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review article molecular neuroscience published 03 november 2011 doi 10.3389/fnmol.2011.00039 rocking regeneration rho kinase inhibition as molecular target for neurorestoration lars tönges 1 jan-christoph koch 1 mathias bähr 1,2 and paul lingor 1,2 1 2 department of neurology university medicine göttingen göttingen germany dfg research center for the molecular physiology of the brain göttingen germany edited by simone di giovanni university of tuebingen germany reviewed by simone di giovanni university of tuebingen germany kazim sheikh the university of texas health science center at houston usa correspondence paul lingor department of neurology university medicine göttingen robert-koch-strasse 40 37075 göttingen germany e-mail plingor@gwdg.de regenerative failure in the cns largely depends on pronounced growth inhibitory signaling and reduced cellular survival after a lesion stimulus one key mediator of growth inhibitory signaling is rho-associated kinase rock which has been shown to modulate growth cone stability by regulation of actin dynamics recently there is accumulating evidence the rock also plays a deleterious role for cellular survival in this manuscript we illustrate that rock is involved in a variety of intracellular signaling pathways that comprise far more than those involved in neurite growth inhibition alone although rock function is currently studied in many different disease contexts our review focuses on neurorestorative approaches in the cns especially in models of neurotrauma promising strategies to target rock by pharmacological small molecule inhibitors and rnai approaches are evaluated for their outcome on regenerative growth and cellular protection both in preclinical and in clinical studies keywords rho kinase rock neuroprotection neurorestoration cell survival axon outgrowth introduction numerous factors are responsible for the failure of neurons to regenerate in the adult mammalian cns and to restore functionality in traumatic and degenerative diseases of the nervous system axonal degeneration and neuronal death clearly limit the regenerative substrate but even when the structural components are only partially lesioned such as in the case of spinal cord injury where neuron cell death is negligible the extracellular environment and the nearly inexistent regenerative capacity of adult neurons counteract a successful functional restoration harel and strittmatter 2006 yiu and he 2006 liu et al 2011 inhibitory signaling of myelin components and other repulsive guidance cues converges upon rho-associated kinase rock which propagates this signal down to the cytoskeleton influencing neurite growth and regeneration this review will focus on rock as a promising molecular target for neurorestorative strategies in the cns and highlight recent insights into rock-mediated effects on survival and regeneration rho kinase rock the rock proteins are serine/threonine kinases with a large homology to other agc kinases such as myotonic dystrophy protein kinase dmpk dmpk-related cdc42-binding kinase mrck and citron kinase pearce et al 2010 structurally rocks have a catalytic kinase domain at the n-terminus which is followed by a coiled-coil region including the rho-binding site and a regulatory pleckstrin homology domain at the c-terminus with cysteine-rich repeats the pleckstrin homology domain is likely to be involved in the stabilization of membrane association of rock the c-terminus acts in an autoinhibitory manner on the kinase activity by interaction with the n-terminus riento and ridley 2003 this interaction is disrupted by binding of active rho and thus increases kinase activity amano et al 1999 two highly homologous isoforms are known rock1 which is mostly expressed in heart lung and other non-neuronal tissue and rock2 which is preferentially found in brain spinal cord and muscle and shows an increased expression with age hashimoto et al 1999 komagome et al 2000 duffy et al 2009 in the bovine brain rock2 is abundantly expressed in neuronal cells in the gray matter in comparison to the white matter here intense immunoreactivity for rock2 has been demonstrated in cortical hippocampal and cerebellar purkinje cell neurons hashimoto et al 1999 preliminary results of an examination of human brain autopsies suggest that rock2 protein expression is not exclusively confined to neuronal cells which however express the protein to a much higher level than non-neuronal cells own unpublished data the sequence homology between rock1 and rock2 is 65 and even 92 in the kinase domain whereas the c-terminus shows a higher divergence nakagawa et al 1996 matsui et al 1998 despite their structural similarities rock1 and rock2 may have distinct roles in tissues where both isoforms are expressed to a comparable extent however an experimental in-depth dissection of differential roles of rock1 and rock2 in the same cell type has only been performed in non-neuronal cell culture models so far yoneda et al 2005 taking advantage of gene knockout technologies it has been found that in murine development rock1 is not able to completely compensate for the loss of the rock2 isoform as a consequence mice deficient for the rock2 gene exhibit severe hypercoagulable states with increased thrombus formation placental dysfunction and intrauterine growth retardation which commonly leads to fetal or early postnatal death in the offspring thumkeo et al 2003 in a different rock2 knockout model mice were normal in gross brain anatomy but were severely altered in synaptic spine morphology frontiers in molecular neuroscience www.frontiersin.org november 2011 volume 4 article 39 1

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tönges et al rock as target for neurorestoration basal synaptic transmission and hippocampal ltp this was found to be attributed to dysfunctions of the actin cytoskeleton and the actin-binding protein cofilin zhou et al 2009 a study that compared haploinsufficient rock1 and rock2 mice focused on vascular biology and examined the extent of neointima formation in the carotid artery here rock1 haploinsufficiency was associated with reduced neointima formation as well as with decreased vascular smooth muscle cell proliferation and decreased levels of proinflammatory adhesion molecule expression noma et al 2008 upstream activators and downstream targets of rock the extracellular environment of the cns is highly repulsive toward regenerating axons which is largely attributed to the presence of inhibitory molecules on oligodendrocytes myelin and scar tissue among these we find nogo myelin-associated glycoprotein mag and oligodendrocyte myelin glycoprotein omgp which are regularly present on the surface of oligodendrocytes wang et al 2002 nogo mag and omgp have been shown to confer their inhibitory activity via a trimeric receptor complex comprising nogo receptor 1 ngr1 lingo-1 and p75ntr or troy mi et al 2004 and also via the paired immunoglobulin-like receptor b pirb as recently reported atwal