Rac IN NEURONAL DEVELOPMENT

The three mammalian Rac isoforms that exist (Rac1, Rac2, and Rac3) are ~90% homologous (Didsbury et al., 1989; Haataja et al., 1997). However, the differential expression patterns of these three isoforms of Rac indicate that they may have tissue-specific functions during nervous system development. While Rac1 is ubiquitously expressed, Rac2 is mainly expressed in the hematopoietic system, and Rac3 is expressed in the brain. Intriguingly, Rac1 is expressed diffusely in the murine brain during development, while Rac3 expression peaks during times of neuronal maturation and synaptogenesis (Bolis et al., 2003). Rac1-deficient mice fail to complete gastrulation and are embryonic lethal at E8.5, demonstrating defects in cell adhesion and migration (Sugihara et al., 1998). Although Rac2- and Rac3-deficient mice are viable and fertile, certain strains of these knockouts display signs of cell type-specific defects in the hematopoietic system and brain, respectively (Pedersen and Brakebusch, 2012). Here, we will focus on the function of Rac1 and Rac3 in regulating neuronal development.

While loss of function mutations in Drosophila Rac genes result in defects in axonal growth, guidance, and branching (Hakeda-Suzuki et al., 2002; Ng et al., 2002), the involvement of Rac in vertebrate nervous system development is less clear. Given that genetic inactivation of Rac1 results in embryonic lethality in mice, more recent studies have focused on the effects of tissue-specific deletion of Rac1 on nervous system development, confirming an important role for Rac1 in neurite outgrowth and axonal pathfinding, as well as neuronal migration. Indeed, conditional deletion of Rac1 in whole murine brain increased apoptosis and impaired axonal pathfinding and neuronal migration in CGNs (Tahirovic et al., 2010). Consistent with a critical role for Rac1 in axon guidance during development of the vertebrate CNS, restricted deletion of Rac1 in the ventral telencephalon of mice prevents axonal migration across the midline of the corpus callosal and hippocampal commissures but does not affect axonal outgrowth, indicating that Rac1 primarily controls axon guidance rather than outgrowth in these brain regions (Chen et al., 2007). Nonetheless, Rac1 regulates mammalian neuronal development in a cell type-specific manner as the Rac-specific GEF, Tiam1, has been shown to enhance both neurite outgrowth and axon formation via its activation of Rac1 (Kunda et al., 2001).

More recent studies have uncovered an important function for Rac1 GTPase in neuronal proliferation. For example, deletion of Rac1 in the neural crest enhanced cell-cycle exit and diminished both the self-renewal and proliferation of late stage neural crest-derived stem cells (Fuchs et al., 2009). However, genetic deletion and virus-based loss of function studies demonstrated that Rac1 is not required for the proliferation of neural stem cell/progenitor cells (NS) in the hippocampus during embryonic development, but is an important regulator of late dendritic growth and spine maturation (Vadodaria et al., 2013). Nonetheless, the involvement of Rac1 in mediating proliferation in additional brain regions has also been suggested in recent literature. For instance, in the murine forebrain, loss of Rac1 reduced proliferation while increasing cell-cycle exit and premature differentiation of progenitor cells in the subventricular zone (Leone et al., 2010). Interestingly, a novel function was recently described for Rac1 in mediating learning-evoked neurogenesis in the hippocampus of adult mice. While previous reports have demonstrated that increased production of adult-born neurons in the hippocampus occurs in a Rac1-independent manner leading up to learning events, Haditsch et al. (2013) revealed that neuronal loss of active Rac1 attenuated proliferation and accumulation of neural progenitors during learning events. Future work will be required to further elucidate the involvement of Rac1 in mediating neural progenitor proliferation during nervous system development.

