Development and validation of LC-MS/MS method for determining the metabolic stability, pharmacokinetics, and dose proportionality of a novel anti-inflammatory cofilin inhibitor.

A novel small molecule cofilin inhibitor (SZ-3) has recently become the focus of investigation for targeting neuroinflammation in different neurodegenerative diseases. In the present study, the metabolic stability, blood-brain barrier (BBB) penetration, and tissue concentration of SZ-3 were evaluated to support our future studies. In silico drug metabolism prediction was investigated using the StarDrop WhichP450 module. LC-MS/MS method was developed and validated to quantify the SZ-3 for in-vitro and in-vivo studies. The in-vitro metabolic stability was performed using human liver microsomes (HLMs), and the in-vivo pharmacokinetics were investigated in mice after a single intraperitoneal (IP) injection or oral (P.O.) administration, followed by a collection of blood and brain samples at different time points. The dose-proportionality was also evaluated after a single IP injection of three ascending doses (5, 10, and 25 mg/kg). In-vitro results showed that SZ-3 has a moderate intrinsic clearance (Clint) value of 17.42 ml/min/mg with a half-life (t1/2) value of 39.77 mins, indicative of good bioavailability. In vivo study revealed that SZ-3 was rapidly absorbed, entered the brain, and yielded a good concentration of the unbound drug after IP and oral administration. However, the higher maximum concentration (Cmax) values of IP and P.O. (2244 ng/ml and 1069 ng/g, respectively) revealed that the IP administration led to higher blood and brain concentrations than the P.O. Furthermore, Cmax and area under the curve (AUC) of SZ-3 increased in a dose-proportional manner between the three ascending doses. These results will guide us in optimizing the dosing regimen for future SZ-3 pharmacological studies targeting neuroinflammation.

Effects of neural-derived estradiol on actin polymerization and synaptic plasticity-related proteins in prefrontal and hippocampal cells of mice.

Neural-derived 17ß-estradiol (E2) plays an important role in the synaptic plasticity of the hippocampus and prefrontal cortex, but the mechanism is not well defined. This study was designed to explore the effect and mechanism of neural-derived E2 on synaptic plasticity of the hippocampus and prefrontal cortex. Primary cultured hippocampal and prefrontal cells in mice were randomly divided into the DMSO (D), aromatase (Rate-limiting enzymes for E2 synthesizes) inhibitor letrozole (L), and ERs antagonist (MPG) treated groups. After intervention for 48 h, the cell was collected, and then, the expressions of AMPA-receptor subunit GluR1 (GluR1), synaptophysin (SYN), p-21-Activated kinase (PAK) phosphorylation, Rho kinase (ROCK), p-Cofilin, F-actin, and G-actin proteins were detected. Letrozole or ER antagonists inhibited the expression of GluR1, F-actin/G-actin, p-PAK and p-Cofilin proteins in prefrontal cells significantly. And the expressions of GluR1 and F-actin/G-actin proteins were declined in hippocampal cells markedly after adding letrozole or ERs antagonists. In conclusion, neural-derived E2 and ERs regulated the synaptic plasticity, possibly due to promoting actin polymerization in prefrontal and hippocampal cells. The regional specificity in the effect of neural-derived E2 and ERs on the actin polymerization-related pathway may provide a theoretical basis for the functional differences between the hippocampus and prefrontal cortex.

Upregulated neuregulin-1 protects against optic nerve injury by regulating the RhoA/cofilin/F-actin axis.

OBJECTIVE: In recent years, the roles of Neuregulin-1 (NRG-1) in optic nerve injury and retinal cells have been investigated. However, the molecular mechanism by which NRG-1 affects optic nerve injury remains elusive and merits deeper exploration. Hence, this study examined the specific function of NRG-1 in the RhoA/cofilin/F-actin axis in optic nerve injury. METHODS: Retinal cells were isolated and identified for subsequent experimental uses. Reverse transcription quantitative polymerase chain reaction and Western blot assays were performed to measure NRG-1 expression in retinal cells which were cultured under elevated pressure. TUNEL staining was used to detect the cell apoptosis rate, and Western blot assay was performed to detect the expression of related genes. The axon growth was examined by immunofluorescence. The effects of NRG-1 on RhoA activity, cofilin phosphorylation, and F-actin were detected by Western blot assay. In other studies we established a rat model of acute optic nerve injury, and tested for beneficial effects of NRG-1 in vivo. RESULTS: High expression of NRG-1 was evident in the retinal tissues of rats with optic nerve injury. Overexpressing NRG-1 successfully inhibited RhoA activity and the phosphorylation of cofilin and promoted F-actin expression. In cell experiments, overexpressed NRG-1 suppressed the apoptosis of retinal cells and promoted axon growth through the RhoA/cofilin/F-actin axis. In animal experiments, overexpressed NRG-1 relieved retinal injury. CONCLUSION: Our results strongly suggest that overexpressed NRG-1 is highly effective in the protection of normal optic nerve function by suppressing RhoA activity and the phosphorylation of cofilin and rescuing F-actin function.