et al 2008 after ligand binding the p75ntr receptor component activates the small gtpase rhoa which has been identified as a main binding partner of rock ishizaki et al 1996 additionally g-protein-coupled receptor gpcr stimulation by lysophosphatidic acid lpa or sphingosine-1 phosphate s1p results in the transformation of gdp-bound rhoa gtpase into the gtp-bound form which is the active one after binding to the rho-binding domain active rhoa increases the kinase activity of rock by release of its autoinhibition after this activation rock translocates to peripheral membranes leung et al 1995 following lesion repulsive guidance molecules which direct axonal outgrowth during embryogenesis can be upregulated and act as regeneration inhibitors this has been shown for sema5a goldberg et al 2004 and several ephrins/eph receptors summarized in goldshmit et al 2006 although ephrins and semaphorins employ a different receptor parts of their downstream signaling similarly converge on the rho/rock cascade via ephexin and rac1 respectively liu and strittmatter 2001 shamah et al 2001 in contrast to the reversible action of rhoa caspases have been shown to irreversibly activate rock by truncation and generation of a constitutively active form rock activity in this context was necessary and sufficient for the apoptotic process by formation of membrane blebs and re-localization of fragmented dna coleman et al 2001 sebbagh et al 2001 similar to caspases granzyme b is able to proteolytically cleave and activate rock sebbagh et al 2005 numerous downstream target proteins have been identified which are regulated by rock phosphorylation many of these are involved in regulation of cell shape and motility but others participate in cell cycle and survival pathways myosin light chain mlc is a substrate of activated rock and its phosphorylation results in actomyosin contraction amano et al 1996 in addition rock can inactivate mlc phosphatase mlcp indirectly regulating mlc phosphorylation which results in conformational changes of myosin required for contraction of actin filaments kimura et al 1996 being a serine/threonine kinase rock can activate lim kinase-1 limk1 which then inactivates cofilin/actin depolymerizing factor adf by phosphorylation yang et al 1998 as a consequence cofilin is no longer able to severe filamentous actin f-actin and to depolymerize actin from the pointed ends which promotes actin polymerization initiation of growth cone collapse and reduced axonal outgrowth or growth arrest ng and luo 2004 rock2 has also been shown to phosphorylate the so-called erm proteins ezrin radixin and moesin matsui et al 1998 erm proteins act as molecular bridges between the plasma membrane and the actin cytoskeleton and therefore play essential roles in axon growth and regeneration arpin et al 2011 phosphorylation by rock reduces the head-to-tail association of erm proteins which stabilizes their open and active conformation adducin is a protein that binds to f-actin promoting the association of spectrin and f-actin rock-mediated phosphorylation of adducin enhances its f-actin-binding potential and thus regulates membrane ruffling and cell motility fukata et al 1999 elongation factor-1 alpha ef-1 is another substrate of rock that has been shown to be phosphorylated in vitro which results in decreased f-actin-binding activity of ef-1alpha izawa et al 2000 further targets of rock phosphorylation are collapsin response mediator protein 2 crmp2 vimentin glial fibrillary acidic protein gfap neurofilament tau and map2 kosako et al 1997 goto et al 1998 hashimoto et al 1998 arimura et al 2000 amano et al 2003 one of the most interesting and recently described downstream targets of rock is the phosphatase and tensin homolog pten which is directly activated by phosphorylation through rock in hek cells and leukocytes li et al 2005 the pten gene product is a phosphatase that acts as a tumor suppressor and is involved in cell cycle regulation pten negatively regulates akt/pkb signaling by antagonizing activated phosphatidylinositol 3,4,5 trisphosphate pip3 which is a canonical pathway employed by growth factors to regulate neuronal survival suppressing pten thus fosters pip3 signaling via phosphatidylinositol-dependent kinase 1/2 pdk1/2 to activate akt/pkb which in turn activates ras homolog enriched in brain rheb through inhibition of the tuberous sclerosis complex 1/2 tsc1/2 activated rheb stimulates mtor complex 1 mtorc1 that finally leads to an enhancement of general translation with an increase in protein synthesis and cell growth guertin and sabatini 2007 park et al 2010 however there is also a much closer link of rock to mtor that has been elucidated in fibroblasts here rock1 has been identified as mediator of the cell anchorage signal to the extracellular matrix by physically interacting with tsc2 this in turn relays the signal to rheb and finally mtor park et al 2011 a summary of the most important signaling pathways involving rock is presented in figure 1 erm proteins crosslink actin filaments with plasma membranes they co-localize with cd44 at actin filament-plasm a membrane interaction sites associating with cd44 via their n-terminal domains and with actin filaments via their c-terminal domains ver os genes up and down regulated inhibition of rock small molecule inhibitors of rock a plethora of different pharmacological inhibitors of rock have been synthesized which structurally belong to different chemical groups mueller et al 2005 among the best characterized frontiers in molecular neuroscience www.frontiersin.org november 2011 volume 4 article 39 2

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tönges et al rock as target for neurorestoration figure 1 the rock signaling pathway with focus on survival and regeneration nogo mag omgp present on the surface of oligodendrocytes confer their inhibitory activity via a trimeric receptor complex comprising ngr1 lingo-1 and p75ntr to activate rhoa sema5a and several ephrins can also contribute to rhoa activation via their plexin or eph receptors respectively additionally g protein-coupled receptor gpcr stimulation by lysophosphatidic acid lpa or sphingosine-1 phosphate s1p results in the activation of the small gtpase rhoa gtp-bound rhoa further activates rock to phosphorylate several substrates that are involved in regulation of cell shape and motility but also in survival pathways caspase-3 and granzyme b are able to proteolytically cleave and thus activate rock myosin light chain mlc is a substrate of activated rock and its phosphorylation results in actomyosin contraction in addition rock can inactivate mlc phosphatase mlcp and thus indirectly regulate mlc phosphorylation being a serine/threonine kinase rock can activate lim kinase-1 limk1 that then inactivates cofilin/actin depolymerizing factor adf by phosphorylation promoting actin filament stabilization the phosphorylation of erm proteins leads to actin-membrane linkage adducin is a protein that after activation binds to f-actin promoting the association