Mild and repetitive very mild axonal stretch injury triggers cystoskeletal mislocalization and growth cone collapse

Abstract

Diffuse axonal injury is a hallmark pathological consequence of non-penetrative traumatic brain injury (TBI) and yet the axonal responses to stretch injury are not fully understood at the cellular level. Here, we investigated the effects of mild (5%), very mild (0.5%) and repetitive very mild (2×0.5%) axonal stretch injury on primary cortical neurons using a recently developed compartmentalized in vitro model. We found that very mild and mild levels of stretch injury resulted in the formation of smaller growth cones at the tips of axons and a significantly higher number of collapsed structures compared to those present in uninjured cultures, when measured at both 24 h and 72 h post injury. Immunocytochemistry studies revealed that at 72 h following mild injury the axonal growth cones had a significantly higher colocalization of βIII tubulin and F-actin and higher percentage of collapsed morphology than those present following a very mild injury. Interestingly, cultures that received a second very mild stretch injury, 24 h after the first insult, had a further increased proportion of growth cone collapse and increased βIII tubulin and F-actin colocalization, compared with a single very mild injury at 72 h PI. In addition, our results demonstrated that microtubule stabilization of axons using brain penetrant Epothilone D (EpoD) (100 nM) resulted in a significant reduction in the number of fragmented axons following mild injury. Collectively, these results suggest that mild and very mild stretch injury to a very localized region of the cortical axon is able to trigger a degenerative response characterized by growth cone collapse and significant abnormal cytoskeletal rearrangement. Furthermore, repetitive very mild stretch injury significantly exacerbated this response. Results suggest that axonal degeneration following stretch injury involves destabilization of the microtubule cytoskeleton and hence treatment with EpoD reduced fragmentation. Together, these results contribute a better understanding of the pathogenesis of mild and repetitive TBI and highlight the therapeutic effect of microtubule targeted drugs on distal part of neurons using a compartmentalized culturing model.

Proteomic Profiling of Mouse Brains Exposed to Blast-Induced Mild Traumatic Brain Injury Reveals Changes in Axonal Proteins and Phosphorylated Tau

“…Loss of axonal microtubule was found after stretch injury and progressive disassembly of microtubules along the breakage points [35]. Stabilization of microtubule resulted in a significant reduction in the number of fragmented axons following injury [34]. Consistent with previous reports [36], our studies also revealed depolymerization of microtubules.…”

“…Diffuse axonal injury is a hallmark pathological consequence of non-penetrative mTBI [34]. In neuronal culture model, very mild stretch injury to a localized region of the cortical axon is able to trigger a degenerative response characterized by growth cone collapse and significant abnormal cytoskeleton rearrangement, resulting in the formation of smaller growth cones at the tips of axons and a significantly higher number of collapsed structures at both 24 h and 72 h post injury [34]. Axonal degeneration following stretch injury involves destabilization of the microtubule cytoskeleton.…”

The cytoskeleton as a modulator of tension driven axon elongation

“…In organotypic cerebellar slices, a biaxial strain of 30% increases axonal amyloid precursor protein accumulation, which supports disrupted axonal transport, and the formation of axonal swellings is observed (Chierto et al., 2019). Of note, the microtubule‐stabilizing drug epothilone D is capable of reducing axon fragmentation after stretch injury in cortical neurons (Yap et al., 2017). In addition to disrupting axonal physiology, axon stretch injury can cause the formation of periodic swellings in dendrites, which could also be the consequence of microtubule rupture and impaired axonal transport (Monnerie et al., 2010).…”

Microfluidic platforms for the study of neuronal injury in vitro

“…Furthermore, these abnormalities in cytoskeletal structure could be decreased through the application of the potentially clinically therapeutic microtubule stabilizing agent, Epothilone D (Brunden et al, 2012). While these results exemplify the utility of their device for studying neuronal responses to discrete axonal stretch injury, their device lacked optical transparency for high resolution live time-lapse analysis and the ability to investigate synaptically connected axons from two different neuronal populations (Yap et al, 2017). The injury pad layer was reversibly sealed to the glass coverslip to allow access to the injured neurons for subsequent transmission electron microscopy (TEM) analysis (Table 2).…”

“…Conversely, the mild stretch injury led to significant axonal degeneration at both 1 and 3 days following injury (Yap et al, ). Recently, this in vitro model of axonal stretch injury was used to investigate axonal responses to single and repetitive stretch injuries in a fluidically isolated microenvironment (Yap et al, ). The study compared the morphology and cytoskeletal profile of growth cones on the tips of the axons following mild (5% strain), very mild (0.5% strain), and repetitive very mild (2 × 0.5% strain) axonal stretch injury.…”