Targeting ROCK/LIMK/cofilin signaling pathway in cancer.

Rho-associated coiled-coil-containing protein kinase (ROCK)/Lin11, Isl-1 and Mec-3 kinase (LIMK)/cofilin-signaling cascades are stimulated by receptor tyrosine kinases, G protein-coupled receptors, integrins and its ligands, growth factors, hormones, fibronectin, collagen, and laminin. Activated signaling cascades can cause transit from normal cells to cancer cells by modulating actin/filament dynamics. In various cancers including breast, prostate, and colorectal cancers, high expression or activity of each cascade protein is significantly associated with poor survival rate of patients as well as aggressive metastasis. Silencing ROCK, LIMK, or cofilin can abrogate their activities and inhibit cancer cell growth, invasion, and metastasis. Therefore ROCK/LIMK/cofilin signaling proteins might be good candidates to develop cancer prevention strategies or therapeutics. Currently, netarsudil, a ROCK inhibitor, is only used in clinical patients for glaucoma or ocular hypertension, but not for cancer. In this review, we will discuss comprehensive ROCK/LIMK/cofilin signaling pathway in cancers and its inhibitors for developing cancer therapy.

Cofilin is a cAMP effector in mediating actin cytoskeleton reorganization and steroidogenesis in mouse and human adrenocortical tumor cells.

cAMP pathway plays a major role in the pathogenesis of cortisol-producing adrenocortical adenomas (CPA). cAMP-induced steroidogenesis is preceded by actin cytoskeleton reorganization, a process regulated by cofilin activity. In this study we investigated cofilin role in mediating cAMP effects on cell morphology and steroidogenesis in adrenocortical tumor cells. We demonstrated that forskolin induced cell rounding and strongly reduced phosphorylated (P)-cofilin/total cofilin ratio in Y1 (-52 ± 16%, p < 0.001) and human CPA cells (-53 ± 18%, p < 0.05). Cofilin silencing significantly reduced both forskolin-induced morphological changes and progesterone production (1.3-fold vs 1.8-fold in controls, p < 0.05), whereas transfection of wild-type or S3A (active), but not S3D (inactive) cofilin, potentiated forskolin effects on cell rounding and increased 3-fold progesterone synthesis with respect to control (p < 0.05). Furthermore, cofilin dephosphorylation by a ROCK inhibitor potentiated forskolin-induced cell rounding and steroidogenesis (2-fold increase vs forskolin alone). Finally, we found a reduced P-cofilin/total cofilin ratio and increased cofilin expression in CPA vs endocrine inactive adenomas by western blot and immunohistochemistry. Overall, these results identified cofilin as a mediator of cAMP effects on both morphological changes and steroidogenesis in mouse and human adrenocortical tumor cells.

Septin 9 Exhibits Polymorphic Binding to F-Actin and Inhibits Myosin and Cofilin Activity.

Septins are a highly conserved family of proteins in eukaryotes that is recognized as a novel component of the cytoskeleton. Septin 9 (SEPT9) interacts directly with actin filaments and functions as an actin stress fiber cross-linking protein that promotes the maturation of nascent focal adhesions and cell migration. However, the molecular details of how SEPT9 interacts with F-actin remain unknown. Here, we use electron microscopy and image analysis to show that SEPT9 binds to F-actin in a highly polymorphic fashion. We demonstrate that the basic domain (B-domain) of the N-terminal tail of SEPT9 is responsible for actin cross-linking, while the GTP-binding domain (G-domain) does not bundle F-actin. We show that the B-domain of SEPT9 binds to three sites on F-actin, and the two of these sites overlap with the binding regions of myosin and cofilin. SEPT9 inhibits actin-dependent ATPase activity of myosin and competes with the weakly bound state of myosin for binding to F-actin. At the same time, SEPT9 significantly reduces the extent of F-actin depolymerization by cofilin. Taken together, these data suggest that SEPT9 protects actin filaments from depolymerization by cofilin and myosin and indicate a mechanism by which SEPT9 could maintain the integrity of growing and contracting actin filaments.