While in vitro and in vivo evidence suggests a crucial function for Rac1 in nervous system development, to date, relatively few studies have highlighted a similar function for Rac3. For example, genetic disruption of Rac3 in mice did not lead to obvious developmental defects and histopathological and immunohistochemical analysis did not reveal any abnormalities in the gross morphology of Rac3^-/- brains when compared to wild type mice. However, Rac3 knockout mice learned to perform significantly better on a rotarod test than wild type mice, indicating that loss of Rac3 improves motor learning and retention of coordinated memory behaviors (Corbetta et al., 2005). Rac3 may perform overlapping functions with Rac1 during nervous system development as neuronal-specific knockout of both Rac1 and Rac3 in mice led to behavioral defects, epilepsy, and premature death at postnatal day 9. Further analysis of brain tissue in Rac1^-/-/Rac3^-/- mice revealed developmental abnormalities in the hippocampus when compared to wild type brains (Corbetta et al., 2009). Thus, evidence suggests that both Rac1 and Rac3 are important for proper CNS development.

AMYOTROPHIC LATERAL SCLEROSIS

Amyotrophic lateral sclerosis is a devastating neuromuscular disorder characterized by loss of motor neurons of the motor cortex, brain stem, and upper and lower spinal cord. Approximately 10% of ALS cases appear to be caused by a genetic component, while the remaining 90% are considered sporadic in nature. In 1993, the glycine to alanine substitution at position 93 in Cu, Zn- superoxide dismutase (SOD1) was one of eleven mutations initially identified to be causative in familial ALS. Although the G93A SOD1 mutation remains the best characterized to date, mutations in additional genes such as Alsin (ALS2), Tar DNA-binding protein 43 (TDP43), and fused in sarcoma (FUS) have also been linked to other familial forms of ALS. Recent evidence suggests that dysregulation of Rho GTPases may be a factor underlying both genetic and sporadic ALS.

Recessive mutations in a Rac GEF, alsin, have been reported in juvenile forms of ALS, primary lateral sclerosis, and infantile-onset ascending hereditary spastic paralysis (Hadano et al., 2007). When overexpressed in cultured human cells, alsin mutants are rapidly degraded, suggesting disease progression occurs through a mechanism consistent with alsin loss-of-function (Yamanaka et al., 2003). Although alsin can function as a GEF for both Rab5 and Rac1, alsin colocalizes with Rac1 in the growth cones of neurons and regulates Rac1 signaling and neurite outgrowth through its intrinsic GEF activity, thus suggesting a link between Rac1 disruption and neurodegenerative disease (Topp et al., 2004; Kanekura et al., 2005; Tudor et al., 2005). These studies highlight that a loss of Rac activity may underlie the progression of early onset forms of ALS.

The critical role of Rac in maintaining neuronal survival is evidenced by the fact that disruptions in Rac signaling have been shown to induce apoptosis in a variety of diverse neuronal cell types, including primary motor neurons (Kanekura et al., 2005; Jacquier et al., 2006; Loucks et al., 2006; Stankiewicz et al., 2012). In spinal motor neurons isolated from embryonic rats, siRNA knockdown of alsin induces neurite retraction and cell death that is abrogated by expression of constitutively active Rac1 (Jacquier et al., 2006). Furthermore, alsin antagonizes NSC34 motor neuronal death induced by the expression of ALS-causing SOD1 mutants (mSOD1) via activation of a pro-survival Rac1/PI3K/Akt signaling cascade (Kanekura et al., 2005). Intriguingly, accumulating evidence suggests that SOD1 may regulate the activity of Rac1 GTPase. In particular, neuronal apoptosis induced via expression of either G93A or H80R mSOD1 in SH-SY5Y dopaminergic cells correlated with a decrease in Rac1 GTPase activity. Indeed, expression of a constitutively active Rac1 GTPase mutant abrogated mSOD1-induced neuronal death while expression of dominant negative Rac1 GTPase alone induced apoptosis (Pesaresi et al., 2011). Future studies should be aimed at determining whether expression of mutant forms of SOD1 similarly decrease Rac1 activation in motor neurons in particular.

In addition to mutations that code for alsin and SOD1, recent evidence also suggests that ARHGEF16, a GEF for Rac GTPase, is hypermethylated and downregulated in postmortem sporadic ALS spinal cord samples when compared to samples from neurologically normal controls (Figueroa-Romero et al., 2012). Thus, dysregulation of Rac GTPase may be a common factor underlying both familial and sporadic forms of ALS. While accumulating evidence has highlighted a critical function for Rac GTPase in maintaining neuronal survival, the involvement of Rac in the etiology of ALS has only been examined recently and the exact mechanism by which disruptions in Rac activity may induce neuronal apoptosis remains poorly understood. Therefore, alterations in Rac activity in motor neurons and mouse models of ALS warrants further investigation.

