b的突变导致脂体改变，促进细胞外小泡的过度生产，从而将蛋白质聚集体从外周组织传播到大脑，并可能在大脑中的细胞之间传播。为了检验这一假说并探索其内在机制，我们提出了三个目标。首先，我们将测试GBA1b突变体中脑蛋白质聚集的非细胞自主挽救是否由细胞外小泡介导，以及这一途径是否影响其他常见神经退行性疾病中的蛋白质聚集，包括阿尔茨海默病。其次，我们将测试GBA1b突变体是否通过阻止GBA1b突变体中EV的产生以及通过将EV从GBA1b突变体移植到WT苍蝇来促进内源性蛋白质聚集体的传播，以及在阿尔茨海默病和普里恩病中看到的那些蛋白质聚集体的传播。第三，我们将进行蛋白质组、脂体学和细胞生物学实验，以探索GBA1b的突变如何改变细胞外小泡的形成和组成。鉴于越来越多的证据表明，阿尔茨海默病和其他神经退行性疾病的外周影响大脑蛋白质聚集体的扩散，我们预计我们的发现将促进我们对这一现象的理解，并为这些疾病的治疗干预创造新的机会。

Accumulation of brain protein aggregates is a hallmark of Alzheimer’s disease and many other neurodegenerative disorders. Brain protein aggregation was originally assumed to be a cell-autonomous phenomenon in Alzheimer’s disease, but increasing evidence indicates that non–cell-autonomous processes also play major roles by promoting the spread of toxic aggregation-prone proteins. Although the principal components of the brain protein aggregates that accumulate in Alzheimer’s disease victims have been detected in extracellular vesicles (EVs), the mechanisms underlying the spread of protein aggregates in Alzheimer’s disease are poorly understood. Recent work in our lab suggests that the GBA gene plays an important role in this process. GBA encodes the lysosomal enzyme glucocerebrosidase, which catalyzes the conversion of the sphingolipid glucosylceramide to glucose and ceramide. Mutations in GBA cause several neurodegenerative diseases characterized by the accumulation of protein aggregates in the brain. To study the mechanisms by which mutations in GBA cause disease, we created a Drosophila model of glucocerebrosidase deficiency by deleting the Drosophila GBA ortholog, GBA1b. GBA1b mutants accumulate ubiquitinated protein aggregates, show age-related neurodegeneration, have changes in the turnover and abundance of EV proteins and exhibit a six-fold elevation in EV abundance. Furthermore, ectopic expression of GBA1b in peripheral tissues such as muscle or gut rescued protein aggregation in the brain. These findings lead us to hypothesize that mutations in GBA1b result in lipidomic alterations promoting the overproduction of extracellular vesicles that spread protein aggregates from peripheral tissues to the brain and possibly between cells in the brain. To test this hypothesis and explore the underlying mechanisms, we propose three aims. First, we will test whether the non–cell-autonomous rescue of brain protein aggregation in GBA1b mutants is mediated by extracellular vesicles, and whether this pathway influences the protein aggregates seen in other common neurodegenerative diseases, including Alzheimer’s disease. Second, we will test whether GBA1b mutants promote the spread of endogenous protein aggregates, as well as those seen in Alzheimer’s and prion disease, by blocking the production of EVs in GBA1b mutants and by transplanting EVs from GBA1b mutants to WT flies. Third, we will perform proteomic, lipidomic and cell biological experiments to explore how mutations in GBA1b alter extracellular vesicle formation and composition. Given the increasing evidence of peripheral influences on the spread of brain protein aggregates in Alzheimer’s disease and other neurodegenerative disorders, we anticipate that our findings will advance our understanding of this phenomenon and also create novel opportunities for therapeutic intervention in these diseases.