Rho-associated kinase signaling is required for osteopontin-induced cell invasion through inactivating cofilin in human non-small cell lung cancer cell lines.

Osteopontin (OPN) is involved in tumor progression such as invasion and metastasis, and poor prognosis of lung cancer. However, how OPN affects the invasive behavior of lung cancer is not well defined. Here, we examined the underlying molecular mechanism of OPN-induced invasion in human non-small cell lung cancer (NSCLC) cell lines including A549 cells. OPN markedly increased the phosphorylation of LIM kinase 1 and 2 (LIMK1/2), and cofilin without affecting their total forms. The expression of Rho-associated kinase 1 (ROCK1), but not p21-activated kinase 1 and 2 (PAK1/2), was increased by OPN treatment as an upstream effector of LIMK/cofilin. The phosphorylation of cofilin by OPN was suppressed when cells were pretreated with ROCK inhibitor Y27632 by Western blot. Moreover, it verified that OPN inactivated cofilin through ROCK signaling in other NSCLC cell lines. OPN induced the phosphorylation of FAK and AKT. FAK inhibitor FAKi-14 and PI3K inhibitor wortmannin decreased the expressions of ROCK1, and phosphorylation of LIMK1/2 and cofilin. OPN caused a significant increase in the lamellipodia formation and cell invasion, and these are suppressed by FAK inhibitor FAKi-14, PI3K inhibitor wortmannin and ROCK inhibitor Y27632. Taken together, these results suggest that OPN triggers ROCK signaling mediated by FAK/PI3K/AKT pathway, which in turn induces the lamellipodia formation to allow the invasion of lung cancer cells through inactivating cofilin.

JG6, a novel marine-derived oligosaccharide, suppresses breast cancer metastasis via binding to cofilin.

Cofilin, an actin-binding protein which disassembles actin filaments, plays an important role in invasion and metastasis. Here, we discover that JG6, an oligomannurarate sulfate, binds to cofilin, suppresses the migration of human breast cancer cells and cancer metastasis in breast cancer xenograft model. Mechanistically, JG6 occupies actin-binding sites of cofilin, thereby disrupting cofilin modulated actin turnover. Our results highlight the significance of cofilin in cancer and suggest JG6, a cofilin inhibitor, to treat metastatic cancer.

RanBP9 overexpression down-regulates phospho-cofilin, causes early synaptic deficits and impaired learning, and accelerates accumulation of amyloid plaques in the mouse brain.

Loss of synaptic proteins and functional synapses in the brains of patients with Alzheimer's disease (AD) as well as transgenic mouse models expressing amyloid-ß protein precursor is now well established. However, the earliest age at which such loss of synapses occurs, and whether known markers of AD progression accelerate functional deficits is completely unknown. We previously showed that RanBP9 overexpression leads to enhanced amyloid plaque burden in a mouse model of AD. In this study, we found significant reductions in the levels of synaptophysin and spinophilin, compared with wild-type controls, in both the cortex and the hippocampus of 5- and 6-month old but not 3- or 4-month old APΔE9/RanBP9 triple transgenic mice, and not in APΔE9 double transgenic mice, nor in RanBP9 single transgenic mice. Interestingly, amyloid plaque burden was also increased in the APΔE9/RanBP9 mice at 5-6 months. Consistent with these results, we found significant deficits in learning and memory in the APΔE9/RanBP9 mice at 5 and 6 month. These data suggest that increased amyloid plaques and accelerated learning and memory deficits and loss of synaptic proteins induced by RanBP9 are correlated. Most importantly, APΔE9/RanBP9 mice also showed significantly reduced levels of the phosphorylated form of cofilin in the hippocampus. Taken together these data suggest that RanBP9 overexpression down-regulates cofilin, causes early synaptic deficits and impaired learning, and accelerates accumulation of amyloid plaques in the mouse brain.

Instantaneous inactivation of cofilin reveals its function of F-actin disassembly in lamellipodia.