In a manner dissimilar to motor neurons, several studies indicate that mutant forms of SOD1 associated with the pathology of ALS have an opposing function in microglia, ultimately resulting in enhanced activation of Rac1 GTPase in a redox-sensitive manner. For example, Harraz et al. (2008) demonstrated that wild type SOD1 binds to Rac1 and inhibits its intrinsic GTPase activity under reducing conditions, ultimately increasing active Rac1 signaling. In the presence of H₂O₂, the interaction between wild type SOD1 and Rac1 GTPase was uncoupled. Intriguingly, ALS causing mutations in SOD1 prevent the enzyme from redox uncoupling, resulting in enhanced and persistent activation of Rac1 GTPase in microglia cells. Inevitably, aberrant Rac1 GTPase activity leads to increased activation of Nox2, a catalytic subunit of NADPH oxidase, resulting in an overproduction of damaging ROS. In agreement with Rac1-dependent activation of NADPH oxidase as a contributing factor underlying motor neuron degeneration in the G93A mSOD1 mouse, inhibition of, or genetic deletion, of Nox2 slows disease progression and increases survival in this particular mouse model of familial ALS (Wu et al., 2006; Marden et al., 2007; Valdmanis et al., 2008). Furthermore, Apolloni et al. (2013) recently identified extracellular ATP as a potential mechanism by which Rac1 GTPase activity may be enhanced in microglia of G93A mSOD1 mice. In primary microglia cells isolated from G93A mSOD1 mice, stimulation of the P2X₇ receptor by its agonist, 2′-3′-O-(benzoyl–benzoyl) ATP, enhanced Nox2 activation and the production of ROS in a Rac1-dependent manner when compared to microglia derived from non-transgenic animals. Thus, recent experimental evidence suggests that G93A mSOD1 may stimulate Rac1 GTPase activation in a P2X₇-dependent manner to enhance the release of toxic ROS from microglia.

Recent studies also implicate aberrant Rho GTPase activity as an underlying factor in the pathology of ALS. For example, although the significance remains unknown, the Rho GEF, RGNEF, displays enhanced cytoplasmic inclusions in spinal motor neurons from both sporadic and familial ALS patients (Keller et al., 2012; Droppelmann et al., 2013). However, future studies will be required to identify the importance of RGNEF-positive spinal motor neuron cytoplasmic inclusions. Nonetheless, in accordance with enhanced Rho GTPase activity as a causative factor to motor neuronal death in ALS disease progression, ROCK activity is increased in the G93A mSOD1 mouse model of ALS, as well as sporadic ALS patients (Hu et al., 2003; Capitanio et al., 2012; Conti et al., 2014). Tönges et al. (2014) demonstrated that inhibition of ROCK preserved neuromuscular junctions and extended the survival of G93A mSOD1 mice via a mechanism consistent with reduced microgliosis and decreased release of pro-inflammatory cytokines and chemokines such as TNFα and IL-6. When examining motor neuron death in G93A mSOD1 mice, Takata et al. (2013) demonstrated that administration of fasudil slowed disease progression and reduced motor neuron loss by a mechanism consistent with reduced activation of ROCK and PTEN and restored activation of the pro-survival kinase Akt. While the above studies indicate that a gain of Rho GTPase activity may underlie the progression of ALS, new studies will need to be carried out to determine the precise mechanism by which unopposed activation of Rho GTPase may contribute to the motor neuronal death observed in ALS.

ALZHEIMER’S DISEASE

Alzheimer’s disease is the most common form of dementia and is characterized by extensive loss of neurons in the hippocampus and cerebral cortex. The onset of AD correlates with an aberrant accumulation of extracellular amyloid-β (Aβ) plaques which are the products of proteolytic cleavage of the membrane protein amyloid precursor protein (APP). Aβ plaques have been demonstrated to be at least partially responsible for the dysregulation of actin polymerization that underlies synaptic dysfunction observed during AD progression (Bloom, 2014). Accumulating evidence suggests that dysregulated Rho family GTPase activity may correlate with Aβ production and thereby contribute to the loss of actin polymerization and dendritic spines, which underlies the pathogenesis of AD.