of spectrin and f-actin and thus assembling the actin network another downstream target of rock is pten which negatively regulates akt/pkb signaling by antagonizing pip3 if active akt in turn activates rheb because of inhibition of tsc1/2 and subsequently stimulates mtorc1 which finally leads to an enhancement of general translation with an increase in protein synthesis and cell rock1 may also physically interact with tsc2 and most frequently used substances is fasudil a member of the isoquinoline series its main metabolite is hydroxyfasudil which shows a good bioavailability in various animal models in vivo even after oral administration hattori et al 2004 wang et al 2005b y-27632 is a more specific rock inhibitor frequently used in biological and pharmacological experiments and a member of the 4-aminopyridine series over the years these substances have been developed further based on indazole amide or urea backbones mueller et al 2005 liao et al 2007 in order to optimize kinase selectivity cell activity microsomal stability and pharmacogenetic properties new pharmacological variants of the early rock inhibitors or even new pharmacological classes have been generated among them are chroman-3-amides chen et al 2008 azaindol-based inhibitors schirok et al 2008 and tetrahydroisoquinoline derivatives fang et al 2010 dominant-negative mutants and rnai-based approaches next to pharmacological inhibitors of rock the expression of a dominant-negative mutant protein has been shown to act as a functional rock inhibitor both in vitro and in vivo in a spinal cord lesion model wu et al 2009 approaches using the rnai technology to down regulate rock expression have been effectively applied in vitro and allow for an isoform and cell type-specific view on the roles of rock1 and rock2 lock and hotchin 2009 studies in fibroblasts and vascular smooth muscle cells have shown that both rock1 and rock2 seem to have distinct non-redundant functions one example is the disassembly of stress fibers that is observed after knock-down of rock1 but not of rock2 in fibroblasts in contrast this seems to be mediated mainly by rock2 in smooth muscle cells yoneda et al 2005 wang et al 2009 furthermore rock1 is cleaved and thereby constitutively activated by caspase-3 at a conserved sequence at its c-terminus that is not found in rock2 sebbagh et al 2001 if a cell specific regulation of rock isoform expression in animal models in vivo is required viral vector mediated transfer methods of shrna expressing plasmids leading to rnai-mediated knock-down of the target gene are a valuable option important considerations for viral vector design include size of the transgene route of delivery tropism duration and regulation of gene expression and side effects for a review see davidson and breakefield frontiers in molecular neuroscience www.frontiersin.org november 2011 volume 4 article 39 3

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tönges et al rock as target for neurorestoration 2003 studying the role of rock2 in cell survival paradigms after optic nerve on lesion we were able to almost selectively infect retinal ganglion cells by aav vectors this enabled us to cell specifically regulate rock2 expression whereas the vector delivery via intravitreal injection was easy to handle vector dosage in vivo was much more difficult and required an elaborate dose finding study unpublished personal observation targeting the rock pathway by modulation of upstream activators and downstream targets as described in the previous section a plethora of upstream activators and downstream effectors of rock are known therefore rock can also be regulated in an indirect manner however under these circumstances significant off-target effects have to be considered an obvious possibility to disrupt rock inhibitory signaling is provided by the use of the exoenzyme c3 transferase which is an adp ribosyl transferase that selectively ribosylates rhoa rhob and rhoc proteins on asparagine residue 41 rendering them inactive it is available as a cell permeable recombinant protein and has been successfully applied in regeneration models of the on and the spinal cord dergham et al 2002 bertrand et al 2005 lordfontaine et al 2008 recently this compound has been studied in a human clinical trial of patients with acute spinal cord injury where its local application to the injury site in the first 7 days after injury led to an improvement in the asia motor score after 1 year in patients with cervical spinal cord lesions if the patients encountered a thoracic spinal cord lesion the same treatment was not effective most probably because this patient group very often suffered from accompanying severe trauma to surrounding tissues creating an environment that allows for more systemic absorption of the locally applied c3 transferase fehlings et al 2011 looking even more upstream in the inhibitory cascade therapeutic approaches blocking nogo function are most advanced the nogo-a extracellular peptide residues 1-40 nep1-40 can be used to antagonize nogo receptor signaling alternatively nogo receptor fragments that bind nogo-a or antibodies that block the nogo receptor are available for an overview see schwab 2010 a monoclonal antibody directed against nogo-a is currently under clinical investigation in spinal cord injury patients nct00406016 closely related downstream targets of rock are lim kinase limk and cofilin apart from rna interference approaches which can be applied to knock-down a target on the mrna level pharmacological inhibitors of limk have recently been generated ross-macdonald et al 2008 harrison et al 2009 they can effectively activate cofilin by lowering its phosphorylation and thereby enable it to severe filamentous actin again scott et al 2010 however their characterization is still incomplete and an application in neuronal tissue has not been demonstrated so far effects of rock inhibition neurite growth and regeneration because rock is involved in the regulation of the cytoskeleton through downstream regulation of actin myosin and associated proteins its effects on cellular functions such as motility and changes of cellular shape have been intensively studied rho-associated kinase inhibition was shown to enhance neurite outgrowth in pc-12 or ntera-2 cells in vitro zhang et al 2006 lingor et al 2007 the situation is more complex in primary neuronal cells where the effect of rock inhibitor treatment seems to depend on the age of the cultures neurite outgrowth was promoted best if the cultures were of embryonic or early postnatal age lehmann et al 1999 borisoff et al 2003 monnier et al 2003 in contrast adult retinal ganglion cells rgc grown on cns myelin required the addition of a growth promoting factor such as cntf or elevation of camp to promote neurite outgrowth through rock inhibition ahmed et al 2009 this supports the hypothesis that the low intrinsic growth capacity of adult neurons is an important limiting factor for neurite regeneration which cannot be overcome through regulation of the