Unconventional protein secretion-mediated protein quality control in health and diseases

路易体（Lewy bodies，LB）是帕金森病（Parkinsons disease，PD）的典型标志。这些内含物的主要成分是α-突触核蛋白（α-syn），一种具有错误折叠和聚集内在倾向的蛋白质。在PD患者中，首先在嗅球和背侧运动核中观察到的α-syn包涵体逐渐扩散到整个大脑。进一步的研究发现，健康的胚胎多巴胺神经元移植到PD患者发展LB点表明神经元到神经元的α-syn传输的诱人的可能性。随后的工作证实，合成的a-syn预形成的原纤维（PFF）可以被神经元吸收，引起内源性a-syn错误折叠成不溶性路易样内含物。总的来说，这些研究导致了PD的朊病毒假说，其中错误折叠的-syn提供了用于接种新聚集体、传播错误折叠的a-syn和相关细胞毒性的模板。因此，-syn传播是在开发新的PD疗法中待探索的可行的新靶点。 突触核蛋白的细胞间传递包括两个关键步骤：供体神经元分泌突触核蛋白和受体神经元摄取突触核蛋白。 错误折叠相关蛋白分泌（MAPS）是最近发现的蛋白质质量控制过程，其选择性地输出错误折叠的胞质蛋白，包括α-syn。通过MAPS的分泌需要膜定位的去泛素化酶USP 19，其将异常多肽募集到内质网（ER）表面以促进其并入与ER紧密结合的晚期内体。 当晚期内体与质膜融合时，错误折叠的蛋白质被分泌到细胞外环境。 释放的错误折叠蛋白的命运目前尚不清楚。 我们最近的研究表明，哺乳动物细胞可以通过内吞作用内化错误折叠蛋白，但目前还不清楚它们是否具有一个或多个错误折叠蛋白的受体。 内化的蛋白质是否能在被溶酶体降解之前造成损伤也不清楚。 该建议是阐明的生理相关性的MAPS途径的特征之间的相互作用分泌的错误折叠的蛋白质和靶细胞。 使用基于邻近的连接方法，我们使用纯化的Tau作为诱饵来鉴定由微管相关蛋白Tau形成的候选蛋白质聚集体可以在人脑中以刻板模式传递，这与统称为Tau病的几种神经退行性疾病的进展相关。这一过程需要从神经元释放的Tau与靶细胞上的细胞表面受体相互作用，但对特别是Tau参与神经胶质细胞的潜在机制和下游病理生理后果知之甚少。使用空间分辨蛋白质组学作图策略，我们确定整合素V/1，整合素家族的异二聚体，作为受体，不仅介导Tau纤维进入星形胶质细胞，而且激活整合素信号转导后Tau结合。我们表明，不同的Tau物种差异激活整合素信号转导，与丝状Tau是一个更强大的刺激。这导致NFB活化和促炎细胞因子和趋化因子的差异上调。此外，神经毒性星形胶质细胞标志物的亚组也优先由丝状Tau以整联蛋白依赖性方式诱导，导致星形胶质细胞释放神经毒性因子。总之，这些发现建立了一个范例，即星形胶质细胞可以通过丝状Tau经由整合素受体直接转化为神经毒性状态，其作为转导未折叠蛋白病理的传感器，可以为Tau病提供新的治疗靶点。 在帕金森病中，突触特异性的、易错误折叠的蛋白-突触核蛋白（-Syn）的细胞-细胞传递包括两个表征不佳的细胞过程：通过非常规蛋白分泌释放-Syn和通过网格蛋白依赖性内吞作用摄取带有-Syn的原纤维。使用CRISPR介导的遗传筛选，我们将SLC 35 B2鉴定为非神经元细胞和神经元中预形成的Syn原纤维（PFF）的内吞作用的关键调节剂。SLC 35 B2介导细胞表面硫酸乙酰肝素蛋白聚糖（HSPG）的合成，其带有线性负电荷的糖部分可以插入寡聚化-Syn中由成簇的赖氨酸残基形成的沟中。有趣的是，-Syn纤维和其他携带聚阳离子的HSPG货物的摄取也涉及肌球蛋白马达。这种肌球蛋白与肌动蛋白丝一起作用，在富含网格蛋白的质膜结构域维持膜动力学。在没有肌球蛋白的情况下，一部分网格蛋白包被的小凹不能从质膜上切断，导致巨大的网格蛋白阳性瘢痕在细胞表面上积累。我们的数据表明，肌动蛋白介导的膜动力学促进细胞表面网格蛋白包被的小凹的成熟和功能，以促进一系列HSPG货物的内吞作用。

Proteinaceous inclusions termed Lewy bodies (LBs) is a classical hallmark of Parkinsons disease (PD). The primary component of these inclusions is alpha-synuclein (a-syn), a protein with an intrinsic propensity to misfold and aggregate. In PD patients, alpha-syn inclusions first observed in the olfactory bulb and the dorsal motor nucleus, progressively spread throughout the brain. Further findings that healthy embryonic dopamine neurons transplanted into PD patients developed LBs points suggest the tantalizing possibility of neuron-to-neuron transmission of a-syn. Subsequent work comfirmed that synthetic a-syn pre-formed fibrils (PFFs) can be taken up by neurons, eliciting the misfolding of endogenous -syn into insoluble Lewy-like inclusions. Collectively, these studies led to the prion hypothesis of PD, wherein misfolded -syn provides a template for seeding new aggregates, propogating misfolded a-syn and associated cytotoxicity. Thus, that propagation of -syn is a viable new target to be explored in the development of new PD therapies. The intercellular transmission of synuclien consists of two key steps: the secretion of synuclein from a donor neuron and its uptake by a recipient neuron. Misfolding-associated protein secretion (MAPS) is a recently discovered protein quality control process that selectively exports misfolded cytosolic proteins including a-syn. Secretion through MAPS requires the membrane localized deubiquitinase USP19, which recruits aberrant polypeptides to endoplasmic reticulum (ER) surface to facilitate their incorporation into to late endosomes that are in tight association with the ER. Misfolded proteins are secreted to the extracellular milieu when late