Although diminished Rac1 expression is observed in AD brains obtained from patients early in disease (Zhao et al., 2006), recent evidence suggests a positive correlation between Rac GTPase activity and APP expression during later stages of AD disease progression. For example, in primary hippocampal cells, inhibition of Rac utilizing NSC23766 or expression of a dominant negative Rac1 mutant reduced APP mRNA and APP protein expression, indicating that Rac1 activates APP transcription (Wang et al., 2009). In addition, Rac1 may also contribute to the formation of toxic Aβ fragments as inhibition of Rac1 diminished gamma secretase-dependent cleavage of APP in COS-7 cells (Boo et al., 2008). Interestingly, while these data highlight that Rac1 may contribute to APP transcription and the generation of toxic Aβ fragments, recent reports also suggest that Aβ peptides can in turn, induce the activation of Rac1. For example, addition of Aβ42 to hippocampal cultures resulted in upregulation of the Rac-specific GEF Tiam1, enhanced activation of Rac1, downstream activation of the Rac effectors PAK1 and LIMK, and increased actin polymerization (Mendoza-Naranjo et al., 2007, 2012). Thus, Rac1 GTPase may function in a positive feedback loop with APP and/or Aβ fragments to regulate the expression and activities of one another.

Further evidence suggests that alterations of a Rac1-dependent signaling cascade links expression of toxic Aβ fragments to disruption of actin polymerization and dendritic spines. Indeed, it has previously been reported that expression of the downstream Rac effector, PAK, is decreased in hippocampal sections, while levels of phosphorylated (active) PAK were elevated in intraneural aggregates during AD progression (Zhao et al., 2006). In human AD and Tg2576 mice, phosphorylated (active) PAK translocated from the cytosol to intracellular granule bodies that also stain positive for Rac1 and Aβ. In primary hippocampal cells, expression of Aβ induced a similar redistribution of PAK and resulted in a reduction in dendrites (Ma et al., 2008). Therefore, dysregulation of PAK signaling may underlie some synaptic defects observed in AD.

Although these previous studies indicate that enhanced Rac1 activity may contribute to AD progression, the global reduction of Rac activity in the brains of human AD patients reveals that the involvement of Rac GTPase in the progression of AD is complex. In accordance with reduced Rac GTPase activity, it was previously reported that the mRNA and protein expression levels of the Rac GEF, Kalirin-7, are reduced in the hippocampus of human AD brains. Kalirin-7 is known to play a critical role in dendritic spine maintenance. Intriguingly, Kalirin-7 associates with iNOS to downregulate its activity in AtT-20, SH-SY5Y, C6, and Neuro-2A cells. In the human AD hippocampus, elevated expression of iNOS correlates with diminished association with Kalirin-7 (Youn et al., 2007). Thus, these data suggest that reduced Kalirin-7 levels may contribute to the pathogenesis of AD through enhanced activity of iNOS. Future studies will be critical in determining whether reduced levels of Kalirin-7 contribute to diminished Rac GTPase activity during early stages of AD progression.

In addition to Rac GTPase, aberrant Rho GTPase activity has also been linked to the pathology of AD. Indeed, RhoA is decreased in human AD brains and in the brains of transgenic mice overexpressing the Swedish double mutant of APP. Moreover, while RhoA was decreased in synapses of AD mice, its expression was increased in degenerating neurites consistent with the involvement of RhoA in neurite retraction (Huesa et al., 2010). Addition of Aβ to human neuroblastoma cells resulted in reduced neurite length concomitant with enhanced activation of RhoA and diminished activation of Rac1 (Petratos et al., 2008). Activation of RhoA has also been correlated with production of Aβ fragments as activation of Rho and its downstream effector, ROCK, increased production of toxic Aβ fragments by gamma secretase-dependent cleavage of APP (Zhou et al., 2003). Thus, aberrant activation of Rho GTPase may contribute to AD pathology through enhanced neurite retraction, as well as, increased production of toxic Aβ fragments.

