Mechanisms of Neuronal Maintenance and Protection

 描述(由申请人提供)：神经变性可由各种遗传、表观遗传和环境因素触发。健康的神经元能够在动物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。我们实验室和其他实验室之前的工作发现，果蝇和哺乳动物中的NMNAT蛋白是强大和多功能的神经保护因子。然而，目前尚不清楚神经元是否以及如何调节由NMNAT等维持因子介导的神经保护过程。神经元如何将NMNAT划分为两个不同的功能-管家(NAD合成)和神经保护，以及在正常和不利条件下如何调节这种划分以实现神经保护是本提案的重点。我们的初步数据表明，果蝇nmnat基因被交替剪接到两个mRNA变体(RA和RB)中，导致两组功能不同的蛋白质：PA/PC和PB/PD，前者具有更高的酶活性，后者具有增强的伴侣功能。我们假设转录后调控，包括选择性剪接，在NMNAT的神经保护和酶作用之间发挥切换作用，神经元在正常和疾病条件下使用这一切换机制来调节神经保护反应。我们建议在果蝇中描述假想的开关机制，然后将研究扩展到哺乳动物神经元中的人NMNAs。首先，我们将描述果蝇和人类NMNAT蛋白变体的生化和细胞特性，并说明选择性剪接在nmnat基因转录后调控中的作用。接下来，我们将确定选择性剪接是一种为神经元提供保护的应激反应，并进一步揭示microRNAs在调节神经保护RNA变体的丰度和调节NMNAT的神经保护效果方面的功能作用。最后，我们将研究人类NMNAT蛋白变体，并表征选择性剪接的NMNAT变体在培养的DRG外植体和体内皮质脊髓径迹神经元中的神经保护功能，并确定NMNAT变体在变性条件下不同神经保护作用的关键机制。对果蝇和人类NMNAs的研究--一种“双模式”方法--将揭示进化上保守的调节机制，并确定与加强人类神经保护相关的策略。

 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 animal, suggesting the existence of a maintenance mechanism that allows neurons to sustain, mitigate or even repair damage. Previous work in our lab and others has found NMNAT proteins in Drosophila and mammals to be robust and versatile neuroprotective factors. However, it remains unclear whether and how neurons regulate the neuroprotective processes mediated by maintenance factors such as NMNAT. How neurons partition NMNAT into two distinct functions - housekeeping (NAD synthesis) and neuroprotection, and how such partitioning is regulated under normal and adverse conditions to achieve neuroprotection are the foci of this proposal. Our preliminary data indicate that Drosophila nmnat gene is alternatively spliced into two mRNA variants (RA and RB) that lead to two sets of functionally distinct proteins: PA/PC, with higher enzyme activity, and PB/PD, with enhanced chaperone function. We hypothesize that post-transcriptional regulation, including alternative splicing, functions as a switch between the neuroprotective and enzymatic roles of NMNAT, and that neurons use this switch mechanism to regulate the neuroprotective response under normal and disease conditions. We propose to characterize the hypothesized switch mechanism in Drosophila and then extend the study to human NMNATs in mammalian neurons. First, we will characterize the biochemical and cellular properties of Drosophila and human NMNAT protein variants, and illustrate the role of alternative splicing in post- transcriptional regulation of nmnat genes. Next, we will identify alternative splicing as a stress response that affords protection to neurons, and further reveal the functional role of microRNAs in regulating the abundance of neuroprotective RNA variants and modulating the neuroprotective efficacy of NMNAT. Finally, we will study human NMNAT protein variants and characterize the neuroprotective function of alternatively spliced NMNAT variants in cultured DRG explants and in vivo in cortical spinal track neurons, and identify key mechanisms underlying the divergent neuroprotective effects of NMNAT variants in degenerative conditions. Studies on Drosophila and human NMNATs - a "two-model" approach - will reveal the evolutionarily conserved regulatory mechanisms and identify strategies relevant to enhancing neuroprotection in humans.