Cofilin is a key regulator of the actin cytoskeleton. It can sever actin filaments, accelerate filament disassembly, act as a nucleation factor, recruit or antagonize other actin regulators, and control the pool of polymerization-competent actin monomers. In cells these actions have complex functional outputs. The timing and localization of cofilin activity are carefully regulated, and thus global, long-term perturbations may not be sufficient to probe its precise function. To better understand cofilin's spatiotemporal action in cells, we implemented chromophore-assisted laser inactivation (CALI) to instantly and specifically inactivate it. In addition to globally inhibiting actin turnover, CALI of cofilin generated several profound effects on the lamellipodia, including an increase of F-actin, a rearward expansion of the actin network, and a reduction in retrograde flow speed. These results support the hypothesis that the principal role of cofilin in lamellipodia at steady state is to break down F-actin, control filament turnover, and regulate the rate of retrograde flow.

Cytochalasin D acts as an inhibitor of the actin-cofilin interaction.

Cofilin, a key regulator of actin filament dynamics, binds to G- and F-actin and promotes actin filament turnover by stimulating depolymerization and severance of actin filaments. In this study, cytochalasin D (CytoD), a widely used inhibitor of actin dynamics, was found to act as an inhibitor of the G-actin-cofilin interaction by binding to G-actin. CytoD also inhibited the binding of cofilin to F-actin and decreased the rate of both actin polymerization and depolymerization in living cells. CytoD altered cellular F-actin organization but did not induce net actin polymerization or depolymerization. These results suggest that CytoD inhibits actin filament dynamics in cells via multiple mechanisms, including the well-known barbed-end capping mechanism and as shown in this study, the inhibition of G- and F-actin binding to cofilin.

cPLA2α-mediated actin rearrangements downstream of the Akt signaling is required for Cronobacter sakazakii invasion into brain endothelial cells.

Cronobacter sakazakii (C. sakazakii) is an opportunistic pathogen that causes sepsis and meningitis in neonate. The molecular mechanism involved in the pathogenesis of C. sakazakii meningitis remains unclear. In this study, we found that C. sakazakii invasion was significantly decreased in human brain microvascular endothelial cells (HBMEC) treated with cytosolic phospholipases A(2)α (cPLA(2)α) inhibitor. Increased phosphorylation of cPLA(2)α was observed in HBMEC infected with C. sakazakii, which was prevented by treatment with cPLA(2)α inhibitor. cPLA(2)α knockdown in HBMEC significantly attenuated C. sakazakii invasion into HBMEC. Immunofluorescence demonstrated that the rearrangements of actin filaments in HBMEC induced by C. sakazakii were effectively blocked by either treatment with cPLA(2)α inhibitor or transfection with cPLA(2)α siRNA. Interestingly, we found that C. sakazakii infection promoted the aggregation of phosphorylated cPLA(2)α, which was associated with depolymerized actin filaments in HBMEC. Furthermore, our data revealed that cPLA(2)α acts downstream of Akt signaling pathway in HBMEC stimulated with C. sakazakii. Taken together, our results illustrated that cPLA(2)α-mediated actin filament rearrangements downstream of Akt activation is required for C. sakazakii invasion into brain endothelial cells.

Overexpression of HSP70 inhibits cofilin phosphorylation and promotes lymphocyte migration in heat-stressed cells.

Hyperthermia adversely affects cell structure and function, but also induces adaptive responses that allow cells to tolerate these stressful conditions. For example, heat-induced expression of the molecular chaperone protein HSP70 can prevent stress-induced cell death by inhibiting signaling pathways that lead to apoptosis. In this study, we used high-resolution two-dimensional gel electrophoresis and phosphoprotein staining to identify signaling pathways that are altered by hyperthermia and modulated by HSP70 expression. We found that in heat-shocked cells, the actin-severing protein cofilin acquires inhibitory Ser3 phosphorylation, which is associated with an inhibition of chemokine-stimulated cell migration. Cofilin phosphorylation appeared to occur as a result of the heat-induced insolubilization of the cofilin phosphatase slingshot (SSH1-L). Overexpression of HSP70 reduced the extent of SSH1-L insolubilization and accelerated its resolubilization when cells were returned to 37°C after exposure to hyperthermia, resulting in a more rapid dephosphorylation of cofilin. Cells overexpressing HSP70 also had an increased ability to undergo chemotaxis following exposure to hyperthermia. These results identify a critical heat-sensitive target controlling cell migration that is regulated by HSP70 and point to a role for HSP70 in immune cell functions that depend upon the proper control of actin dynamics.

Arabidopsis actin-depolymerizing factors (ADFs) 1 and 9 display antagonist activities.