HUNTINGTON’S DISEASE (HD)

Huntington’s disease (HD) is a progressive and fatal autosomal dominant disorder characterized by loss of cells in the striatum and cortex, resulting in involuntary body movements (chorea), cognitive decline, psychiatric conditions, and eventually death. HD is caused by a mutation in the Huntingtin gene which results in expansion of a CAG nucleotide repeat. Mutant huntington (Htt) forms toxic gain-of-function aggregates that underlie the etiology of HD. Although relatively few studies have examined the involvement of Rho GTPases in the progression of HD, recent efforts have linked dysregulated Rho family GTPase signaling to both the production of toxic ROS and mutant Htt aggregation in the degenerating CNS.

In a similar manner to other neurodegenerative diseases, the production of ROS is believed to play a role in the etiology of HD. Compared to neurologically normal controls, HD patients show enhanced NADPH oxidase activity and an increase in ROS in the cortex and striatum of postmortem brain samples. In the 140Q/140Q mouse model of HD, NADPH oxidase activity in cortex and striatum correlated with enhanced production of ROS and neurite swellings, and was prevented by treatment with NOX inhibitors. Similarly, mutant HD mice bred with NOX2 knockout mice showed enhanced survival concomitant with decreased NADPH oxidase activity and lowered production of ROS (Valencia et al., 2013). Given the important involvement of Rac1 GTPase in activation of NADPH oxidase activity, this study indicates that aberrant Rac1 activation may contribute to production of ROS and thereby the pathology of HD. In addition to the role of Rac1 in contributing to the production of ROS through activation of NADPH oxidase, Rac1 interacts with mutant Htt in a yeast two-hybrid screen and siRNA-mediated knockdown of Rac1 reduces caspase-3/7 activity in striatal cells expressing mutant Htt (Tourette et al., 2014). Thus, enhanced Rac1 activation may contribute to the pathogenesis of HD through either activation of NADPH oxidase in microglia or direct binding to mutant Htt in susceptible neuronal populations.

The downstream Rac effector, PAK, has also been implicated in the pathogenesis of HD. PAK1 binds to Htt in vitro and in vivo and colocalizes with mutant Htt in human HD brains. Overexpression of PAK1 enhances the aggregation of mutant Htt and Htt-induced toxicity of HeLa cells independent of its kinase activity (Luo et al., 2008). In addition to PAK1, a recent study utilizing a yeast two-hybrid screen identified PAK2 as a modulator of Htt-induced toxicity (Tourette et al., 2014). Finally, the PAK-interacting exchange factor (-pix), a GEF for Rac and Cdc42, promotes mutant Htt aggregation in the MG251 neuronal glioblastoma cell line (Eriguchi et al., 2010). Thus, Rac and its interacting proteins may underlie the neurotoxicity and aggregation of mutant Htt in HD.

Intriguingly, recent studies have also highlighted an important role for ROCK activation in the etiology of HD. Inhibition of ROCK utilizing Y-27632 has been demonstrated to have neuroprotective effects in vitro and in a Drosophila model of HD. More recently, Y-27632 has also been shown to improve rotarod performance and reduce soluble mutant Htt levels in the R6/2 mouse model of HD. However, inhibition of ROCK did not have a significant effect on brain weight, inclusion number or size, striatal medium spiny neuron number, clasping behavior, or lifespan (Li et al., 2009a). ROCK may underlie HD progression through the phosphorylation and inhibition of profilin, which when overexpressed, reduces the aggregation of polyglutamine-expanded Htt and androgen receptors in primary neurons (Shao et al., 2008). These data indicate that enhanced activation of Rho/ROCK signaling may also contribute to the pathogenesis of HD.

PARKINSON’S DISEASE

Parkinson’s disease is the most common neurodegenerative movement disorder and is caused by progressive degeneration of nigrostriatal dopaminergic neurons of the midbrain. Clinically, PD manifests as a progressive locomotor, cognitive, and behavioral disorder. In the CNS, PD results in the formation of Lewy bodies containing accumulation of -synuclein. The most common genetic cause of familial PD is due to mutation in leucine-rich repeat kinase 2 (LRRK2; Macleod et al., 2006). Although several genetic mutations have been identified in PD, exposure to environmental toxins has also been strongly associated with development of PD.

