Regenerative and degenerative responses to axonal injury

项目概要/摘要： 轴突在大脑和身体中的远距离神经元之间形成连接，因此容易受到 伤害和压力。该项目研究了一种进化上保守的应激反应途径， 在轴突损伤和压力的多种情况下激活。由双亮氨酸拉链控制的途径 激酶DLK，在果蝇中称为Wallenda（Wnd），参与神经元的结构可塑性机制 让神经回路适应轴突损伤这些反应包括轴突再生，神经元死亡， 以及这个项目新发现的突触缺失本项目的长期目标是（1）了解 导致DLK信号传导激活的机制，以及（2）了解 由DLK监管。该项目结合了对果蝇和小鼠的研究，重点是运动神经元（MN） 周围神经损伤（PNI）。对于第一个目标，目标1测试DLK/Wnd信号传导的假设 受到完整突触连接的抑制，因此在突触连接后被激活。 损失这些实验建立在对果蝇突触丢失的互补范例的观察基础上 神经肌肉接头（NMJ）突触：（a）对分支轴突的损伤表明，只有完全的 去除所有传出连接能够激活Wnd信号传导;（B）多个细胞骨架突变 导致NMJ突触回缩和退化的神经元也导致Wnd信号传导激活。拟议 实验将区分突触相互作用如何与轴突运输过程交叉， 激活WND。目的2研究DLK调控的下游反应，使结构 可塑性，并侧重于新的表型DLK在小鼠脊髓：突触输入的损失， 轴突切断的MN的细胞体（称为“突触剥离”）依赖于MN中的DLK功能。在 此外，在突触丧失之前，激活的小胶质细胞向MN细胞体的募集需要 DLK在轴突切断的MN中的功能。目的2将测试DLK信号转导门控蛋白的分泌的假设。 在小胶质细胞中募集特异性反应以促进突触丢失的分子信号。实验将 评估从RiboTag中鉴定的候选分泌和免疫分子的需求 翻译谱分析方法是轴突切断的MN中DLK调节的强靶标。实验将 还通过分离的神经元的单细胞RNA-seq鉴定了轴突切断的MN中DLK门控的小胶质细胞反应。 小胶质细胞总的来说，这项工作有望为神经元-小胶质细胞相互作用提供新的线索， 神经系统损伤，并通过特定的透镜的结构可塑性和突触损失的机制， 特异性轴突损伤信号通路。

Project Summary/Abstract: Axons form connections between neurons over great distances in the brain and body, hence are vulnerable to damage and stress. This project studies an evolutionarily conserved stress response pathway that becomes activated in multiple scenarios of axonal damage and stress. The pathway, governed by the dileucine zipper kinase DLK, known as Wallenda (Wnd) in Drosophila, engages structural plasticity mechanisms in neurons that allow circuits to adapt to axon damage. These responses include axonal regeneration, neuronal death, and, newly discovered in this project, synapse loss. The long-term goals of this project are (1) to understand the mechanisms that lead to DLK signaling activation, and (2) to understand the cellular pathways that are regulated by DLK. The project combines studies in both Drosophila and mice, focusing on motoneuron (MN) responses to peripheral nerve injury (PNI). For the first goal, Aim 1 tests a hypothesis that DLK/Wnd signaling is restrained by the presence of an intact synaptic connection, hence becomes activated following synapse loss. The experiments build upon observations in complementary paradigms of synapse loss at Drosophila neuromuscular junction (NMJ) synapse: (a) injuries to branched axons demonstrate that only complete removal of all efferent connections are capable of activating Wnd signaling; (b) multiple cytoskeletal mutations that lead to retraction and degeneration of NMJ synapses also lead to Wnd signaling activation. The proposed experiments will distinguish how synaptic interactions intersect with the process of axonal transport to control the activation of Wnd. Aim 2 studies the downstream responses regulated by DLK that enable structural plasticity, and focuses on new phenotypes for DLK in the mouse spinal cord: the loss of synaptic inputs on the cell bodies of axotomized MNs (termed `synaptic stripping') is dependent upon DLK function in MNs. In addition, the recruitment of activated microglia to the MN cell body, which precedes the synapse loss, requires DLK function in axotomized MNs. Aim 2 will test a hypothesis that DLK signaling gates the secretion of molecular signals that recruit specific responses in microglia to facilitate synapse loss. The experiments will evaluate the requirement of candidate secreted and immune molecules that were identified from a RiboTag translational profiling approach to be strong targets of DLK regulation in axotomized MNs. The experiments will also identify the microglial responses gated by DLK in axotomized MNs through single cell RNA-seq of isolated microglia. Taken together, this work is expected to shed new light on neuron-microglial interactions relevant to nervous system injury, and mechanisms structural plasticity and synapse loss through the specific lens of a specific axonal damage signaling pathway.