Viral vector-mediated downregulation of RhoA increases survival and axonal regeneration of retinal ganglion cells.

Jan Christoph Koch,Lars Tã¶Nges,Mathias Bã¤Hr,Uwe Michel,Paul Lingor

doi:10.3389/fncel.2014.00273

Abstract

The Rho/ROCK pathway is a promising therapeutic target in neurodegenerative and neurotraumatic diseases. Pharmacological inhibition of various pathway members has been shown to promote neuronal regeneration and survival. However, because pharmacological inhibitors are inherently limited in their specificity, shRNA-mediated approaches can add more information on the function of each single kinase involved. Thus, we generated adeno-associated viral vectors (AAV) to specifically downregulate Ras homologous member A (RhoA) via shRNA. We found that specific knockdown of RhoA promoted neurite outgrowth of retinal ganglion cells (RGC) grown on the inhibitory substrate chondroitin sulfate proteoglycan (CSPG) as well as neurite regeneration of primary midbrain neurons (PMN) after scratch lesion. In the rat optic nerve crush (ONC) model in vivo, downregulation of RhoA significantly enhanced axonal regeneration compared to control. Moreover, survival of RGC transduced with AAV expressing RhoA-shRNA was substantially increased at 2 weeks after optic nerve axotomy. Compared to previous data using pharmacological inhibitors to target RhoA, its upstream regulator Nogo or its main downstream target ROCK, the specific effects of RhoA downregulation shown here were most pronounced in regard to promoting RGC survival but neurite outgrowth and axonal regeneration were also increased significantly. Taken together, we show here that specific knockdown of RhoA substantially increases neuronal survival after optic nerve axotomy and modestly increases neurite outgrowth in vitro and axonal regeneration after optic nerve crush.

Highlights

Neuronal cell death, axonal degeneration and regenerative failure are the central pathological hallmarks of most traumatic and neurodegenerative diseases of the central nervous system (CNS)
Transduction with associated viral vectors (AAV).Ras homologous member A (RhoA)-short hairpin RNA (shRNA) resulted in a significant downregulation of RhoA expression levels to 35 ± 0.18% compared to AAV.EGFP-shRNA which was set 100% (Figure 1B)
In contrast to retinal ganglion cells (RGC) cultured on laminin, neurite growth on chondroitin sulfate proteoglycan (CSPG)-coating was significantly reduced in untreated RGC and RGC transduced with AAV.EGFP-shRNA (689 ± 120 μm)

Summary

Introduction

Axonal degeneration and regenerative failure are the central pathological hallmarks of most traumatic and neurodegenerative diseases of the central nervous system (CNS). The Rho/ROCK/LIMK-pathway appears to be an excellent candidate to fulfill these criteria (Mueller et al, 2005; Tönges et al, 2011a) It is not clear which pathway members are most suitable to be targeted to address specific pathological mechanisms. The Rho/ROCK/LIMK-pathway is activated by several extracellular growth-inhibitory signals, including Nogo, ephrins (Shamah et al, 2001) and semaphorins (Dontchev and Letourneau, 2003; Lin et al, 2007). Their signaling is mediated through different transmembrane receptors, e.g., the ephrin receptor and the trimeric NogoR/p75/Lingo receptor complex. To its functions in developmental axon guidance (Luo, 2000), the Rho/ROCK/LIMK-cascade plays an important role after

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Conclusion