Expression of disease-causing LRRK2 mutants induces neurite retraction and dopaminergic apoptotic cell death in primary neurons and in the rodent CNS (Macleod et al., 2006). Interestingly, wild type LRRK2 interacts with and increases the activity of Rac1 in vitro; however, the disease-causing mutants, G2019S and R1441C display weakened Rac1 binding while the Y1699C and I2020T mutations confer increased binding of Rac1. Thus, the distinct LRRK2 mutations exert differential effects on Rac1 binding. Consistent with diminished Rac1 activity playing a role in PD pathology, expression of Rac1 rescues SH-SY5Y cells from neurite retraction and cell death caused by disease-causing LRRK2 PD mutants (Chan et al., 2011). Therefore, mutations in LRRK2 may induce neurite retraction and cell death in dopaminergic neurons principally through diminished Rac1 GTPase activity.

Rotenone is an environmental toxin that induces dopaminergic loss and parkinsonian features when chronically administered to rodents (Betarbet et al., 2000; Alam and Schmidt, 2002). It has been previously reported that rotenone induces the production of ROS not only by inhibition complex I, but also through the activation of NADPH oxidase (Gao et al., 2002, 2003). In phagocytes, rotenone directly interacted with the catalytic subunit of NADPH oxidase, NOX2, to enhance activation of NADPH oxidase and the formation of ROS. Rac1 enhanced the activation of rotenone-dependent ROS formation and inhibition of Rac1 utilizing NSC23766 decreased ROS release from microglia (Zhou et al., 2012). In a similar manner, inhibition of NADPH oxidase utilizing apocynin relieved PD symptoms in a marmoset model of the disease (Philippens et al., 2013). These data indicate that aberrant activation of Rac in microglia may contribute to enhanced production of ROS and underlie the death of neighboring dopaminergic neurons in PD.

However, the involvement of Rac in mediating neuronal survival may be much more complex in PD progression as it has also been demonstrated that hippocampal and dopaminergic neurons exposed to rotenone showed decreased active Rac1-GTP and enhanced RhoA-GTP (Sanchez et al., 2008). Thus, in a manner similar to the antagonistic relationship between Rac and Rho in regulating the survival of motor neurons, diminished Rac activity and enhanced Rho activity may contribute to neuronal death during the progression of some forms of PD, particularly those forms that are linked to environmental toxin exposure. Further, Rac1 may contribute to PD disease progression in a cell type-specific manner, where loss of Rac GTPase activity may contribute to the death of neurons while increased Rac-GTP activity in microglia may contribute to the formation of toxic ROS.

In addition to rotenone, the neurotoxic 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is taken up by dopaminergic neurons and induces PD symptoms through inhibition of mitochondrial complex I of the electron transport chain, leading to enhanced ROS production and neuronal apoptosis. When administered to mice, MPTP induces cellular polarization of microglia and leads to the formation of neuron-glia contacts, which precede the phagocytosis of dopaminergic cell bodies. This process may be mediated by ROCK activation as inhibition of ROCK utilizing fasudil prevented microglia motility and activation of microglia in the substantia nigra of MPTP-treated mice (Barcia et al., 2012). Indeed, inhibition of ROCK has shown to enhance survival of dopaminergic neurons and attenuate axonal loss in the MPTP mouse model of PD (Tönges et al., 2012). These data indicate that dysregulation of Rho family GTPases may underlie both genetic and environmental cases of PD.

