Targeting a Dominant Negative Rho Kinase to Neurons Promotes Axonal Outgrowth and Partial Functional Recovery After Rat Rubrospinal Tract Lesion

Abstract

Many axonal growth inhibitors that contribute to the usual failure of axon regeneration in the central nervous system (CNS) exert their effects via the RhoA-Rho kinase (ROCK) signal pathway. In this study, we investigated whether lentiviral vector (LV)-mediated neuron-specific expression of a dominant negative mutant of ROCK (DNROCK) could promote axon outgrowth in vitro and in vivo. Dissociated adult rat dorsal root ganglion (DRG) neurons were seeded on solubilized myelin proteins and transduced with either LV/DNROCK or LV/green fluorescent protein (GFP). DNROCK-expressing neurons were shown to have a greater chance of generating neurites and a longer mean length of neurite than GFP-expressing neurons. In the in vivo studies, lentiviruses were injected into the adult rat red nucleus followed by unilateral rubrospinal tract (RST) transection at the fourth cervical level. Rats in the DNROCK group showed better functional recovery in the affected hindlimbs and forelimbs than those in the GFP group. Examination of the spinal cord sections revealed more rubrospinal axonal profiles growing to the spinal cord caudal to the lesion in the DNROCK group than in the GFP group. These results indicate that blocking the RhoA-ROCK signal pathway by expressing DNROCK can enhance regenerative axonal sprouting and lead to partial recovery of limb function.

Expression of a dominant-negative Rho-kinase promotes neurite outgrowth in a microenvironment mimicking injured central nervous system

“…These results are consistent with those reported in our previously published study [33] . In the current study, we tested whether a different promoter of DNROCK could affect its effects on neurite outgrowth, stress fiber formation, and growth cone morphology.…”

Preferential and Bidirectional Labeling of the Rubrospinal Tract with Adenovirus-GFP for Monitoring Normal and Injured Axons

“…SCI inevitably affects the RST, an important descending motor pathway, either partially or completely. Therefore, axonal regeneration, plasticity, and sprouting of the RST following SCI have been studied extensively (Harvey et al, 2005;Koda et al, 2004;Kwon et al, 2002;Liu et al, 1999;Ruitenberg et al, 2003;Wu et al, 2009;Xiao et al, 2005;Martin, 1989, 1991). Using Adv-GFP to study the remodeling and regeneration of the RST is advantageous since it can specifically label RST axons that traverse through the lesion area both anterogradely and retrogradely.…”

Enhancing intrinsic growth capacity promotes adult CNS regeneration

“…A few critical molecules mediating these inhibitory effects have also been identified, such as Nogo, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (Omgp), chondroitin sulfate proteoglycans (CSPGs), semaphorins and ephrins [6,7]. However, blocking genetically or pharmacologically the activities of such inhibitory molecules results in limited sprouting, especially in the case of long-distance axon regeneration [6,8,9]. Furthermore, even provided with permissive substrates, majority of the adult neurons fail to regenerate their axons after injury [10].…”