Increased Phosphorylation Of Cofilin Research Articles

Spine impairment in mice high-expressing neuregulin 1 due to LIMK1 activation

The genes encoding for neuregulin1 (NRG1), a growth factor, and its receptor ErbB4 are both risk factors of major depression disorder and schizophrenia (SZ). They have been implicated in neural development and synaptic plasticity. However, exactly how NRG1 variations lead to SZ remains unclear. Indeed, NRG1 levels are increased in postmortem brain tissues of patients with brain disorders. Here, we studied the effects of high-level NRG1 on dendritic spine development and function. We showed that spine density in the prefrontal cortex and hippocampus was reduced in mice (ctoNrg1) that overexpressed NRG1 in neurons. The frequency of miniature excitatory postsynaptic currents (mEPSCs) was reduced in both brain regions of ctoNrg1 mice. High expression of NRG1 activated LIMK1 and increased cofilin phosphorylation in postsynaptic densities. Spine reduction was attenuated by inhibiting LIMK1 or blocking the NRG1–LIMK1 interaction, or by restoring NRG1 protein level. These results indicate that a normal NRG1 protein level is necessary for spine homeostasis and suggest a pathophysiological mechanism of abnormal spines in relevant brain disorders.

Receptor dynamics regulates actin polymerization state through phosphorylation of cofilin in mast cells

Aggregation of IgE bound to the high-affinity IgE receptor (FcεRI) by a multivalent antigen induces mast cell activation, while disaggregation of aggregated FcεRI by monomer hapten immediately terminates degranulation mediated by dephosphorylation of Syk and mediates a decrease in intracellular Ca2+ concentration ([Ca2+]i). The actin polymerization state is intimately involved in mast cell activation mediated by FcεRI aggregation. However, the relation between aggregation-disaggregation of FcεRI and actin rearrangement in mast cells is not well understood. The addition of a multivalent antigen rapidly depolymerized actin filaments, while the subsequent addition of monomer hapten rapidly recovered actin polymerization. Whereas cofilin, an actin-severing protein, was temporally dephosphorylated several minutes after a multivalent antigen stimulation and the addition of monomer hapten rapidly increased cofilin phosphorylation level within 30 s. The removal of extracellular Ca2+ instead of monomer hapten addition did not restore cofilin phosphorylation, suggesting that the significant decrease in [Ca2+]i by monovalent hapten was not a critical reason for the actin rearrangement. Additionally, monovalent hapten did not completely reduce [Ca2+]i in mast cells pretreated with jasplakinolide, an inhibitor of actin depolymerization. These results suggest that the multivalent antigen-induced actin depolymerization mediated by cofilin dephosphorylation, and the subsequent addition of monovalent hapten in the F-actin severing state efficiently elicited actin re-polymerization by cofilin phosphorylation.

Synaptotoxicity in Alzheimer's Disease Involved a Dysregulation of Actin Cytoskeleton Dynamics through Cofilin 1 Phosphorylation.

Amyloid-β (Aβ) drives the synaptic impairment and dendritic spine loss characteristic of Alzheimer's disease (AD), but how Aβ affects the actin cytoskeleton remains unknown and contentious. The actin-binding protein, cofilin-1 (cof1), is a major regulator of actin dynamics in dendritic spines, and is subject to phospho-regulation by multiple pathways, including the Rho-associated protein kinase (ROCK) pathway. While cof1 is implicated as a driver of the synaptotoxicity characteristic of the early phases of AD pathophysiology, questions remain about the molecular mechanisms involved. Cofilin-actin rods are observed in neurons exposed to Aβ oligomers (Aβo) and in tissue from AD patients, and others have described an increased cofilin phosphorylation (p-cof1) in AD patients. Here, we report elevated p-cof1 of the postsynaptic enriched fraction of synaptosomes from cortical samples of male APP/PS1 mice and human AD cases of either sex. In primary cortical neurons, Aβo induced rapid actin stabilization and increased p-cof1 in the postsynaptic compartment of excitatory synapses within 30 min. Fluorescence recovery after photobleaching of actin-GFP and calcium imaging in live neurons expressing active or inactive cof1 mutants suggest that cof1 phosphorylation is necessary and sufficient for Aβo-induced synaptic impairment via actin stabilization before the reported formation of cofilin-actin rods. Moreover, the clinically available and well-tolerated ROCK inhibitor, fasudil, prevented Aβo-induced actin stabilization, synaptic impairment, and synaptic loss by blocking cofilin phosphorylation. Aβo also blocked the LTP-induced insertion of the AMPAR subunit, GluA1, at the postsynaptic density, in a fasudil-sensitive manner. These data support an important role for ROCKs and cofilin in mediating Aβ-induced synaptic impairment.SIGNIFICANCE STATEMENT We report that amyloid-β oligomers rapidly induce aberrant stabilization of F-actin within dendritic spines, which impairs synaptic strength and plasticity. Activation of the Rho-associated protein kinase (ROCK) pathway results in phosphorylation of cof1 and is sufficient to mediate Aβo-induced actin stabilization synaptic impairment and synaptic loss. Further, the ROCK inhibitor, fasudil, prevents cofilin phosphorylation, acute synaptic disruption, and synaptotoxicity in primary cortical neurons. Together, the herein presented data provide strong support for further study of the ROCK pathway as a therapeutic target for the cognitive decline and synaptotoxicity in Alzheimer's disease.