CHARCOT MARIE-TOOTH DISEASE (CMTD)

Charcot marie-tooth disease (CMTD) is an inherited neurological disorder that presents as progressive muscle weakness and decreased muscle size. The first subtype of CMTD is characterized by demyelination and results in lower nerve conduction velocities while the second subtype is due to a defect within the axons themselves. Recent studies have highlighted that dysregulated Rho family GTPase activity may underlie demyelination and axonal abnormalities. Recently, the T332I mutation was identified in ARHGEF10 in patients exhibiting slowed nerve conduction velocities and thin myelination of peripheral nerves. ARHGEF10 is a GEF for RhoA that is highly expressed in the peripheral nervous system and exhibits enhanced activity when harboring the T332I mutation (Verhoeven et al., 2003; Chaya et al., 2011). Consistent with activation of RhoA, T332I ARHGEF10 induces cell contraction through Rho/ROCK signaling in Schwann cells from rat sciatic nerve. Thus, ARHGEF10 may contribute to the pathology of CMTD by enhanced activation of RhoA-dependent cell contraction in the peripheral nervous system (Chaya et al., 2011).

SPINAL CORD INJURY (SCI)

The spinal cord connects the CNS to the PNS to control sensory and motor functions. Following spinal cord injury (SCI), this connection can be disrupted to elicit neurological deficits due to impaired transmission between the CNS and PNS. The symptoms of SCI vary widely dependent upon the location and severity of injury but neurological defects can range from pain to paralysis. Given the extensive involvement of Rho GTPases in regulating axonal outgrowth and pathfinding, it is not surprising that members of the Rho GTPase family have emerged as key modulators of axonal regeneration following SCI.

Axonal regeneration following SCI is impeded by increased expression of the inhibitory myelin-associated proteins, Nogo, myelin-associated glycoprotein (MAG), and oligodendrocytes-myelin glycoprotein (OMgp). Early studies in CGNs demonstrated that NogoA and MAG mediate their inhibitory effects on neurite outgrowth through disruption of the delicate balance between antagonistic Rho/ROCK and Rac signaling pathways. In particular, the inhibitory proteins enhance activation of Rho/ROCK signaling and diminish activation of Rac1 (Niederöst et al., 2002). Indeed, in numerous in vivo models of SCI, administration of either Rho GTPase inhibitors (e.g., C3 transferase) or ROCK inhibitors (e.g., fasudil, Y-27632) is protective following traumatic nerve injury (Fournier et al., 2003; Boato et al., 2010; Impellizzeri et al., 2010; Boomkamp et al., 2012). In vivo, treatment with fasudil improved motor performance while reducing histological damage, the production of proinflammatory cytokines, and activation of caspases following application of vascular clips to the dura via a laminectomy in mice (Impellizzeri et al., 2010). In a similar manner, lentiviral injection of dominant negative ROCK into the adult rat red nucleus protected against damage induced by unilateral rubrospinal tract transection at the fourth cervical level (Wu et al., 2009). Moreover, ROCKII^-/- mice subjected to dorsal root nerve crush injury recovered use of the affected forepaw more quickly and demonstrated enhanced regeneration of sensory axons across the dorsal root entry zone when compared to controls (Duffy et al., 2009). Collectively, these studies strongly suggest that enhanced Rho/ROCK signaling impedes axonal growth and regeneration following SCI injury.

In opposition to Rho, recent studies indicate that activation of Rac may be a critical feature which promotes axonal regeneration following SCI. Wallerian degeneration occurs in response to axonal transection and involves axonal degeneration and fragmentation of the myelin sheath. Jung et al. (2011) recently determined that a dominant negative Rac1 mutant or siRNA directed against Rac1 prevents myelin fragmentation in vivo following sciatic nerve axotomy in mice. However, constitutively active Rac1 failed to induce myelin fragmentation on uninjured nerves, indicating that Rac1 is an upstream regulator, but is not sufficient to induce myelin degradation following sciatic nerve injury in mice. In contrast to this prior study indicating Rac1 has an essential role in myelin fragmentation following nerve injury, Lorenzetto et al. (2013) demonstrated that injection of cell-penetrating constitutively active Rac1 mutants following optic nerve crush improved survival and axonal regeneration while preventing dendrite degeneration. The pro-survival effects of constitutively active Rac1 may have been mediatated through PAK and ERK1/2 signaling, as both showed enhanced activation in retinal ganglion cells (Lorenzetto et al., 2013). Therefore, although the exact mechanism is presently unclear, these studies indicate that the disrupted balance between Rac and Rho GTPases is a critical determinant of axonal regeneration following traumatic nerve injury.