inhibitory cascade alone although there seems to be an intrinsic cellular rock activity that is present even if cells are cultured on a growth permissive substrate lingor et al 2008 rock activity is considerably increased and outgrowth is inhibited when upstream activators of the rock signaling pathway such as cns myelin components e.g nogo-66 mag omgp are present alabed et al 2006 along with their strong outgrowth promoting potential for rgc in vitro intravitreally applied y-27632 and dimethylfasudil were shown to facilitate axon regeneration after on crush in vivo lingor et al 2007 2008 however the therapeutic window in both models was narrow and an attenuation of beneficial effects occurred at higher concentrations although the lower k i value for p160rock of more potent rock inhibitors like dimethylfasudil is preferred from a pharmacological point of view the probability to observe off-target effects on other kinases seems to be higher too davies et al 2000 consequently treatment with very high concentrations of fasudil or high concentrations of dimethylfasudil resulted even in shorter neurite lengths in vitro in vivo application of an intermediate concentration of dimethylfasudil was most effective for axonal regeneration lingor et al 2007 if a peripheral nerve graft is transplanted onto the stump of the cns on a pns environment can be created which is semipermissive for regeneration while a peripheral nerve does not express nogo-a it still shows expression of other inhibitory molecules that can activate the rock signaling cascade e.g mag sema-iii pasterkamp et al 1998 gupta et al 2006 however in this regeneration paradigm treatment with y-27632 failed to significantly promote regeneration of on axons although a combination treatment with cntf resulted in an improved regenerative effect the overall outcome was limited lingor et al 2008 this may be due to an already robust basal regenerative response in the peripheral environment in a sciatic nerve crush lesion model rhoa and its downstream target rock are activated in corresponding motoneurons interestingly here the systemic application of fasudil increased the regeneration of large diameter axons that led to a significantly faster increase of amplitudes of distally evoked compound muscle action potentials hiraga et al 2006 the expression of rhoa in adult dorsal root ganglia sensory neurons rises dramatically after lesion but it is even more important that activated gtp-bound rhoa which is undetectable in intact ganglia is strongly upregulated in both drg neurons and axons after injury consequently application of fasudil promoted axonal regeneration of transected drg axons into the distal stump ex vivo cheng et al 2008 frontiers in molecular neuroscience www.frontiersin.org november 2011 volume 4 article 39 4

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tönges et al rock as target for neurorestoration crystallins have been shown to promote regeneration of retinal ganglion cells in vivo and are likely to represent one of the mediators of lens injury-induced regenerative response in the lesioned on fischer et al 2008 interestingly application of -crystallin to the vitreous resulted in a decreased rhoa and rock activity and a reduction of p-cofilin and p-mlc which was associated with increased axonal regeneration in vivo thus pro-regenerative effects of crystallins may at least partially be attributed to the regulation of the rhoa/rock/cofilin pathway wang et al 2011 because -crystallin interacts with 6-integrin membrane receptor complexes and alters their cellular signaling menko and andley 2010 a speculative link to rhoa consists in the prevention of the gdp to gtp exchange of rhoa through integrin membrane complex binding of guanine nucleotide exchange factors wettschureck and offermanns 2002 in the spinal cord lesion model a mutant rho-binding pleckstrin homology protein was expressed in the rubrospinal tract using a lentiviral vector that leads to an abolishment of the rho-binding activity of rock and thus functionally serves as a dominant-negative mutant of rock this resulted in a better functional recovery of lesioned animals and in an increased axonal sprouting of rubrospinal tract axons wu et al 2009 comparing treatment effects of fasudil y-27632 and c3 exoenzyme a rhoa inhibitor fasudil was most efficient in improving the locomotor bbb score after moderate spinal cord injury at the t9/t10 region sung et al 2003 however if fasudil was not applied directly after spinal cord lesion but only in a delayed treatment paradigm 4 weeks later it could not stimulate axonal sprouting or recover hind limb function anymore nishio et al 2006 this was most probably due to the strong scarring activity after lesion that forms a rigid obstacle at 4 weeks after lesion and cannot be surpassed by axonal fibers even if they are disinhibited by fasudil as the transplantation of bone marrow stromal cells bmsc has been shown to promote functional recovery after spinal cord injury if transplanted into the lesion site fasudil was additionally applied in order to augment the bmsc effects on regeneration here bmsc co-treatment with fasudil significantly increased the number of sprouting spinal cord axons but was not able to improve the therapeutic effects of bmsc alone on locomotor function chiba et al 2010 in a similar study on spinal cord injury assessing open field locomotor activity both fasudil and bmsc treatment alone were not able to improve the outcome compared with sham treated control animals however the combination treatment showed a significantly enhanced locomotion at 8 and 9 weeks after lesion furuya et al 2009 optimal rock inhibitor dosage is also important in spinal cord lesion models comparing two different dosages of intrathecally applied y-27632 in a cervical 4/5 dorsal column transection model only the high dose of y-27632 was able to stimulate sprouting of the dorsal ascending axons whereas low dose-treated animals did not benefit from treatment the low dose-treated group even suffered from deficits in a functional footprint analysis in comparison to controls and showed less corticospinal tract axonal sprouting chan et al 2005 one possible reason for this could be that the low dosage did not provide enough outgrowth disinhibition to overcome the inhibitory milieu or perhaps did not even reach the site of the growing axonal cones additionally a relatively higher local concentration close to the scar could have elicited disproportionate undesirable effects of scar tissue including astroglia in view of all these data on neurite outgrowth and axonal regeneration it becomes obvious that the regenerative effect in vitro is usually more pronounced than in vivo this can be due to the fact that frequently only one substrate is used to inhibit neurite regeneration of cultured neuronal cells in vitro even if preparations of whole cns myelin are applied its integrity is disrupted and a tight physical contact is not maintained the abundance of cns myelin components extracellular matrix