Tankyrase-Binding Protein TNKS1BP1 Regulates Actin Cytoskeleton Rearrangement and Cancer Cell Invasion

Tankyrase, a PARP that promotes telomere elongation and Wnt/β-catenin signaling, has various binding partners, suggesting that it has as-yet unidentified functions. Here, we report that the tankyrase-binding protein TNKS1BP1 regulates actin cytoskeleton and cancer cell invasion, which is closely associated with cancer progression. TNKS1BP1 colocalized with actin filaments and negatively regulated cell invasion. In TNKS1BP1-depleted cells, actin filament dynamics, focal adhesion, and lamellipodia ruffling were increased with activation of the ROCK/LIMK/cofilin pathway. TNKS1BP1 bound the actin-capping protein CapZA2. TNKS1BP1 depletion dissociated CapZA2 from the cytoskeleton, leading to cofilin phosphorylation and enhanced cell invasion. Tankyrase overexpression increased cofilin phosphorylation, dissociated CapZA2 from cytoskeleton, and enhanced cell invasion in a PARP activity-dependent manner. In clinical samples of pancreatic cancer, TNKS1BP1 expression was reduced in invasive regions. We propose that the tankyrase-TNKS1BP1 axis constitutes a posttranslational modulator of cell invasion whose aberration promotes cancer malignancy. Cancer Res; 77(9); 2328-38. ©2017 AACR.

Identification of sennoside A as a novel inhibitor of the slingshot (SSH) family proteins related to cancer metastasis

Phospho-cofilin (p-cofilin), which has a phosphate group on Ser-3, is involved in actin polymerization. Its dephosphorylated form promotes filopodia formation and cell migration by enhancing actin depolymerization. Protein phosphatase slingshot homologs (SSHs), known as dual-specificity phosphatases, catalyze hydrolytic removal of the Ser-3 phosphate group from phospho-cofilin. Aberrant SSH activity results in cancer metastasis, implicating SSHs as potential therapeutic targets for cancer metastasis. In this study, we screened 658 natural products purified from traditional oriental medicinal plants to identify three potent SSH inhibitors with submicromolar or single-digit micromolar Ki values: gossypol, hypericin, and sennoside A. The three compounds were purified from cottonseed, Saint John’s wort, and rhubarb, respectively. Sennoside A markedly increased cofilin phosphorylation in pancreatic cancer cells, leading to impaired actin dynamics in pancreatic cancer cells with or without EGF stimulation and reduced motility and invasiveness in vitro and in vivo. Collaboratively, these results demonstrate that sennoside A is a novel inhibitor of SSHs and suggest that it may be valuable in the development of pharmaceutical drugs for treating cancer metastasis.

