Genesis of axonal swellings in traumatic injury

Abstract 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 early effects on axonal homeostasis are poorly understood. Here, we describe the cytoskeletal changes, transport effects and the molecular events that occur in the axon immediately after a traumatic injury. Method We developed an in vitro system to study in real‐time the effects of traumatic injury on the axon of mature human neurons. We also generated an algorithm which allowed us to assess the dynamics of the AxS formation in real‐time and examine the role of Ca 2+ in the swellings. Through mass spectrometry analysis on isolated axons we described the axonal proteome and identified the immediate phosphorylation changes ocurring in the axon after a traumatic injury. We also used superresolution microscopy to characterize the earliest cytoskeletal changes in the axon and their impact on the movement of several axonal cargoes. Result As a result of the mechanical stress we observed the appereance of AxS, which were temporally correlated to Ca 2+ levels increase in the axon. Interestingly, by pharmacologically blocking different sources of Ca 2+ we observed that Ca 2+ plays a role in sustaining the AxS and not in their formation. Proteomic and phosphoproteomic analysis revealed a significant regulation in proteins related to axon development after injury. These proteins were mainly interactors or components of the cytoskeleton, while no association to axonal degeneration or impairment in normal processes was found. We also predicted a general increase in kinases activity after injury, among them ROCK, ERK, JNK, PKC and GSK. A detailed analysis of cytoskeletal arrangement revealed modifications in the actin‐spectrin periodic structure and a mesh‐like organization of microtubules and neurofilaments in the AxS. We finally observed that the structure of swellings allows cargos to cross and that axonal transport of different cargos were not interrupted during mechanical stress. Conclusion These observations indicate that the mechanical stress to the axon generates segments of cytoskeletal reorganization that result from Ca 2+ increase and correlate with regulation through phosphorylation of cytoskeletal and cytoskeletal interacting proteins.