Regenerative and Degenerative Responses to Axonal Injury

 描述(申请人提供)：轴突损伤后的功能修复需要在受损轴突的近端和远端做出不同的反应。近端轴突必须启动新的轴突生长，然而在许多情况下，这不会发生，特别是在成年哺乳动物的中枢神经系统中。此外，必须清除远端轴突残端。这是通过细胞自主启动的轴突碎裂过程发生的，该过程被称为“沃勒式变性”。虽然这种变性在清除不可修复的受损轴突方面起到了有益的作用，但轴突的丧失通常是神经疾病和神经退行性疾病的有害特征。该项目的长期目标是了解检测轴突损伤的细胞机制，并促进变性和修复的二分性结果。该实验室之前的工作，使用果蝇模型，描绘了一条保守的分子途径，调节受损轴突的再生和退化。目的1验证由初步数据提出的假说，即哺乳动物中的WND及其DLK同系物直接受cAMP信号下游的PKA调控。由于cAMP信号的升高与三叉神经核的成功再生相关，这一机制可能有助于深入了解WND/DLK激活的促再生结果如何具体偏向。目的2验证轴突保护机制的新假说，特别是Nmnat可减弱损伤触发的内质网钙内流，并进一步将钙稳态与受损轴突中的ATP降解和线粒体运输联系起来。这些方法利用了果蝇幼虫的遗传和活体成像技术，允许在活体动物受损的轴突和突触内操纵和跟踪亚细胞事件(包括细胞内钙离子、ATP和线粒体运输的变化)。此外，该项目还在小鼠背根神经节培养中启动了补充研究，以研究受DLK调控的单个细胞事件中的轴突再生。这项工作有望揭示影响轴突损伤的再生和退行性反应的重要细胞机制。

 DESCRIPTION (provided by applicant): Functional repair after axonal damage requires disparate responses in the proximal and distal parts of the damaged axon. The proximal axon must initiate new axonal growth, however in many cases this fails to occur, particularly in the adult mammalian CNS. In addition, the distal axonal stump must be cleared out of the way. This occurs via a cell-autonomously initiated axonal fragmentation process termed `Wallerian degeneration'. While this degeneration plays a beneficial role in clearing irreparably damaged axons, the loss of axons is generally a deleterious feature of neuropathies and neurodegenerative diseases. The long term goals of this project are to understand the cellular mechanisms that detect axonal damage and facilitate the dichotomous outcomes of degeneration verses repair. Previous work in the lab, using a Drosophila model, has delineated a conserved molecular pathway that regulates both the regeneration and degeneration of damaged axons. The current grant focuses upon two important components of this pathway: (1) Wnd/DLK, a conserved axonal kinase whose can mediate either regenerative or degenerative responses to axonal injury, hence has been coined a `dual-edged sword', and (2) the NAD+ biosynthetic enzyme, Nmnat [2, 3], whose rapid turnover in distal axons appears to play an important role in initiating degeneration, however the cellular mechanism for Nmnat's protective function(s) is not known. Aim 1 tests a hypothesis, raised by preliminary data, that both Wnd and its DLK homologue in mammals are regulated directly by PKA downstream of cAMP signaling. Because elevated cAMP signaling correlates with successful regeneration in the PNS, this mechanism may yield insight into how pro-regenerative outcomes of Wnd/DLK's activation can be specifically biased. Aim 2 tests a new hypothesis for the protective mechanism in axons, specifically that Nmnat attenuates intracellular Ca2+ influx from ER stores that are triggered by injury, and further links calcium homeostasis to ATP rundown and mitochondrial trafficking in damaged axons. The approaches take advantage of genetic and live imaging techniques in Drosophila larvae which allow for subcellular events (including changes in intracellular Ca2+, ATP, and mitochondrial trafficking) to be manipulated and tracked within injured axons and synapses in live animals. In addition, the project initiates complementary studies in mouse DRG cultures to study axonal regeneration in the context of individual cellular events regulated by DLK. This work is expected to reveal important cellular mechanisms that influence both regenerative and degenerative responses to axonal damage.