Nonproteolytic Polyubiquitin Chains at the Synapse

 描述（由申请人提供）：突触和神经回路的组装、功能和可塑性改变是基本上所有神经精神和神经系统疾病的认知、记忆和情感缺陷的基础。调节突触蛋白质周转和突触重塑的一个充分研究的机制是常规的泛素-蛋白酶体途径，通过该途径，多聚泛素链与蛋白质底物缀合（可能通过泛素的赖氨酸48（K48））并标记它们用于蛋白酶体降解。在中枢神经系统（CNS）中一个被极大忽视的泛素修饰是K63连接的多聚泛素化，这是一种非常规的连接机制，通常认为它不靶向蛋白酶体降解;相反，它调节蛋白质支架，运输和活性。尽管它在介导先天性和适应性免疫的细胞信号传导机制中的重要性已被公认，但实际上对这种蛋白酶体独立的多聚泛素化过程在CNS，特别是突触中的作用一无所知。尽管如此，K63连接的泛素化是大鼠大脑中第二丰富的连接，仅略低于K48连接。此外，新出现的证据表明K63相关的泛素化与许多大脑疾病之间存在联系。因此，有必要研究这种非蛋白水解的多聚泛素拓扑结构在神经元和突触中的机制和作用。我们的初步研究表明，K63连接的多泛素化是一个基本的机制，调节突触组装和可塑性，并确定突触后支架蛋白PSD-95作为第一底物。我们还确定了PSD相关的，K63连接特异性酶机制，控制PSD-95在突触泛素化。本R 01申请的目标是定义PSD-95 K63多聚泛素化的分子细节和功能后果（目标1），描述K63多聚泛素化在突触发育、功能、重塑和可塑性中的作用（目标2），并表征缺乏K63连接特异性E3泛素连接酶TRAF 6和去泛素化酶CYLD的小鼠的行为改变（目标3）。将采用分子、生物化学、电生理和行为方法的组合。这些研究代表了一个根本性的重要概念突破，为研究突触和电路功能以及可塑性开辟了新的途径。该项目有可能揭示新的范式转变原则，这些原则管理神经免疫相互作用，这可能有助于神经发育障碍（如精神分裂症和自闭症谱系障碍）中的异常大脑布线和损伤后成人大脑中的神经回路修复。所获得的信息将促进这些脑疾病的新的治疗策略的发展。

 DESCRIPTION (provided by applicant): Altered assembly, function, and plasticity of synapses and neural circuits underlie cognitive, memory, and emotional deficits of essentially all neuropsychiatric and neurological diseases. A well-investigated mechanism that regulates synaptic protein turnover and synapse remodeling is the conventional ubiquitin-proteasome pathway, by which polyubiquitin chains conjugate to protein substrates (likely through lysine 48 (K48) of ubiquitin) and tag them for proteasomal degradation. A vastly overlooked ubiquitin modification in the central nervous system (CNS) is K63-linked polyubiquitination, an unconventional linkage mechanism generally believed not to target proteasomal degradation; rather, it regulates protein scaffolding, trafficking, and activity. Despite its recognized importace in cellular signaling mechanisms that mediate innate and adaptive immunity, virtually nothing is known about the role of this proteasome-independent polyubiquitination process in the CNS, especially synapses. Nonetheless, K63-linked ubiquitination is the second most abundant linkage in the rat brain, only slightly behind the K48 linkage. In addition, emerging evidence points to a link between K63-linked ubiquitination and a number of brain disorders. Thus, there is a need to investigate the mechanisms and roles of this nonproteolytic polyubiquitin topology in neurons and at synapse. Our preliminary studies indicate that K63-linked polyubiquitination is a fundamental mechanism regulating synapse assembly and plasticity and identify the postsynaptic scaffolding protein PSD-95 as the first substrate. We also identify a PSD-associated, K63-linkage-specific enzyme machinery that controls PSD-95 ubiquitination at synapses. The goals of this R01 application are to define the molecular details and functional consequences of PSD-95 K63 polyubiquitination (Aim 1), to delineate the role of K63 polyubiquitination in synapse development, function, remodeling, and plasticity (Aim 2), and to characterize the behavioral alterations in mice lacking K63-linkage-specific E3 ubiquitin ligase TRAF6 and deubiquitinase CYLD (Aim 3). A combination of molecular, biochemical, electrophysiological, and behavioral approaches will be employed. The proposed studies represent a fundamentally important conceptual breakthrough that opens up new avenues for investigation of synapse and circuit function and plasticity. The project has the potential to uncover new paradigm-shifting principles that govern neuro-immune interactions, which may contribute to abnormal brain wiring in neurodevelopmental disorders (such as schizophrenia and autism spectrum disorders) and neural circuit repair in the adult brain following injury. The information obtained will facilitate development of novel treatment strategies for these brain disorders.