scar tissue and infiltration of inflammatory cells in combination with enhanced mechanical repression due to reactive edematous swelling and physical displacement of axonal tracts as seen in acute trauma models is only present in animal models in vivo sandvig et al 2004 yiu and he 2006 busch and silver 2007 as a consequence many encouraging findings in vitro have not been successfully transferred to the in vivo setting therefore it is clear that rock inhibition by itself will not be sufficient to target all pathophysiological obstacles in regenerative approaches and thus will have to be complemented by combination treatments cell survival the role of rock in cell death and survival has been addressed only recently as several rock substrates have been shown to be involved in the regulation of these processes for review see shi and wei 2007 rock inhibition by y-27632 ha-1077 ha-1004 and h-8 strongly promotes survival in ntera-2 cells a pluripotent human embryonic carcinoma cell line barbaric et al 2011 and application of dimethylfasudil leads to protection from apoptotic cell death in serum-deprived organotypic retinal cell cultures tura et al 2009 purkinje cell survival in organotypic cultures of mouse cerebella can be increased by both dimethylfasudil and y-27632 julien et al 2008 in addition to improved cell survival of oxygen­glucose deprived pc-12 cells fasudil application also triggered neurogenesis in the subventricular zone in a hypoxia/reoxygenation injury model yamashita et al 2007 ding et al 2010b we have evaluated the effects of rock inhibition on rgc in a paradigm of primary rgc cell death in vitro the application of y-27632 is able to robustly increase survival and neurite outgrowth these effects can be additively increased by the combination treatment with cntf or regarding cell survival through co-treatment with the cdk5-inhibitor indolinone a lingor et al 2008 bermel et al 2009 other intracellular signaling cascades employed by rock in survival signaling have been described only recently an important finding came from dissociated prostate stem cells which undergo apoptosis upon dissociation lacking extracellular matrix contact this has been shown to be associated with rhoa/rock activation increased pten and reduced akt activities treatment with y-27632 reduced pten activation and improved stem cell survival even though pten activity alone was not sufficient to mediate cell death in this paradigm zhang et al 2011 the distinguished role of pten for neuronal cell survival was underlined in studies where a conditional deletion of pten or tsc1 was used to increase mtor activity in rgc after on lesion park et al 2008 this resulted frontiers in molecular neuroscience www.frontiersin.org november 2011 volume 4 article 39 5

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tönges et al rock as target for neurorestoration in a strong increase of surviving rgc and at the same time was associated with an enhanced regenerative response of on axons apart from the experimental evidence linking inhibition of rock and cell survival there are complementary findings that demonstrate increased apoptosis upon rock activation for example increased phosphorylation of the rock effector mlc and actomyosin contraction in apoptotic cells regulate apoptotic membrane blebbing mills et al 1998 rock may also promote apoptosis through increased ezrin phosphorylation that leads to activation and clustering of fas a component of the receptor-associated death-inducing signaling complex piazzolla et al 2005 this signaling cascade can be inhibited by treatment with y-27632 as shown in jurkat cells hebert et al 2008 simultaneous modulation of neurite outgrowth and cell survival ­ pros and cons whereas the blocking of rock as a central target in neurite outgrowth inhibitory signaling is a rather straightforward approach a similarly powerful handle is more difficult to find in regard of cell survival regulation as described before cell survival responses after rock inhibition are most probably indirectly mediated and have been shown to involve mapk and akt signaling pathways lingor et al 2008 however there has been no study in neuronal cells so far which has identified such an intermediate signaling partner the lack of knowledge concerning these intermediate signaling partners in cell protective signaling renders the precalculation of combinatorial treatments a difficult task in the case of the co-application of y-27632 and cntf in the on crush lesion paradigm we noted a differential impact of cntf and y-27632 on mitogen-activated protein kinase mapk akt and signal transducer and activator of transcription 3 stat3 the mapk pathway was activated in an additive manner by both compounds to a lesser extent this held true also for the akt-pathway as demonstrated by akt phosphorylation however the application of y-27632 markedly reduced the cntf-evoked activation of stat3 thus inhibition of rock in rgc attenuates some of the cntf-mediated effects via stat3 while the mapk and aktpathway are triggered in an additive manner these data suggest that the opposing actions of cntf and y-27632 on the phosphorylation of stat3 could be responsible for the lack of an additive effect on rgc survival in the corresponding on axotomy model in vivo lingor et al 2008 also axonal regeneration after on crush was less pronounced than expected in this combinatorial treatment paradigm here the attenuation of stat3 activation caused by rock inhibition could be responsible because stat3 signaling has been associated with axon regeneration in rgc kretz et al 2005 and other neuronal cells in the cns and pns qiu et al 2005 bareyre et al 2011 in view of these data on axonal regeneration and cell survival it is important to point out that it would be highly preferable to modulate both parameters by targeting only one substrate in the case of combination therapies however the selection of a suitable combination partner has to be done carefully rock inhibition in glial and inflammatory cells in the cns after systemic application of pharmacological substances a cell specific targeting is less predictable thus inhibition of rock will likely also modulate glial and inflammatory cell responses which in turn may affect the regenerative and cell survival response concerning glial cells it has been shown that fasudil upregulates glutamate transport via eaat1/2 subsequent to actin remodeling in murine cultured astrocytes lau et al 2011a and dimethylfasudil reduced reactive astrocytic gliosis in the rodent retina after on crush tura et al 2009 furthermore a transcriptomic profiling of astrocytes treated with fasudil did not only reveal an upregulation of genes involved in cellular shape and motility but also of genes involved in cell survival such as bdnf lau et al 2011b enhanced neurogenesis in the subventricular zone upon treatment with fasudil was attributed to an increased production of granulocyte colony-stimulating factor g-csf from astrocytes ding et al 2010b microglial inflammatory responses seem