We provide evidence that one of the 11 Arabidopsis actin-depolymerizing factors (ADFs), namely ADF9, does not display typical F-actin depolymerizing activity. Instead, ADF9 effectively stabilizes actin filaments in vitro and concomitantly bundles actin filaments with the highest efficiency under acidic conditions. Competition experiments show that ADF9 antagonizes ADF1 activity by reducing its ability to potentiate F-actin depolymerization. Accordingly, ectopic expression of ADF1 and ADF9 in tobacco cells has opposite effects. ADF1 severs actin filaments/bundles and promotes actin cytoskeleton disassembly, whereas ADF9 induces the formation of long bundles. Together these data reveal an additional degree of complexity in the comprehension of the biological functions of the ADF family and illustrate that antagonist activities can be displayed by seemingly equivalent actin-binding proteins.

Defining a core set of actin cytoskeletal proteins critical for actin-based motility of Rickettsia.

Many Rickettsia species are intracellular bacterial pathogens that use actin-based motility for spread during infection. However, while other bacteria assemble actin tails consisting of branched networks, Rickettsia assemble long parallel actin bundles, suggesting the use of a distinct mechanism for exploiting actin. To identify the underlying mechanisms and host factors involved in Rickettsia parkeri actin-based motility, we performed an RNAi screen targeting 115 actin cytoskeletal genes in Drosophila cells. The screen delineated a set of four core proteins-profilin, fimbrin/T-plastin, capping protein, and cofilin--as crucial for determining actin tail length, organizing filament architecture, and enabling motility. In mammalian cells, these proteins were localized throughout R. parkeri tails, consistent with a role in motility. Profilin and fimbrin/T-plastin were critical for the motility of R. parkeri but not Listeria monocytogenes. Our results highlight key distinctions between the evolutionary strategies and molecular mechanisms employed by bacterial pathogens to assemble and organize actin.

Screening of novel dominant negative mutant actins using glycine targeted scanning identifies G146V actin that cooperatively inhibits cofilin binding.

A number of studies suggested that the structure of actin filaments changes by interaction with actin-binding proteins such as cofilin and myosin, and that the conformational changes of the actin subunits within a filament are cooperative. To understand the functions of these cooperative conformational changes induced by actin-binding proteins, we sought to obtain dominant negative mutant actins impaired in cooperative conformational changes. A series of mutant actin genes in which glycine residues in actin were systematically substituted by valine residues were constructed, and were expressed individually in yeast cells that carry a wild-type endogenous actin gene. Six dominant negative actin mutations were identified on the basis of growth inhibition. Among them, G146V mutation was chosen for further biochemical analysis because the Gly146 residue is located at the strategic hinge position connecting the large and small domains of an actin molecule. We found that G146V actin filaments hardly bind cofilin, consistent with a previous suggestion that cofilin binding causes conformational changes of actin around Gly146 (Galkin et al. [3]). Notably, copolymer that consists of 1:10 mixture of the mutant and wild-type actin molecules showed significantly reduced affinity for cofilin, suggesting that G146V mutant actin affects the conformation of neighboring wild-type actin within a filament, and inhibits cofilin binding.

Lentiviral Nef proteins utilize PAK2-mediated deregulation of cofilin as a general strategy to interfere with actin remodeling.

Nef is an accessory protein and pathogenicity factor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) which elevates virus replication in vivo. We recently described for HIV type 1(SF2) (HIV-1(SF2)) the potent interference of Nef with T-lymphocyte chemotaxis via its association with the cellular kinase PAK2. Mechanistic analysis revealed that this interaction results in deregulation of the actin-severing factor cofilin and thus blocks the chemokine-mediated actin remodeling required for cell motility. However, the efficiency of PAK2 association is highly variable among Nef proteins from different lentiviruses, prompting us to evaluate the conservation of this actin-remodeling/cofilin-deregulating mechanism. Based on the analysis of a total of 17 HIV-1, HIV-2, and SIV Nef proteins, we report here that inhibition of chemokine-induced actin remodeling as well as inactivation of cofilin are strongly conserved activities of lentiviral Nef proteins. Of note, even for Nef variants that display only marginal PAK2 association in vitro, these activities require the integrity of a PAK2 recruitment motif and the presence of endogenous PAK2. Thus, reduced in vitro affinity to PAK2 does not indicate limited functionality of Nef-PAK2 complexes in intact HIV-1 host cells. These results establish hijacking of PAK2 for deregulation of cofilin and inhibition of triggered actin remodeling as a highly conserved function of lentiviral Nef proteins, supporting the notion that PAK2 association may be critical for Nef's activity in vivo.

