Promoting integrin-mediated neurite outgrowth for the enhancement of neuroregenerative therapy

Abstract

Spinal cord injury (SCI) involves damage to axons, biological wires which mediate communication throughout the central nervous system. Functional recovery from SCI is supported by axon regeneration, however this is incomplete in the central nervous system. Therefore, regenerative therapeutics are required. Delivery of α9 integrin can promote regeneration of injured dorsal root axons, although growth into the spinal cord is limited. Integrin signalling is mediated by the integrin adhesion complex (IAC), which is therefore a target to enhance integrin-mediated axon regeneration. For example, regeneration into the dorsal column is significantly enhanced by co-expression of α9 integrin with the integrin activating protein kindlin-1. The central hypothesis of this project states that the IAC is a target to enhance axon regeneration following SCI.

This project specifically targets three IAC proteins with the potential to enhance integrin-mediated neurite outgrowth: myosin-X, an integrin activating motor protein that promotes actin-mediated protrusion; talin, an integrin activator that mediates mechanotransduction; and focal adhesion kinase (FAK), an IAC signalling checkpoint during growth cone migration. Each protein is overexpressed in a cellular model of neurite outgrowth which is translatable to SCI. Adult DRG neurons are used to model the sensory neurons impacted by SCI. Neuronal differentiation of PC12 pheochromocytoma cells is used as a higher throughput model, to validate approaches to promoting neurite outgrowth. Cells are grown on combinations of substrates which model the extracellular matrix in SCI, and resulting neurite outgrowth is quantified.

Results demonstrate that myosin-X significantly promotes neurite outgrowth in PC12 cells differentiated on inhibitory substrates, and that this effect is integrinindependent. In addition, talin domains R11-12 are required for efficient neurite outgrowth in PC12 cells, providing a rationale for the use of truncated talin constructs to facilitate talin overexpression in neurons. Finally, overexpression of the dominant negative FAK isoform, FAK-related non-kinase, significantly promotes neurite outgrowth of adult DRG neurons, and this effect is not replicated by pharmacological FAK inhibition.

In summary, targeting the IAC promotes neurite outgrowth in both adult DRG neurons and differentiating PC12 cells. Therefore, the IAC is a promising target to promote integrin-mediated axon regeneration following SCI. Furthermore, evidence suggests that such therapies will synergise with delivery of α9 integrin to enhance established therapeutic approaches.

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Identifiers

ORCID for Mathew James Davis-Lunn: orcid.org/0000-0003-0677-9660

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

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