to be suppressed by rock inhibition as has been shown in various experimental paradigms the injection of lysophosphatidylcholine lpc a major phospholipid component of the atherogenic oxidized ldl is known to increase astrocyte and microglia accumulation in neuronal tissue and also elevates inos expression if fasudil is systemically applied it is able to attenuate these lpc induced responses in the rat striatum and also protects from neuronal cell loss sheikh et al 2009 in addition fasudil protection of hippocampal neurons against hypoxia­reoxygenation injury was shown to be mainly due to the suppression of microglial inflammatory responses ding et al 2010a however if c3 transferase is used to inactivate rho gtpases being upstream of rock this causes a transformation of microglial cells rather toward an activated phenotype in culture and triggers the release of nitric oxide and various proinflammatory cyto and chemokines ­ an effect that is not observed with the rock inhibitors y-27632 or fasudil hoffmann et al 2008 the role of rock inhibition has also been studied in classical animal models of inflammatory cns disease for example both a parenteral and an oral administration of fasudil prevented the development of experimental autoimmune encephalomyelitis eae in mice together with a down-regulation of interleukin17 and a decrease in the interferon-gamma/interleukin-4 ratio fasudil significantly reduced the specific proliferation of lymphocytes overall the infiltration of inflammatory cells was markedly decreased and acute axonal transections were attenuated as shown by immunohistochemical analysis sun et al 2006 the fasudilmediated attenuation of eae development was confirmed in a more recent study using a different induction model of the disease here the t-cell proliferation was markedly reduced in addition to a down-regulation of interleukin-17 yu et al 2010 this could be due to a decreased phosphorylation of interferon regulatory factor 4 irf4 which is known to regulate the synthesis of il17 and il-21 and implicates rock in the development of other autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus biswas et al 2010 hints for a possible influence of rock on t-cell migration comes from a recent study in which microtubules were disrupted with nocodazole preventing the formation of lamellipodia as a consequence the migratory behavior is reduced because cells can migrate only by membrane blebbing it was found that the nocodazole-induced microtubule depolymerization was associated with a stimulation of rhoa activity and that the resulting defect in cell migration could be frontiers in molecular neuroscience www.frontiersin.org november 2011 volume 4 article 39 6

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tönges et al rock as target for neurorestoration rescued by rock inhibition with y-27632 leading to an increased microtubule stability takesono et al 2010 therapeutic strategies using rock inhibition from a therapeutic point of view the experimental use of small molecule inhibitors of rock is well established many experimental studies in animals focus on their vasodilator effect in the vascular system which results from the inhibition of rock in vascular smooth cells as one example rock inhibition is able to increase cerebral blood flow via endothelial mechanisms and is protective in stroke rikitake et al 2005 shin et al 2007 however rock inhibition also ameliorates post myocardial infarct heart failure by suppressing left ventricular remodeling hattori et al 2004 reduces cardiac hypertrophy in apolipoprotein e deficient mice wang et al 2005a improves monocrotaline-induced fatal pulmonary hypertension in rats abe et al 2004 and is preventive for ischemia/reperfusion-induced acute renal failure teraishi et al 2004 in regard to neuroprotection y-27632 has been applied to animal models of huntington s disease and spinal muscular atrophy where it improved rotarod performance and prolonged survival respectively li et al 2009 bowerman et al 2010 recently rock inhibitors were also tested in preclinical models of neuropathic and nociceptive pain boyce-rustay et al 2010 spinal cord lesion furuya et al 2009 sexual dysfunction li et al 2011 and multiple sclerosis sun et al 2006 all with encouraging results in regard of disease modulation because of these strong preclinical data rock inhibition has already been tested in human studies almost exclusively using fasudil taking advantage of the strong vasodilator effect it was possible to successfully suppress both coronary artery vasospasm and cerebral vasospasm that often appears after aneurysmal subarachnoid hemorrhage sah masumoto et al 2002 zhao et al 2006 furthermore fasudil significantly improved the patient s clinical outcome if applied in the first 48 h after acute ischemic stroke shibuya et al 2005 overall fasudil showed a safe clinical profile without the occurrence of serious adverse effects and has been approved for the treatment of cerebral vasospasm after sah in japan suzuki et al 2008 iwabuchi et al 2011 currently a phase ii clinical trial is ongoing that evaluates the effect of rock inhibition with fasudil for the prevention of carotid stenosis nct00670202 other clinical phase i/ii trials investigate the treatment of glaucoma nct00846989 or spinal cord injury nct00500812 an already terminated phase i clinical study for glaucoma treatment with snj-1656 confirmed the japanese safety data obtained for sah did not show any systemic adverse effects and reported only minor side effects in a 7-day repeated instillation trial tanihara et al 2008 however it has to be considered that references abe k shimokawa h morikawa k uwatoku t oi k matsumoto y hattori t nakashima y kaibuchi k sueishi k and takeshit a 2004 long-term treatment with a rho-kinase inhibitor improves monocrotaline-induced fatal pulmonary hypertension in rats circ res 94 385­393 ahmed z berry m and logan a 2009 rock inhibition promotes adult retinal ganglion cell neurite outgrowth only in the presence of growth promoting factors mol cell neurosci 42 128­133 alabed y z grados-munro e ferraro g b hsieh s h and fournier a e 2006 neuronal responses to myelin are mediated systemic adverse or off-target effects after ocular application are less likely to occur because the substance remains rather in a confined local compartment and does not reach comparable systemic concentrations the use of isoform specific targeting with sirna as a therapeutic tool is still limited because of the difficult delivery and only moderate stability of the molecules in a therapeutic setting in vivo therefore the development of small molecule inhibitors for isoform specific inhibition of rock is greatly awaited as one example slx-2119 represents one of the first isoform specific pharmacological inhibitors of rock targeting selectively rock2 schueller et al 2006 summary and conclusion rho-associated kinase signaling is a major obstacle for successful regeneration in the cns once this deleterious cascade is activated after acute traumatic lesion