Rho-Associated Kinase II (ROCKII) Limits Axonal Growth after Trauma within the Adult Mouse Spinal Cord

Abstract

Rho GTPases are thought to mediate the action of several axonal growth inhibitors in the adult brain and spinal cord. RhoA has been targeted pharmacologically in both humans and animals to promote neurite outgrowth and functional recovery following CNS trauma. However, rat spinal cord injury studies suggest a complicated and partial benefit of inhibiting Rho or its downstream effector, Rho-associated kinase (ROCKII). This limited benefit may reflect inhibition of other kinases, poor access, or a minimal role of ROCKII in vivo. Therefore, we studied ROCKII mutant mice to probe this pathway genetically. ROCKII−/− dorsal root ganglion neurons are less sensitive to inhibition by Nogo protein or by chondroitin sulfate proteoglycan in vitro. We examined adult ROCKII−/− mice in two injury paradigms, cervical multilevel dorsal rhizotomy and midthoracic dorsal spinal cord hemisection. After dorsal root crush injury, the ROCKII−/− mice recovered use of the affected forepaw more quickly than did controls. Moreover, multiple classes of sensory axons regenerated across the dorsal root entry zone into the spinal cord of mice lacking ROCKII. After the spinal cord injury, ROCKII−/− mice showed enhanced local growth of raphespinal axons in the caudal spinal cord and corticospinal axons into the lesion site. Improved functional recovery was not observed by Basso Mouse Scale score following dorsal hemisection, likely due to developmental defects in the nervous system. Together, these findings demonstrate that the ROCKII gene product limits axonal growth after CNS trauma.

Myelin-derived ephrinB3 restricts axonal regeneration and recovery after adult CNS injury

“…Adult dorsal root ganglion neurons were cultured and analyzed for outgrowth in the presence or absence of myelin fractions, as described (30,44).…”

“…Mouse thoracic spine level T6 spinal cord injury was achieved in ephrinB3 −/− and ephrinB3 +/+ littermates after dorsal laminectomy (30,37,44,45). Either dorsal hemisection or near-total transection was performed.…”

“…Video analysis of limb kinematics has been described for rats (32). CST axons were traced with BDA, and RST axons were immunostained (30,44).…”

Construction and identification of pIRES2-NGF-VEGF165 bicistronic eukaryotic expression vector

“…This proportion was significantly increased to 37% after 14 days of continuous NGF infusion through catheters placed in the dorsal spinal cord (Jones et al, 2001). These regenerating axons penetrate spinal cord white matter and grow up to 3 mm past the dorsal root entry zone (Duffy et al, 2009). Additional experiments confirmed this NGF-driven regrowth of injured sensory fibers into the spinal cord and revealed the regenerating axons as positive for calcitonin gene-related peptide, a marker for small diameter, unmyelinated peptidergic axons (Lykissas et al, 2007).…”

Chondroitin Sulfates in Axon Regeneration and Plasticity

“…The ability of CSPGs to affect intracellular signaling has been well documented. Studies have identified the roles of the small GTPase Rho and its downstream effector Rho kinase (ROCK) in CSPG mediated growth cone collapse and inhibition of neurite outgrowth (80)(81)(82). CSPGs induce calcium transients in dissociated DRG neurons growing across a barrier of laminin and CSPGs (83) and activate calcium-dependent protein kinase C (PKC) (84) leading to phosphorylation of epidermal growth factor receptor (EGFR) kinase (85) and increased the activity of both serine/threonine protein kinase Akt and glycogen synthase kinase-3 (GSK-3β) (86).…”