Regenerative and degenerative responses to axonal injury

项目摘要/摘要： 轴突在大脑和身体的很远的距离上形成神经元之间的连接，因此很容易受到 损伤和压力。该项目研究了进化上保守的应激反应途径，该途径成为 在轴突损伤和应激的多种情况下被激活。这条小路由双亮氨酸拉链控制 DLK在果蝇中被称为Wallenda(WND)，参与神经元的结构可塑性机制 使电路能够适应轴突损伤。这些反应包括轴突再生，神经元死亡， 而且，这个项目中新发现的突触丢失。这个项目的长期目标是：(1)了解 导致DLK信号激活的机制，以及(2)了解 受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)的反应。对于第一个目标，Aim 1测试了一个假设，即DLK/WND信号 受到完整突触连接的限制，因此在突触后被激活 损失。这些实验建立在对果蝇突触丢失的互补范式观察的基础上 神经肌肉接头(NMJ)突触：(A)对分支轴突的损伤表明只有完整的 移除所有传出连接能够激活WND信号；(B)多种细胞骨架突变 这导致NMJ突触的收缩和退化，也导致了WND信号的激活。建议数 实验将区分突触相互作用如何与轴突运输过程相交以控制 WND的激活。目的2研究DLK调节的下游反应，使结构 可塑性，并重点研究了DLK在小鼠脊髓中的新表型：在 MNS轴突切断后的胞体(突触剥离)依赖于MNS中DLK的功能。在……里面 此外，在突触丢失之前，激活的小胶质细胞被招募到MN细胞体需要 DLK在MNS轴突切断中的作用。目标2将检验一个假设，即DLK信号门控分泌 在小胶质细胞中招募特定反应以促进突触丢失的分子信号。这些实验将会 评估从RiboTag中鉴定的候选分泌和免疫分子的需求 翻译侧写方法是DLK在MNS轴突切断后调控的有力靶点。这些实验将会 通过分离的单细胞RNA-SEQ鉴定DLK对MN损伤后的小胶质细胞反应的影响 小胶质细胞。综上所述，这项工作有望为相关的神经元-小胶质细胞相互作用提供新的线索 神经系统损伤，以及结构可塑性和突触丢失的机制 特定的轴突损伤信号通路。

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.