or in chronic neurodegenerative diseases the outcome of axonal outgrowth/regeneration and of cellular survival is limited however if pathological rock activation can be effectively prevented neurological sequelae may be mitigated too whereas the regeneration disinhibiting potential of rock inhibition is well known and has been experimentally characterized in detail cell protective responses have emerged as a new focus after first successful applications of rock inhibition in cell death paradigms in culture models in vitro it has later successfully been transferred to neurologic disease models such as on lesion and spinal cord trauma but also to neurodegenerative diseases models like huntington s disease and spinal muscular atrophy now first hints on the underlying neuroprotective mechanisms emerge some of these seem to be linked to the modulation of the recently discovered rock targets involving pten/akt or tsc1/2 but may also be associated with modulatory effects on glial and inflammatory cells in the cns the development of new pharmacological inhibitors with enhanced specificity and less off-target effects improved bioavailability and compound stability in addition to selective targeting of rock isoforms now enables us to better counteract deregulated rock due to the good clinical safety profile of fasudil in humans this compound is currently under investigation in various clinical trials that focus on the compromised cardiovascular system however based on the many promising findings in cns disease models it has potential to also enter this area of clinical research acknowledgments lars tönges has received grant support from the forschungsförderungsprogramm of the university medicine göttingen paul lingor and mathias bähr were supported by the dfg research center for molecular physiology of the brain cmpb göttingen by rho kinase j neurochem 96 1616­1625 amano m chihara k nakamura n kaneko t matsuura y and kaibuchi k 1999 the cooh terminus of rho-kinase negatively regulates rho-kinase activity j biol chem 274 32418­32424 amano m ito m kimura k fukata y chihara k nakano t matsuura y and kaibuchi k 1996 phosphorylation and activation of myosin by rho-associated kinase rho-kinase j biol chem 271 20246­20249 amano m kaneko t maeda a nakayama m ito m yamauchi t goto h fukata y oshiro n shinohara a iwamatsu a and kaibuchi k 2003 identification frontiers in molecular neuroscience www.frontiersin.org november 2011 volume 4 article 39 7

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in vivo imaging reveals a phase-specific role of stat3 during central and peripheral nervous system axon regeneration proc natl acad sci u.s.a 108 6282­6287 bermel c tonges l planchamp v gillardon f weishaupt j h dietz g p bahr m and lingor p 2009 combined inhibition of cdk5 and rock additively increase cell survival but not the regenerative response in regenerating retinal ganglion cells mol cell neurosci 42 427­437 bertrand j winton m j rodriguezhernandez n campenot r b and mckerracher l 2005 application of rho antagonist to neuronal cell bodies promotes neurite growth in compartmented cultures and regeneration of retinal ganglion cell axons in the optic nerve of adult rats j neurosci 25 1113­1121 biswas p s gupta s chang e song l stirzaker r a liao j k bhagat g and pernis a b 2010 phosphorylation of irf4 by rock2 regulates il-17 and il-21 production and the development of autoimmunity in mice j clin invest 120 3280­3295 borisoff j f chan c c hiebert g w oschipok l robertson g s 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tönges et al rock as target for neurorestoration hebert m potin s sebbagh m bertoglio j breard j and hamelin j 2008 rho-rock-dependent ezrin-radixin-moesin phosphorylation regulates fas-mediated apoptosis in jurkat cells j immunol 181 5963­5973 hiraga a kuwabara s doya h kanai k fujitani m taniguchi j arai k mori m hattori t and yamashita t 2006 rho-kinase inhibition enhances axonal regeneration after peripheral nerve injury j peripher nerv syst 11 217­224 hoffmann a hofmann f just i lehnardt s hanisch u k bruck w kettenmann h ahnert-hilger g and holtje m 2008 inhibition of rho-dependent pathways by clostridium botulinum c3 protein induces a proinflammatory profile in microglia glia 56 1162­1175 ishizaki t maekawa m fujisawa k okawa k iwamatsu a fujita a watanabe n saito y kakizuka a morii n and narumiya s 1996 the small gtp-binding protein rho binds to and activates a 160 kda ser/thr protein kinase homologous to myotonic dystrophy kinase embo j 15 1885­1893 iwabuchi s yokouchi t hayashi m sato k saito n hirata y harashina j nakayama h akahata m ito k kimura h and aoki k 2011 intraarterial administration of fasudil hydrochloride for vasospasm following subarachnoid haemorrhage experience of 90 cases acta neurochir suppl 110 179­181 izawa t fukata y kimura t iwamatsu a dohi k and kaibuchi k 2000 elongation factor-1 alpha is a novel substrate of rho-associated kinase biochem biophys res commun 278 72­78 julien s schnichels s teng h tassew n henke-fahle s mueller b k and monnier p p 2008 purkinje cell survival in organotypic cultures implication of rho and its downstream effector rock j neurosci res 86 531­536 kimura k ito m amano m chihara k fukata y nakafuku m yamamori b feng j nakano t okawa k iwamatsu a and kaibuchi k 1996 regulation of myosin phosphatase by rho and rho-associated kinase rhokinase science 273 245­248 komagome r kimura k and saito m 2000 postnatal changes in rho and rho-related proteins in the mouse brain jpn j vet res 47 127­133 kosako h amano m yanagida m tanabe k nishi y kaibuchi k and inagaki m 1997 phosphorylation of glial fibrillary acidic protein at the same sites by cleavage furrow kinase and rho-associated kinase j biol chem 272 10333­10336 kretz a happold c j marticke j k and isenmann s 2005 erythropoietin promotes regeneration of adult cns neurons via jak2/stat3 and pi3k/akt pathway activation mol cell neurosci 29 569­579 lau c l o shea r d broberg b v bischof l and beart p m 2011a the rho kinase inhibitor fasudil upregulates astrocytic glutamate transport subsequent to actin remodelling in murine cultured astrocytes br j pharmacol 163 533­545 lau c l perreau v m chen m j cate h s merlo d cheung n s o shea r d and beart p m 2011b transcriptomic profiling of astrocytes treated with the rho kinase inhibitor fasudil reveals cytoskeletal and pro-survival responses j cell physiol doi 10.1002/jcp.22838 [epub ahead of print lehmann m fournier a sellesnavarro i dergham p sebok a leclerc n tigyi g and mckerracher l 1999 inactivation of rho signaling pathway promotes cns axon regeneration j neurosci 19 7537­7547 leung t manser e tan l and lim l 1995 a novel serine/threonine kinase binding the ras-related rhoa gtpase which translocates the kinase to peripheral membranes j biol chem 270 29051­29054 li m huang y ma a a lin e and diamond m i 2009 y-27632 improves rotarod performance and reduces huntingtin levels in r6/2 mice neurobiol dis 36 413­420 li w j park k paick j s and kim s w 2011 chronic treatment with an