Myosin II activity regulates neurite outgrowth and guidance in response to chondroitin sulfate proteoglycans

Failure of neurons in the central nervous system (CNS) to regenerate their axons after injury is attributed at least partly to the formation of a glial scar soon after injury. Within the glial scar there is a rapid and large upregulation of chondroitin sulfate proteoglycans (CSPGs) that serve to limit axonal regeneration and recovery of function. As growth cones enter CSPG-rich regions, they stop, turn or even retract to avoid the repulsive CSPGs (Silver & Miller 2004). These inhibitory actions of CSPGs can be modeled in vitro: neurites in culture growing on permissive substrates turn at a CSPG boundary, and neurite outgrowth is reduced when dissociated neurons are plated onto uniform substrates of CSPGs (Snow & Letourneau 1992; Wang et al. 2008). The response of neurons to guidance cues is orchestrated by signal transduction mechanisms that ultimately result in changes in cytoskeletal dynamics, of which the major components are actin and tubulin. After contact with CSPGs, microtubules are rearranged within growth cones to accomplish turning. Application of either cytochalasin B to block actin filament formation or low doses of taxol to stabilize microtubules results in a reduction in growth cone turning to avoid CSPGs, indicating that both actin filaments and dynamic microtubules are required for growth cone turning (Challacombe et al. 1996; Challacombe et al. 1997). We have previously demonstrated that the rate and direction of tubulin polymerization are locally altered only within growth cones that encounter CSPGs (Kelly et al. 2010). These clearly demonstrate the presence of local signaling pathways triggered by CSPGs. However, the underlying mechanisms for cytoskeletal reorganization remain to be elucidated. Non-muscle myosin II (hereafter, myosin II) is an actin binding protein that has been implicated in many aspects of cell adhesion and neuronal motility. It contributes to retrograde actin flow and actin filament reorganization and therefore induces neurite extension/retraction and also controls the direction of growth (Brown & Bridgman 2003; Lin et al. 1996; Medeiros et al. 2006; Wylie et al. 1998). Mammalian neurons express three isoforms of myosin II heavy chains (IIA, IIB and IIC), among which myosin IIB is the predominant form (Golomb et al. 2004). The activity of each of these isoforms is regulated by the phosphorylation of a common pair of regulatory myosin light chains (RMLC) which are bound to the myosin II heavy chains (Vicente-Manzanares et al. 2009). Although myosin II isoforms are similar in structure and are capable of partially substituting for each other (Bao et al. 2007), each isoform has been implicated in the performance of distinct tasks essential for normal neuronal function: myosin IIA has been reported to be responsible for neurite retraction and myosin IIB is required for neurite outgrowth (Wylie & Chantler 2003; Wylie et al. 1998). Myosin IIA mediates growth cone collapse and neurite retraction in response to repulsive guidance molecule (RGMa) (Kubo et al. 2008) and myosin IIB mediates growth cone turning at the border between laminin (an attractive guidance cue) and poly-L-ornithine (PLO) (Turney & Bridgman 2005). Intriguing is the involvement of both myosin IIA and IIB in response to semaphorin 3A: myosin IIA mediates the first phase response which results in growth cone collapse and myosin IIB is required for the second phase response which drives neurite retraction (Brown et al. 2009). In this study, we used a neurite guidance assay and a neurite outgrowth assay to demonstrate that treatment of dissociated cerebellar granule neurons (CGNs) with blebbistatin, a specific inhibitor of myosin II, or knockdown of myosin IIA or IIB reduces the inhibitory activity of CSPGs. In addition to the role of myosin II in growth cone guidance and neurite outgrowth, we demonstrate a novel synergistic effect of CSPGs and blebbistatin on the actin cytoskeleton that appears to promote neurite initiation on CSPGs.