Molecular events regulating axonal structure and function during injury

Background Axonal swellings (AxS) are focal enlargements of axons found in a range of biological and pathological settings, including traumatic brain injury. However, the development of AxS and their effects on axonal homeostasis are poorly understood. Here, using a novel in vitro experimental paradigm we describe cytoskeletal and molecular events responsible of AxS formation and its consequences to axonal homeostasis. Method Human neuronal progenitor cells were seeded into microfluidic chambers and terminally differentiated to neurons. In these chambers, axons grow into microchannels, which are crossed by a perpendicular channel that is connected to a syringe pump. A negative flow caused by the syringe pump subjects axons to bending stress. After full characterization of the axonal response to the stress, we established a protocol that causes injury without reaching axotomy. Detailed morphometric and structural analysis were performed to validate axonal injury. Result Structural analysis of the AxS showed disarrangement of microtubules and neurofilaments, with a disrupted periodicity of Actin and BII‐Spectrin rings underlying the axolemma. The functional impact of these changes was evidenced by the significant dysregulation observed in the axonal transport of APP vesicles during and after injury. Axonal membrane and calcium levels changes were analysed in real‐time, showing an increase in calcium levels that was concomitant to the formation of focal membrane enlargement. To understand the main source of axoplasm calcium increase during injury, extracellular and different intracellular stores of calcium were blocked. Activation of CaMKII derived from the local increase of Ca in the axon can lead to the regulation of multiple targets. We reasoned that myosins (one of CaMKII targets) could be regulating axolemmal changes by exerting a contraction force on the actin rings underlying the axolemma. By pharmacological and molecular intervention we uncover a physiological role of myosins in the regulation of the axonal shaft structure and the AxS formation. Conclusion We created a unique cell culture paradigm to study the response of axons to physical injury and provide novel insight into mechanisms responsible for axonal shaft structure and the formation of AxS.