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

脑蛋白聚集体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。脑蛋白聚集最初被认为是阿尔茨海默病的细胞自主现象，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易感蛋白的传播发挥重要作用。尽管已经在细胞外囊泡（EVs）中检测到阿尔茨海默病患者体内积聚的脑蛋白聚集体的主要成分，但人们对阿尔茨海默病中蛋白质聚集体扩散的机制知之甚少。我们实验室最近的研究表明，GBA基因在这一过程中起着重要作用。GBA编码溶酶体酶葡脑苷酶，该酶催化鞘脂糖基神经酰胺转化为葡萄糖和神经酰胺。GBA突变可引起几种以脑内蛋白质聚集体积累为特征的神经退行性疾病。为了研究GBA突变导致疾病的机制，我们通过删除果蝇GBA同源基因GBA1b，创建了一个葡萄糖脑苷酶缺乏症的果蝇模型。GBA1b突变体积累泛素化蛋白聚集体，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丰度升高6倍。此外，GBA1b在肌肉或肠道等外周组织中的异位表达挽救了大脑中的蛋白质聚集。这些发现使我们假设GBA1b的突变导致脂质组学改变，促进细胞外囊泡的过度产生，细胞外囊泡将蛋白质聚集体从外周组织传播到大脑，并可能在大脑细胞之间传播。为了验证这一假设并探索潜在的机制，我们提出了三个目标。首先，我们将测试GBA1b突变体中脑蛋白聚集的非细胞自主拯救是否由细胞外囊泡介导，以及该途径是否影响其他常见神经退行性疾病（包括阿尔茨海默病）中观察到的蛋白质聚集。其次，我们将通过阻断GBA1b突变体中EVs的产生，并将GBA1b突变体的EVs移植到WT果蝇中，来测试GBA1b突变体是否促进内源性蛋白聚集物的传播，以及在阿尔茨海默病和朊病毒疾病中看到的EVs。第三，我们将进行蛋白质组学、脂质组学和细胞生物学实验，探索GBA1b突变如何改变细胞外囊泡的形成和组成。鉴于越来越多的证据表明，在阿尔茨海默病和其他神经退行性疾病中，外周影响脑蛋白聚集体的传播，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.