Mechanisms of mutanthuntingtin aggregate engulfment and spreading by phagocytic glia

项目总结 神经退行性疾病的特征是逐渐出现不溶性包裹体。 由于蛋白质在大脑中的错误折叠。许多研究都集中在确定这些机制 蛋白质损伤会导致神经元和胶质细胞功能障碍和丧失。越来越多的证据支持这一假设 与大多数神经退行性疾病相关的蛋白质组合通过模板在大脑中传播 相同蛋白质的适当折叠形式的聚集，类似于传染性的普恩病毒。然而，它仍然是 还不能很好地理解如何在大脑中不同的细胞类型之间传播类似普里恩的聚集体或其相关性 这与神经发病机制有关。我们研究的长期目标是确定蛋白质的潜在机制。 聚集体诱导的毒性，并在变性大脑中的神经元和神经胶质细胞之间扩散。整体而言 本项目的目的是确定吞噬细胞的正常和病理功能，以响应 突变型亨廷顿蛋白(MHTT)聚集体在神经元中的形成。先前的研究表明，神经胶质细胞对 随着时间的推移，神经损伤从有益变为有害，并且有新的证据表明吞噬细胞参与了 总体清理和扩散为当前项目提供了理由。中心假设是 吞噬胶质细胞对吞噬的神经元mHTT聚集体的不完全清除促进形成和生长 大脑中具有播种能力的类Pron物种的储存库。这一假设是基于以下假设提出的 研究发现，胶质细胞介导mHTT、tau和a-突触核蛋白聚合体的Pron样转移，并吞噬 随着年龄的增长和处于疾病状态，清除被吞噬的碎片的效果变得不那么有效。我们最近的研究表明 突触连接神经元之间mHTT聚集体的普里子样转移需要Draper依赖 吞噬作用，一种进化保守的清除大脑碎片的神经胶质途径。中心假说 将在三个特定的目标上进行测试，在体内互补的果蝇和体外哺乳动物细胞中进行 文化典范。在目标1中，神经元mHTT聚集体对神经损伤的神经胶质反应的影响将是 在成年果蝇大脑中进行测量，以深入了解mHTT聚集体吞噬是否会导致神经胶质功能障碍。 在目标2中，基于RNAi的正向遗传筛选将识别被吞噬的神经元的Pron样扩散的修饰物 MHTT聚集在神经胶质细胞质中。在目标3中，哺乳动物Draper同源基因MEGF10在mHTT中的作用 共同培养的神经细胞和星形胶质细胞之间的聚集转移将被确定。这些研究将 确定吞噬作用的基础生物学的新机制，以及与疾病相关的机制 驱动蛋白质聚集导致多种细胞类型的神经退化。拟议的研究是 意义重大，因为它解决了吞噬胶质细胞如何与神经元相互作用以驱动 潜在的许多神经疾病的发病机制。我们的发现将揭示关于 聚集体相关的发病机制是神经退行性变的基础，并具有确定靶点的巨大潜力 治疗这些致命疾病的根本原因的新疗法。

PROJECT SUMMARY Neurodegenerative diseases are characterized by the progressive appearance of insoluble inclusions that arise due to protein misfolding in the brain. Much research has focused on determining mechanisms by which these protein lesions cause dysfunction and loss of neurons and glia. Accumulating evidence supports the hypothesis that protein assemblies associated with most neurodegenerative diseases propagate in the brain by templating the aggregation of properly-folded versions of the same protein, similarly to infectious prions. However, it is still not well understood how “prion-like” aggregates spread between different cell types in the brain or the relevance of this to neuropathogenesis. The long-term goal of our research is to identify mechanisms underlying protein aggregate-induced toxicity and spreading between neurons and glia in the degenerating brain. The overall objective of this project is to determine normal and pathological functions of phagocytic glia in response to formation of mutant huntingtin (mHTT) aggregates in neurons. Prior research showing that glial responses to neural injury shift from beneficial to harmful over time, and emerging evidence that phagocytic glia participate in aggregate clearance and spreading provide rationale for the current project. The central hypothesis is that incomplete clearance of engulfed neuronal mHTT aggregates by phagocytic glia promotes formation and growth of a reservoir of seeding-competent, prion-like species in the brain. This hypothesis was formulated based on findings that glia mediate prion-like transfer of mHTT, tau, and a-synuclein aggregates, and that phagocytic clearance of engulfed debris becomes less effective with age and in the disease state. Our recent work indicates that prion-like transfer of mHTT aggregates between synaptically-connected neurons requires Draper-dependent phagocytosis, an evolutionarily-conserved glial pathway for clearing debris in the brain. The central hypothesis will be tested in three Specific Aims, carried out in complementary in vivo Drosophila and in vitro mammalian cell culture models. In Aim 1, effects of neuronal mHTT aggregates on glial responses to neural injury will be measured in adult fly brains to gain insight into whether mHTT aggregate engulfment leads to glial dysfunction. In Aim 2, RNAi-based forward genetic screens will identify modifiers of prion-like spreading of engulfed neuronal mHTT aggregates to the glial cytoplasm. In Aim 3, roles of the mammalian Draper homolog MEGF10 in mHTT aggregate transfer between co-cultured neuronal and astrocytic cells will be determined. These studies will identify novel mechanisms about the basic biology underlying phagocytosis, and disease-related mechanisms that drive protein aggregate-induced neurodegeneration in multiple cell types. The proposed research is significant because it addresses critical questions of how phagocytic glia interact with neurons to drive pathogenesis in potentially many neurological disorders. Our findings will reveal new information about aggregate-related pathogenesis underlying neurodegeneration and have great potential to identify targets for novel therapies that treat the underlying cause of these fatal disorders.