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Suppression of Rho-kinase activity promotes axonal growth on inhibitory CNS substrates

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Abstract

Several molecules inhibit axonal growth cones and may account for the failure of central nervous system regeneration, including myelin proteins and various chondroitan sulfate proteoglycans expressed at the site of injury. Axonal growth inhibition by myelin and chondroitan sulfate proteoglycans may in part be controlled by Rho-GTPase, which mediates growth cone collapse. Here, we tested in vitro whether pharmacological inhibition of a major downstream effector of Rho, Rho-kinase, promotes axonal outgrowth from dorsal root ganglia grown on aggrecan. Aggrecan substrates stimulated Rho activity and were inhibitory to axonal growth. Y-27632 treatment promoted the growth of axons by 5- to 10-fold and induced “steamlined” growth cones with longer filopodia and smaller lamellipodia. Interestingly, more actin bundles reminiscent of stress fibers in the central domain of the growth cone were observed when grown on aggrecan compared to laminin. In addition, Y-27632 significantly promoted axonal growth on both myelin and adult rat spinal cord cryosections. Our data suggest that suppression of Rho-kinase activity may enhance axonal regeneration in the central nervous system.

Introduction

Axonal regeneration in the adult mammalian central nervous system (CNS) is hampered by several growth-inhibitory molecules found both in the injury scar and in the distal myelinated tracts (for review, see Fitch and Silver 1997b, Fournier and Strittmatter 2001, McGraw et al 2001. An important class of inhibitory molecules is the chondroitin sulfate proteoglycans (CSPGs), found both in myelin (Niederost et al., 1999) and within CNS injury scars Davies et al 1999, Dow et al 1994, Fitch and Silver 1997a, Gates et al 1996, Lemons et al 1999, McKeon et al 1991. Aggrecan is one prominent member of the CSPG family and is expressed in the developing, adult, and injured spinal cord (Lemons et al., 2001). Aggrecan is inhibitory to sensory and retinal neurites grown in vitro Challacombe et al 1997, Snow and Letourneau 1992 and as such is used here as a model to study axonal growth inhibition. Similarly, other CSPGs have been shown to be both inhibitory to neurite outgrowth in vitro Dou and Levine 1997, Gates et al 1996, Lemons et al 1999, McKeon et al 1991, Niederost et al 1999 and correlated with the arrest of axonal growth in vivo Davies et al 1997, Davies et al 1999. Furthermore, the degradation of CSPGs in vivo by treatment with chondroitinase-ABC has recently been shown to promote axonal growth after injury Bradbury et al 2002, Moon et al 2001. Although CSPGs are clearly important to axonal growth inhibition, the extent to which individual inhibitory molecules contribute to axonal regeneration failure is less clear; it is difficult to predict the number and the nature of the inhibitors that should be neutralized to achieve functional regeneration and recovery.

Axonal growth inhibition is accompanied by distinct changes in growth cone behavior and morphology, mediated primarily through the action of the Rho family of small guanosine triphosphatases (GTPases) on the actin cytoskeleton (Luo, 2000). In nonneuronal cells, activation of Rac and Cdc42 induces the formation of lamellipodia and filopodia respectively, while Rho activation induces stress fiber and focal adhesion formation (Nobes and Hall, 1995). The picture is not as clear in neurons; nonetheless, Rho activation has been shown to be at least partly responsible for the effects of several neurite growth inhibitors including Ephrin-A5-induced collapse of retinal axons (Wahl et al., 2000), myelin inhibition of retinal, dorsal root ganglion (DRG), and cortical neurite outgrowth Dergham et al 2002, Jin and Strittmatter 1997, Lehmann et al 1999 and Semaphorin 3A inhibition of DRG neurite outgrowth (Jin and Strittmatter, 1997). As well, it was recently shown that neurite growth inhibition due to CSPG may be overcome by blocking the Rho signaling pathway (Dergham et al., 2002). Since the effects of several inhibitors of regeneration and repulsive guidance cues appear to be mediated by Rho, a viable approach to stimulating axonal growth may be to block growth inhibitory molecules simultaneously by interfering with the Rho pathway (McKerracher, 2001).

Rho has several downstream effectors (Hall, 1998) among which ROCK may be an attractive target for therapeutic intervention. ROCK (Rho-kinase, ROKα, and ROCK-2) is a recently identified serine/threonine kinase that is highly expressed in brain Hashimoto et al 1999, Matsui et al 1996. ROCK mediates the signaling of Rho to the actin cytoskeleton through several downstream targets (Amano et al., 2000). For example, ROCK regulates the phosphorylation of myosin light chain, both by direct phosphorylation of myosin light chain (Amano et al., 1996) and by the inactivation of myosin phosphatase (Kimura et al., 1996). As well, ROCK phosphorylates LIM-kinase Maekawa et al 1999, Ohashi et al 2000, in turn is necessary for Sema3A-induced growth cone collapse mediated via phosphorylation of cofilin (Aizawa et al., 2001). To test whether ROCK signaling is important to inhibiting axonal outgrowth, we have used a cell-permeable pyridine derivative, (+)-R-trans-4-(1-aminoethyl)-N-(4-pyridyl)-cyclohexanecarboxamide (Y-27632) (Ishizaki et al., 2000), to suppress ROCK activity. Whole explants of chick DRG were grown on inhibitory substrates of aggrecan, myelin, and adult spinal cord cryosections in the presence or absence of Y-27632 and examined for axonal outgrowth and growth cone morphology.

Section snippets

ROCK is expressed in chick DRG

The expression of ROCK has recently been demonstrated at high levels in brain (Matsui et al., 1996), including bovine pyramidal neurons, Purkinje cells (Hashimoto et al., 1999), and chick retinal ganglion cells (Wahl et al., 2000). To confirm the presence of ROCK protein in chick DRG, cultures grown on glass coverslips were stained with an antibody to ROCK-2. Labeled growth cones were imaged at high magnification and observed to display distinct expression of ROCK immunoreactivity (Fig. 1A).

Discussion

Numerous axonal growth inhibitors exist in the injured CNS. It is uncertain how many of these inhibitors will need to be neutralized to achieve successful regeneration in adult mammals. The activities of growth cone guidance molecules are mediated through secondary signaling pathways and eventually converge onto the actin cytoskeleton (for review, see Suter and Forscher, 2000). Here, it was our approach to target putative convergence points of multiple signaling pathways to neutralize several

Experimental methods

Reagents were obtained from Canadian Life Technologies (Burlington, ON) unless otherwise indicated. (+)-R-trans-4-(1-Aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide (Y-27632) was synthesized at Merck Frosst as the TFA salt. Unless otherwise indicated, all experiments performed with Y-27632 were completed by using the TFA salt. In some experiments, we examined the activity of Y-27632 obtained commercially from Biomol Research Laboratories (Plymouth Meeting, PA). This commercial source of

Acknowledgements

This research was funded by a grant from the British Columbia Neurotrauma Fund. J.F.B., C.C.M.C., and L.O. were funded by studentships provided by the BCNTF and CIHR. W.T. holds the Rick Hansen Man in Motion Chair in Spinal Cord Research. The authors thank Drs. Timothy P. O’Connor and Matt S. Ramer for critical reading of the manuscript.

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