Essential role of ADF/cofilin for assembly of contractile actin networks in the C. elegans somatic gonad.

The somatic gonad of the nematode Caenorhabditis elegans contains a myoepithelial sheath, which surrounds oocytes and provides contractile forces during ovulation. Contractile apparatuses of the myoepithelial-sheath cells are non-striated and similar to those of smooth muscle. We report the identification of a specific isoform of actin depolymerizing factor (ADF)/cofilin as an essential factor for assembly of contractile actin networks in the gonadal myoepithelial sheath. Two ADF/cofilin isoforms, UNC-60A and UNC-60B, are expressed from the unc-60 gene by alternative splicing. RNA interference of UNC-60A caused disorganization of the actin networks in the myoepithelial sheath. UNC-60B, which is known to function in the body-wall muscle, was not necessary or sufficient for actin organization in the myoepithelial sheath. However, mutant forms of UNC-60B with reduced actin-filament-severing activity rescued the UNC-60A-depletion phenotype. UNC-60A has a much weaker filament-severing activity than UNC-60B, suggesting that an ADF/cofilin with weak severing activity is optimal for assembly of actin networks in the myoepithelial sheath. By contrast, strong actin-filament-severing activity of UNC-60B was required for assembly of striated myofibrils in the body-wall muscle. Our results suggest that an optimal level of actin-filament-severing activity of ADF/cofilin is required for assembly of actin networks in the somatic gonad.

Suppression of the invasive capacity of rat ascites hepatoma cells by knockdown of Slingshot or LIM kinase.

Actin cytoskeletal reorganization is essential for tumor cell migration, adhesion, and invasion. Cofilin and actin-depolymerizing factor (ADF) act as key regulators of actin cytoskeletal dynamics by stimulating depolymerization and severing of actin filaments. Cofilin/ADF are inactivated by phosphorylation of Ser-3 by LIM kinase-1 (LIMK1) and reactivated by dephosphorylation by Slingshot-1 (SSH1) and -2 (SSH2) protein phosphatases. In this study, we examined the roles of cofilin/ADF, LIMK1, and SSH1/SSH2 in tumor cell invasion, using an in vitro transcellular migration assay. In this assay, rat ascites hepatoma (MM1) cells were overlaid on a primary-cultured rat mesothelial cell monolayer and the number of cell foci that transmigrated underneath the monolayer in the presence of lysophosphatidic acid (LPA) was counted. The knockdown of cofilin/ADF, LIMK1, or SSH1/SSH2 expression by small interfering RNAs (siRNAs) significantly decreased the LPA-induced transcellular migration of MM1 cells and their motility in two-dimensional culture. Knockdown of LIMK1 also suppressed fibronectin-mediated cell attachment and focal adhesion formation. Our results suggest that both LIMK1-mediated phosphorylation and SSH1/SSH2-mediated dephosphorylation of cofilin/ADF are critical for the migration and invasion of tumor cells and that LIMK1 is involved in the transcellular migration of tumor cells by enhancing both adhesion and motility of the cells.

Myelin-associated inhibitors regulate cofilin phosphorylation and neuronal inhibition through LIM kinase and Slingshot phosphatase.

Myelin-associated inhibitors (MAIs) signal through a tripartate receptor complex on neurons to limit axon regeneration in the CNS. Inhibitory influences ultimately converge on the cytoskeleton to mediate growth cone collapse and neurite outgrowth inhibition. Rho GTPase and its downstream effector Rho kinase are key signaling intermediates in response to MAIs; however, the links between Rho and the actin cytoskeleton have not been fully defined. We found that Nogo-66, a potent inhibitory fragment of Nogo-A, signals through LIM (LIM is an acronym of the three gene products Lin-11, Isl-1, and Mec-3) kinase and Slingshot (SSH) phosphatase to regulate the phosphorylation profile of the actin depolymerization factor cofilin. Blockade of LIMK1 activation and subsequent cofilin phosphorylation circumvents myelin-dependent inhibition in chick dorsal root ganglion neurons, suggesting that phosphorylation and inactivation of cofilin is critical for neuronal inhibitory responses. Subsequent activation of SSH1 phosphatase mediates cofilin dephosphorylation and reactivation. Overexpression of SSH1 does not mimic the neurite outgrowth inhibitory effects of myelin, suggesting an alternative role in MAI inhibition. We speculate that SSH-mediated persistent cofilin activation may be responsible for maintaining an inhibited neuronal phenotype in response to myelin inhibitors.