Mechanisms of Neuronal Maintenance and Protection.

描述（由申请人提供）：神经退行性变可由多种遗传、表观遗传和环境因素触发。健康的神经元能够在生物体的整个生命过程中保持其完整性，这表明存在一种维护机制，允许神经元维持，减轻甚至修复损伤。最近，我们已经确定了一个神经元的维护因子NMNAT在果蝇的正向遗传筛选。nmnat的缺失导致快速和严重的神经变性，而NMNAT蛋白的过表达提供了针对神经变性的保护。这些结果表明，正常水平的NMNAT维持神经元的稳态，并提供保护的水平增加。NMNAT是一种高度保守的管家酶，NMNAT的神经保护功能也与缓慢沃勒变性的小鼠模型有关。目前，这种维护功能的详细机制和NMNAT在哺乳动物神经元中的保护能力尚不清楚。我们的初步实验表明，除了其NAD合成活性，NMNAT具有分子伴侣功能，参与调节蛋白质的错误折叠和降解。我们假设，像其他分子伴侣一样，NMNAT在压力下上调，减少蛋白质聚集，从而保护神经元免受退行性疾病的影响。在拟议的研究中，我们将使用果蝇和哺乳动物的初级神经元模型表征NMNAT介导的保护过程的生化和细胞机制。在具体目标1中，我们将使用结构-功能分析来定义伴侣功能所需的蛋白质结构域，并表征应激下NMNAT的转录调控。在具体目标2中，我们将首先确定哺乳动物NMNAT亚型在原代神经元中的神经保护活性，然后表征NMNAT在降低蛋白聚集诱导的神经毒性中的作用。在具体目标3中，我们将测试NMNAT蛋白在变性开始后诱导其表达时是否可以发挥保护活性。在最后一项研究中，我们将利用果蝇遗传系统并使用热诱导启动子控制NMNAT的表达。总之，我们在果蝇和哺乳动物模型系统中的研究将有助于揭示NMNAT的功能及其作为分子伴侣的调节，确定人NMNAT在原代神经元中的神经保护特性，并揭示NMNAT在神经元损伤后神经再生中的修复潜力。公共卫生相关性：神经退行性疾病是最难治的疾病之一，因此迫切需要开发有效的治疗方法。我们对神经元维持过程的研究表明，神经元有一个自我防御系统，可以增强以防止神经变性。该提案中的实验将帮助我们更好地了解神经退行性疾病中蛋白质折叠和聚集的分子事件，揭示哺乳动物神经元中潜在的神经保护机制，并有助于设计治疗性治疗。

DESCRIPTION (provided by applicant): Neurodegeneration can be triggered by a variety of genetic, epigenetic, and environmental factors. Healthy neurons are able to maintain their integrity throughout the life of an organism, suggesting the existence of a maintenance mechanism that allows neurons to sustain, mitigate or even repair damage. Recently, we have identified a neuronal maintenance factor NMNAT in a forward genetic screen in Drosophila. Loss of nmnat causes rapid and severe neurodegeneration, whereas over-expression of NMNAT protein offers protection against neurodegeneration. These findings suggest that normal level of NMNAT maintains neuronal homeostasis, and increased level offers protection. NMNAT is a highly conserved housekeeping enzyme, and the neuroprotective function of NMNAT has also been implicated in a mouse model of slow Wallerian Degeneration. Currently, the detailed mechanisms of this maintenance function and the protective capability of NMNAT in mammalian neurons are unclear. Our preliminary experiments suggest that in addition to its NAD synthesis activity, NMNAT has a chaperone function that is involved in regulating protein misfolding and degradation. We hypothesize that like other chaperones, NMNAT is up-regulated under stress, reduces protein aggregation, and thus protects neurons from degenerative conditions. In the proposed research, we will characterize the biochemical and cellular mechanisms underlying the protective process mediated by NMNAT using both Drosophila and mammalian primary neuronal models. In Specific Aim 1, we will use structure- function analysis to define the protein domains that are required for chaperone function, and characterize the transcriptional regulation of NMNAT under stress. In Specific Aim 2, we will first determine the neuroprotective activity of mammalian NMNAT isoforms in primary neurons, and then characterize the role of NMNAT in reducing protein aggregation-induced neurotoxicity. In Specific Aim 3, we will test whether NMNAT proteins can exert protective activity when their expression is induced after the onset of degeneration. For this last study, we will take advantage of the Drosophila genetic system and control the expression of NMNAT using a heat-inducible promoter. In summary, our proposed research in both Drosophila and mammalian model systems will help unmask the function of NMNAT and its regulation as a molecular chaperone, determine the neuroprotective properties of human NMNAT in primary neurons, and reveal the repair potential of NMNAT in neural regeneration after neuronal damage. PUBLIC HEALTH RELEVANCE: Neurodegenerative conditions are among the most intractable of diseases and therefore present an urgent need for developing effective treatments. Our studies on the neuronal maintenance process suggest that neurons have a self-defense system that can be augmented to protect against neurodegeneration. The experiments in this proposal will help us better understand the molecular events of protein folding and aggregation in neurodegenerative conditions, reveal potential neuroprotective mechanisms in mammalian neurons, and aid in the design of therapeutic treatments.