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.