Increasing axonal expression of alpha9 integrin to promote axon regeneration: exosome-mediated delivery and axon-targeting of the alpha9 integrin subunit

Abstract

Following spinal cord injury, lesioned spinal cord axons become disconnected from their anatomical targets leading to loss of motor and sensory function at and below the level of injury. This is compounded by the inability of axons of mature central nervous system neurons to regenerate over long distances in the adult spinal cord and thus to reconnect with their targets. Lack of axon regeneration is a result of the low intrinsic regenerative capacity of adult neurons together with the inhibitory extracellular factors present following spinal cord injury. One of these inhibitory factors are the members of the Tenascin matricellular protein family, which are deposited by reactive perilesional glia following injury. Tenascins family members, such as tenascin-C, repel neurites when presented as a harsh border likely directing regenerating axons away from the spinal cord lesion site. Expression of tenascin-C associated integrin receptors supports axon regeneration. Specifically, the α9β1 integrin heterodimer recognises tenascin-C and overexpression of the α9 integrin subunit promotes adult sensory and motor axon regeneration into spinal cord lesions, however, regeneration is modest and these axons fail to cross the lesion site. Recent studies have shown that primary motor neurons fail to traffic overexpressed α9 integrin into their axons. Such exclusion may limit the ability of α9 integrin to bind to lesion site tenascin-C, thereby limiting its growth-promoting effect. Indeed, experimental interventions that increase anterograde trafficking of integrins also enhance axon outgrowth of cortical neurons in vitro. Nevertheless, these interventions may have integrin-independent effects and therefore the link between increased anterograde integrin trafficking and axon outgrowth remains unclear. We hypothesise that increasing growth cone expression of α9 integrin will enhance the growth-promoting effect of α9 integrin expression on tenascin-C. Using primary neuronal models, we aimed to investigate methods to deliver α9 integrin to axons and the subsequent effect of increased growth cone expression of α9 integrin on axon outgrowth on tenascin-C. These methods included exosome-mediated delivery of α9 integrin and axon-targeting motif modification of α9 integrin. We also assessed whether the growth-promoting effect of PTEN loss-of-function is directly associated with an increase in integrin function or growth cone localisation. We provide evidence that exosome-mediated delivery and axon-targeting motif modification of α9 integrin are viable methods to increase axonal expression of α9 integrin. There was however no evidence of increased growth-promoting function or growth cone expression of α9 integrin in PTEN knockout neurons. The data presented in this thesis provides strong support for further investigation of exosome-mediated delivery and axon-targeting motif modification of α9 integrin in in vivo spinal cord injury models, where α9 integrin is more stringently excluded from motor axons.

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Identifiers

ORCID for Lloyd James Steele-Nicholson: orcid.org/0000-0002-6001-3386

ORCID for Melissa Andrews: orcid.org/0000-0001-5960-5619

ORCID for David Tumbarello: orcid.org/0000-0002-5169-0561

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