Axonal Varicosity Dynamics in Central Neuron Mechanosensation and Injury

项目摘要 关于微机械应力在调节细胞形态和功能中的作用知之甚少。 中枢神经系统（CNS）的神经元。轴突静脉曲张（肿胀或串珠状）扩大， 沿着轴突轴的异质结构，深刻地影响轴突传导和突触 传输它们是被认为代表轴突损伤缓慢积累的关键病理特征 在不可逆变性过程中发生，例如在轻度创伤性脑损伤（mTBI）、阿尔茨海默氏病（Alzheimer's disease） 疾病（AD）和多发性硬化症。在R 01的第一个资助期内，我们发现流体机械 应激立即可逆地诱导培养的CNS神经元无髓鞘轴突的静脉曲张， 进一步可视化了体内静脉曲张诱导。健康成人包括胼胝体在内的大部分脑区 包含有髓和无髓轴突，而髓磷脂似乎保护轴突免受初始的损伤。 机械损伤使用模拟脑震荡的小鼠模型，我们的新研究发现， 静脉曲张形成的无髓轴突的皮质神经元和延迟脱髓鞘的皮质后， 机械冲击我们的新结果还表明，微管（MT）相关蛋白6（MAP 6） 通过MT稳定和Ca 2 +/钙调蛋白结合调节轴突静脉曲张的形成。 基于我们的新发现，我们假设机械冲击立即诱发静脉曲张 在无髓鞘轴突中形成，其受到MAP 6介导的MT稳定化的抑制， 随后促进邻近的脱髓鞘，增加轴突对第二次冲击的脆弱性， 导致反复mTBI的恶性循环并因此恶化行为损伤。为了验证这一 最初的假设，我们将使用多学科的方法，包括mTBI和脱髓鞘小鼠模型， 细胞类型特异性过表达，敲除和拯救，共聚焦和电子显微镜，行为测试， 电生理记录、多功能生物力学测定、髓鞘共培养和最先进的成像 技术.我们将确定（目的1）mTBI诱导的轴突静脉曲张形成和行为学改变是否与mTBI诱导的轴突静脉曲张形成和行为学改变有关。 MAP 6缺失可加重损伤，MAP 6介导的MT稳定可改善损伤（目的2）。 MAP 6如何调节轴突静脉曲张的起始、恢复、定位、异质性和长期命运 部分有髓轴突中不同的信号通路，以及（目的3）MAP 6如何调节少突胶质细胞 机械感觉，以及预先存在的脱髓鞘和/或预先存在的轴突静脉曲张是否增加了机械感觉。 重复机械冲击造成的伤害风险。该项目代表了一个未充分探索的研究领域， 许多开放的问题。这项研究是有意义的，因为它将提供新的机制见解中央 神经元机械感觉和mTBI原发性损伤。

PROJECT SUMMARY Little is known about the role of micromechanical stress in regulating morphology and function of neurons in the central nervous system (CNS). Axonal varicosities (swelling or beading) are enlarged, heterogeneous structures along axonal shafts, profoundly affecting axonal conduction and synaptic transmission. They are a key pathological feature believed to represent slow accumulation of axonal damage that occurs during irreversible degeneration, for example in mild traumatic brain injury (mTBI), Alzheimer’s disease (AD), and multiple sclerosis. In the first funding period of this R01, we discovered that fluid mechanical stress immediately and reversibly induced varicosities in unmyelinated axons of cultured CNS neurons, and we further visualized varicosity induction in vivo. Most brain regions including the corpus callosum in healthy adults contain both myelinated and unmyelinated axons, while myelin appears to protect the axon from initial mechanical injury. Using a mouse model mimicking concussion, our new studies have found immediate varicosity formation in unmyelinated axons of cortical neurons and delayed demyelination in the cortex after mechanical impact. Our new results have also indicated that microtubule (MT)-associated protein 6 (MAP6) regulates axonal varicosity formation through its properties of MT stabilization and Ca2+/calmodulin binding. Based on our new findings, we hypothesize that mechanical impact immediately induces varicosity formation in unmyelinated axons, which is restrained by MAP6-mediated MT stabilization and subsequently promotes adjacent demyelination that increases axon vulnerability to second impact, leading to a vicious cycle in repeated mTBI and hence worsened behavioral impairment. To test this original hypothesis, we will use a multidisciplinary approach including mTBI and demyelination mouse models, cell-type-specific overexpression, knockout and rescue, confocal and electron microscopy, behavioral testing, electrophysiological recording, a versatile biomechanical assay, myelin coculture and state-of-the-art imaging techniques. We will determine (Aim 1) whether mTBI-induced axonal varicosity formation and behavioral impairment can be aggravated by MAP6 deletion and ameliorated by MAP6-mediated MT stabilization, (Aim 2) how MAP6 regulates axonal varicosity initiation, recovery, location, heterogeneity and long-term fate through distinct signaling pathways in partially myelinated axons, and (Aim 3) how MAP6 regulates oligodendrocyte mechanosensation, and whether preexisting demyelination and/or preexisting axonal varicosities increase the risk of injury from repeated mechanical impact. This project represents an underexplored research field with many open questions. This research is significant because it will provide novel mechanistic insights into central neuron mechanosensation and mTBI primary injury.