RhoA activation in axotomy-induced neuronal death

Abstract

After spinal cord injury (SCI) in mammals, severed axons fail to regenerate, due to both extrinsic inhibitory factors, e.g., the chondroitin sulfate proteoglycans (CSPGs) and myelin-associated growth inhibitors (MAIs), and a developmental loss of intrinsic growth capacity. The latter is suggested by findings in lamprey that the 18 pairs of individually identified reticulospinal neurons vary greatly in their ability to regenerate their axons through the same spinal cord environment. Moreover, those neurons that are poor regenerators undergo very delayed apoptosis, and express common molecular markers after SCI. Thus the signaling pathways for retrograde cell death might converge with those inhibiting axon regeneration. Many extrinsic growth-inhibitory molecules activate RhoA, whereas inhibiting RhoA enhances axon growth. Whether RhoA also is involved in retrograde neuronal death after axotomy is less clear. Therefore, we cloned lamprey RhoA and correlated its mRNA expression and activation state with apoptosis signaling in identified reticulospinal neurons. RhoA mRNA was expressed widely in normal lamprey brain, and only slightly more in poorly-regenerating neurons than in good regenerators. However, within a day after spinal cord transection, RhoA mRNA was found in severed axon tips. Beginning at 5 days post-SCI RhoA mRNA was upregulated selectively in pre-apoptotic neuronal perikarya, as indicated by labelling with fluorescently labeled inhibitors of caspase activation (FLICA). After 2 weeks post-transection, RhoA expression decreased in the perikarya, and was translocated anterogradely into the axons. More striking than changes in RhoA mRNA levels, RhoA was continuously active selectively in FLICA-positive neurons through 9 weeks post-SCI. At that time, almost no neurons whose axons had regenerated were FLICA-positive. These findings are consistent with a role for RhoA activation in triggering retrograde neuronal death after SCI, and suggest that RhoA may be a point of convergence for inhibition of both axon regeneration and neuronal survival after axotomy.

Keywords: Apoptosis; Axotomy; Caspase; FLICA; Lamprey; RhoA; Spinal cord injury.