oral rho-kinase inhibitor restores erectile function by suppressing corporal apoptosis in diabetic rats j sex med 8 400­410 li z dong x wang z liu w deng n ding y tang l hla t zeng r li l and wu d 2005 regulation of pten by rho small gtpases nat cell biol 7 399­404 liao j k seto m and noma k 2007 rho kinase rock inhibitors j cardiovasc pharmacol 50 17­24 lingor p teusch n schwarz k mueller r mack h bahr m and mueller b k 2007 inhibition of rho kinase rock increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo j neurochem 103 181­189 lingor p tonges l pieper n bermel c barski e planchamp v and bahr m 2008 rock inhibition and cntf interact on intrinsic signalling pathways and differentially regulate survival and regeneration in retinal ganglion cells brain 131 250­263 liu b p and strittmatter s m 2001 semaphorin-mediated axonal guidance via rho-related g proteins curr opin cell biol 13 619­626 liu k tedeschi a park k k and he z 2011 neuronal intrinsic mechanisms of axon regeneration annu rev neurosci 34 131­152 lock f e and hotchin n a 2009 distinct roles for rock1 and rock2 in the regulation of keratinocyte differentiation plos one 4 e8190 doi:10.1371/journal.pone.0008190 lord-fontaine s yang f diep q dergham p munzer s tremblay p and mckerracher l 2008 local inhibition of rho signaling by cell-permeable recombinant protein ba-210 prevents secondary damage and promotes functional recovery following acute spinal cord injury j neurotrauma 25 1309­1322 masumoto a mohri m shimokawa h urakami l usui m and takeshita a 2002 suppression of coronary artery spasm by the rhokinase inhibitor fasudil in patients with vasospastic angina circulation 105 1545­1547 matsui t maeda m doi y yonemura s amano m kaibuchi k and tsukita s 1998 rho-kinase phosphorylates cooh-terminal threonines of ezrin/radixin/moesin erm proteins and regulates their head-to-tail association j cell biol 140 647­657 menko a s and andley u p 2010 alphaa-crystallin associates with alpha6 integrin receptor complexes and regulates cellular signaling exp eye res 91 640­651 mi s lee x shao z thill g ji b relton j levesque m allaire n perrin s sands b crowell t cate r l mccoy j m and pepinsky r b 2004 lingo-1 is a component of the nogo-66 receptor/p75 signaling complex nat neurosci 7 221­228 mills j c stone n l erhardt j and pittman r n 1998 apoptotic membrane blebbing is regulated by myosin light chain phosphorylation 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tönges et al rock as target for neurorestoration piazzolla d meissl k kucerova l rubiolo c and baccarini m 2005 raf-1 sets the threshold of fas sensitivity by modulating rokalpha signaling j cell biol 171 1013­1022 qiu j cafferty w b mcmahon s b and thompson s w 2005 conditioning injury-induced spinal axon regeneration requires signal transducer and activator of transcription 3 activation j neurosci 25 1645­1653 riento k and ridley a j 2003 rocks multifunctional kinases in cell behaviour nat rev mol cell biol 4 446­456 rikitake y kim h h huang z seto m yano k asano t moskowitz m a and liao j k 2005 inhibition of rho kinase rock leads to increased cerebral blood flow and stroke protection stroke 36 2251­2257 ross-macdonald p de silva h guo q xiao h hung c y penhallow b markwalder j he l attar r m lin t a seitz s tilford c wardwell-swanson j and jackson d 2008 identification of a nonkinase target mediating cytotoxicity of novel kinase inhibitors mol cancer ther 7 3490­3498 sandvig a 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hypertrophy in apolipoprotein e deficient mice eur j pharmacol 512 215­222 wang y x martin-mcnulty b da cunha v vincelette j lu x feng q halks-miller m mahmoudi m schroeder m subramanyam b tseng j l deng g d schirm s johns a kauser k dole w p and light d r 2005b fasudil a rho-kinase inhibitor attenuates angiotensin ii-induced abdominal aortic aneurysm in apolipoprotein e-deficient mice by inhibiting apoptosis and proteolysis circulation 111 2219­2226 wettschureck n and offermanns s 2002 rho/rho-kinase mediated signaling in physiology and pathophysiology j mol med 80 629­638 wu d yang p zhang x luo j haque m e yeh j richardson p m zhang y and bo x 2009 targeting a dominant negative rho kinase to neurons promotes axonal outgrowth and partial functional recovery after rat rubrospinal tract lesion mol ther 17 2020­2030 yamashita k kotani y nakajima y shimazawa m yoshimura s nakashima s iwama t and hara h 2007 fasudil a rho kinase rock inhibitor protects against ischemic neuronal damage in vitro and in vivo by acting directly on neurons brain res 1154 215­224 yang n higuchi o ohashi k nagata k wada a kangawa k nishida e and mizuno k 1998 cofilin phosphorylation by limkinase 1 and its role in rac-mediated actin reorganization nature 393 809­812 yiu g and he z 2006 glial inhibition of cns axon regeneration nat rev neurosci 7 617­627 yoneda a multhaupt h a and couchman j r 2005 the rho kinases i and ii regulate different aspects of myosin ii activity j cell biol 170 443­453 yu j z ding j ma c g sun c h sun y f lu c z and xiao b g 2010 therapeutic potential of experimental autoimmune encephalomyelitis by fasudil a rho kinase inhibitor j neurosci res 88 1664­1672 zhang l valdez j m zhang b wei l chang j and xin l 2011 rock inhibitor y-27632 suppresses dissociation-induced apoptosis of murine prostate stem/progenitor cells and increases their cloning efficiency plos one 6 e18271 doi:10.1371/journal.pone.0018271 zhang z ottens a k larner s f kobeissy f h williams m l hayes r l 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tönges et al rock as target for neurorestoration direct rho-associated kinase inhibition [correction of inhibiton induces cofilin dephosphorylation and neurite outgrowth in pc-12 cells cell mol biol lett 11 12­29 zhao j zhou d guo j ren z zhou l wang s xu b and wang r 2006 effect of fasudil hydrochloride a protein kinase inhibitor on cerebral vasospasm and delayed cerebral ischemic symptoms after aneurysmal subarachnoid hemorrhage neurol med chir tokyo 46 421­428 zhou z meng y asrar s todorovski z and jia z 2009 a critical role of rho-kinase rock2 in the regulation of spine and synaptic function neuropharmacology 56 8­89 conflict of interest statement the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest received 29 august 2011 paper pending published 20 september 2011 accepted 16 october 2011 published online 03 november 2011 citation tönges l koch j-c bähr m and lingor p 2011 rocking regeneration rho kinase inhibition as molecular target for neurorestoration front mol neurosci 4:39 doi 10.3389/fnmol.2011.00039 copyright © 2011 tönges koch bähr and lingor this is an open-access article subject to a non-exclusive license between the authors and frontiers media sa which permits use distribution and reproduction in other forums provided the original authors and source are credited and other frontiers conditions are complied with frontiers in molecular neuroscience www.frontiersin.org november 2011 volume 4 article 39 11

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