The Rho signaling pathway regulates the cytoskeleton and motility and plays an important role in neuronal growth inhibition. Here we demonstrate that inactivation of Rho or its downstream target Rho-associated kinase (ROK) stimulated neurite growth in primary cells of cortical neurons plated on myelin or chondroitin sulfate proteoglycan substrates. Furthermore, treatment either with C3 transferase (C3) to inactivate Rho or with Y27632 to inhibit ROK was sufficient to stimulate axon regeneration and recovery of hindlimb function after spinal cord injury (SCI) in adult mice. Injured mice were treated with a single injection of Rho or Rho-associated kinase inhibitors delivered in a protein adhesive at the lesion site. Treated animals showed long-distance regeneration of anterogradely labeled corticospinal axons and increased levels of GAP-43 mRNA in the motor cortex. Behaviorally, inactivation of Rho pathway induced rapid recovery of locomotion and progressive recuperation of forelimb-hindlimb coordination. These findings provide evidence that the Rho signaling pathway is a potential target for therapeutic interventions after spinal cord injury.
Regeneration in the CNS is blocked by many different growth inhibitory proteins. To foster regeneration, we have investigated a strategy to block the neuronal response to growth inhibitory signals. Here, we report that injured axons regrow directly on complex inhibitory substrates when Rho GTPase is inactivated. Treatment of PC12 cells with C3 enzyme to inactivate Rho and transfection with dominant negative Rho allowed neurite growth on inhibitory substrates. Primary retinal neurons treated with C3 extended neurites on myelin-associated glycoprotein and myelin substrates. To explore regeneration in vivo, we crushed optic nerves of adult rat. After C3 treatment, numerous cut axons traversed the lesion to regrow in the distal white matter of the optic nerve. These results indicate that targeting signaling mechanisms converging to Rho stimulates axon regeneration on inhibitory CNS substrates.
A protein fraction purified from bovine brain myelin, previously called arretin because of its ability to inhibit neurite outgrowth, has been identified as consisting predominantly of oligodendrocyte-myelin glycoprotein (OMgp). We show that it is a potent inhibitor of neurite outgrowth from rat cerebellar granule and hippocampal cells; from dorsal root ganglion explants in which growth cone collapse was observed; from rat retinal ganglion neurons; and from NG108 and PC12 cells. OMgp purified by a different procedure from both mouse and human myelin behaves identically in all bioassays tested.
In normal adult retinas, NGF receptor TrkA is expressed in retinal ganglion cells (RGC), whereas glia express p75NTR . During retinal injury, endogenous NGF, TrkA, and p75 NTR are up-regulated. Paradoxically, neither endogenous NGF nor exogenous administration of wild type NGF can protect degenerating RGCs, even when administered at high frequency. Here we elucidate the relative contribution of NGF and each of its receptors to RGC degeneration in vivo. During retinal degeneration due to glaucoma or optic nerve transection, treatment with a mutant NGF that only activates TrkA, or with a biological response modifier that prevents endogenous NGF and pro-NGF from binding to p75 NTR affords significant neuroprotection. Treatment of normal eyes with an NGF mutant-selective p75 NTR agonist causes progressive RGC death, and in injured eyes it accelerates RGC death. The mechanism of p75 NTR action during retinal degeneration due to glaucoma is paracrine, by increasing production of neurotoxic proteins TNF-␣ and ␣ 2 -macroglobulin. Antagonists of p75 NTR inhibit TNF-␣ and ␣ 2 -macroglobulin up-regulation during disease, and afford neuroprotection. These data reveal a balance of neuroprotective and neurotoxic mechanisms in normal and diseased retinas, and validate each neurotrophin receptor as a pharmacological target for neuroprotection.Neuropathic diseases of the retina that involve the death of retinal ganglion cells (RGCs) 4 are irreversible. This is because RGCs are neurons whose fibers and axons make up the optic nerve (ON) and relay visual input from the retina to the cerebral cortex.Commonly used animal models of neuropathy that cause RGC death include ON axotomy and glaucoma. ON axotomy is an acute model of trauma where the optic nerve is completely severed, causing rapid death of the RGCs (ϳ90% within 2 weeks). Glaucoma is a chronic and progressive optic nerve neuropathy often concomitant with elevated intraocular pressure (IOP) (1). The etiology of RGC death in glaucoma remains unknown.One mechanism is the deprivation of survival signals that neurotrophins provide by acting through the TrkA and TrkB receptors expressed in RGCs (2, 3). Indeed, activation of TrkA (4) or TrkB (5) directly activate pro-survival signals during glaucoma and rescues RGCs from death during ON axotomy or glaucoma. However, it seems paradoxical that whereas TrkA activity is protective, neither endogenous nerve growth factor (NGF) (up-regulated in glaucoma (6)) nor exogenous NGF applied as a drug afford effective RGC neuroprotection during ON axotomy or glaucoma (4, 7).A second mechanism of RGC death in glaucoma is the increased production of tumor necrosis factor-␣ (TNF-␣) (8-10) and ␣ 2 -macroglobulin (␣ 2 M) (11). These neurotoxic factors are produced by activated microglia (12), which express the neurotrophin receptor p75 NTR (7). Indeed, the p75 NTR receptor has been implicated in the acute release of TNF-␣ during acute toxicity leading to RGC death within a few hours after intravitreal injection of glutamate (13) or after activatio...
Spinal cord injury (SCI) leads to robust Rho activation at the lesion site. Here, we demonstrate that BA-210, a cell-permeable fusion protein derived from C3 transferase, formulated in fibrin sealant and delivered topically onto the dura matter, diffuses into the spinal cord and inactivates Rho in a dose-dependent manner. Treatment with BA-210 in rats with thoracic spinal cord contusion increased tissue sparing around the lesion area and led to significant improvement of locomotor function. In mice, BA-210 improved functional outcome when treatment was either applied at the time of injury or delayed by 24 h. In both rats and mice, treatment with BA-210 was well tolerated. Rats gained body weight normally, and BA-210 treatment had no impact on the development of allodynia. Inactivating Rho with BA-210 holds promise for treating patients with SCI.
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