endosomes fuse with the plasma membrane. The fate of the released misfolded proteins is current unknown. Our recent studies suggest that mammalian cells can internalize misfolded proteins via endocytosis, but it is unclear whether they possess one or more receptors for misfolded proteins. Whether internalized proteins can impose damage prior to degradation by the lysosome is also unclear. The proposal is to elucidate the physiological relevance of the MAPS pathway by characterizing the interplay between secreted misfolded proteins and target cells. Using a proximity based ligation approach, we have used purified Tau as a bait to identify candidate Protein aggregates formed by microtubule-associated protein Tau can be transmitted in a stereotypic pattern in human brains, which correlates with the progression of several neurodegenerative diseases collectively termed Tauopathies. This process requires Tau, released from neurons, to interact with a cell surface receptor on a target cell, but little is known about the underlying mechanisms and the downstream pathophysiological consequences particularly for Tau that engages glial cells. Using a spatially resolved proteomic mapping strategy, we identify integrin V/1, a heterodimer of the integrin family, as a receptor that not only mediates Tau fibril entry into astrocytes, but also activates integrin signaling upon Tau binding. We show that distinct Tau species differentially activates integrin signaling, with filamentous Tau being a more robust stimulator. This leads to NFB activation and differential upregulation of pro-inflammatory cytokines and chemokines. Additionally, a sub-group of neurotoxic astrocyte markers are also induced in an integrin-dependent manner preferentially by filamentous Tau, causing astrocytes to release a neurotoxic factor(s). Together, these findings establish a paradigm that astrocytes can be directly converted into a neurotoxic state by filamentous Tau via an integrin receptor, which as a sensor transducing unfolded protein pathology, may provide a new therapeutic target for Tauopathies. In Parkinsons disease, cell-to-cell transmission of synapse specific, misfolding-prone protein -synuclein (-Syn) comprises two poorly characterized cellular processes release of -Syn via unconventional protein secretion and uptake of -Syn-bearing fibrils via Clathrin-dependent endocytosis. Using CRISPR-mediated genetic screen, we identify SLC35B2 as a critical regulator for endocytosis of preformed -Syn fibril (PFF) in both non-neuronal cells and neurons. SLC35B2 mediates the synthesis of cell surface heparan sulfate proteoglycans (HSPG), whose sugar moiety, bearing linearized negative charges, can insert into a groove formed by clustered Lysine residues in oligomerized -Syn. Intriguingly, the uptake of -Syn fibrils and other polycation-carrying HSPG cargos also involves a Myosin motor. This Myosin acts in conjunction with actin filaments maintain membrane dynamics at plasma membrane domains enriched in Clathrin. Without Myosin, a fraction of Clathrin-coated pits fail to be severed from the plasma membranes, causing accumulation of giant Clathrin positive scars on the cell surface. Our data suggest that actin-mediated membrane dynamics facilitates maturation and function of Clathrin-coated pits at the cell surface to promote endocytosis of a collection of HSPG cargos.