Insulin Receptor Substrate-4 Binds to Slingshot-1 Phosphatase and Promotes Cofilin Dephosphorylation

Cofilin plays an essential role in cell migration and morphogenesis by enhancing actin filament dynamics via its actin filament-severing activity. Slingshot-1 (SSH1) is a protein phosphatase that plays a crucial role in regulating actin dynamics by dephosphorylating and reactivating cofilin. In this study, we identified insulin receptor substrate (IRS)-4 as a novel SSH1-binding protein. Co-precipitation assays revealed the direct endogenous binding of IRS4 to SSH1. IRS4, but not IRS1 or IRS2, was bound to SSH1. IRS4 was bound to SSH1 mainly through the unique region (amino acids 335-400) adjacent to the C terminus of the phosphotyrosine-binding domain of IRS4. The N-terminal A, B, and phosphatase domains of SSH1 were bound to IRS4 independently. Whereas in vitro phosphatase assays revealed that IRS4 does not directly affect the cofilin phosphatase activity of SSH1, knockdown of IRS4 increased cofilin phosphorylation in cultured cells. Knockdown of IRS4 decreased phosphatidylinositol 3-kinase (PI3K) activity, and treatment with an inhibitor of PI3K increased cofilin phosphorylation. Akt preferentially phosphorylated SSH1 at Thr-826, but expression of a non-phosphorylatable T826A mutant of SSH1 did not affect insulin-induced cofilin dephosphorylation, and an inhibitor of Akt did not increase cofilin phosphorylation. These results suggest that IRS4 promotes cofilin dephosphorylation through sequential activation of PI3K and SSH1 but not through Akt. In addition, IRS4 co-localized with SSH1 in F-actin-rich membrane protrusions in insulin-stimulated cells, which suggests that the association of IRS4 with SSH1 contributes to localized activation of cofilin in membrane protrusions.

Inhibiting Geranylgeranylation Increases Neurite Branching and Differentially Activates Cofilin in Cell Bodies and Growth Cones

Inhibitors of the mevalonate pathway, including the highly prescribed statins, reduce the production of cholesterol and isoprenoids such as geranylgeranyl pyrophosphates. The Rho family of small guanine triphosphatases (GTPases) requires isoprenylation, specifically geranylgeranylation, for activation. Because Rho GTPases are primary regulators of actin filament rearrangements required for process extension, neurite arborization, and synaptic plasticity, statins may affect cognition or recovery from nervous system injury. Here, we assessed how manipulating geranylgeranylation affects neurite initiation, elongation, and branching in neuroblastoma growth cones. Treatment with the statin, lovastatin (20 μM), decreased measures of neurite initiation by 17.0 to 19.0 % when a source of cholesterol was present and increased neurite branching by 4.03- to 9.54-fold (regardless of exogenous cholesterol). Neurite elongation was increased by treatment with lovastatin only in cholesterol-free culture conditions. Treatment with lovastatin decreased growth cone actin filament content by up to 24.3 %. In all cases, co-treatment with the prenylation precursor, geranylgeraniol (10 μM), reversed the effect of lovastatin. In a prior work, statin effects on outgrowth were linked to modulating the actin depolymerizing factor, cofilin. In our assays, treatment with lovastatin or geranylgeraniol decreased cofilin phosphorylation in whole cell lysates. However, lovastatin increased cofilin phosphorylation in cell bodies and decreased it in growth cones, indicating differential regulation in specific cell regions. Together, we interpret these data to suggest that protein geranylgeranylation likely regulates growth cone actin filament content and subsequent neurite outgrowth through mechanisms that also affect actin nucleation and polymerization.

Membrane ERα attenuates myocardial fibrosis via RhoA/ROCK-mediated actin remodeling in ovariectomized female infarcted rats

We have demonstrated that long-term 17β-estradiol attenuated fibrosis after myocardial infarction, but recent evidence suggests that estrogen mediates more rapid cellular effects. RhoA has been shown to play a role in the control of myocardial fibrosis by regulating the actin-regulatory protein cofilin. We sought to elucidate whether RhoA activity regulates estradiol-induced fibrosis after infarction in ovariectomized female rats. Twenty-four hours after coronary ligation, female Wistar rats were randomized into control or estradiol treatment for 4 weeks starting from 2 weeks after bilateral ovariectomy. There were similar infarct sizes among the infarcted groups. Myocardial fibrosis was significantly reduced, as measured by hydroxyproline content, Sirius Red staining and actin polymerization after administering estradiol. Estradiol treatment was associated with RhoA/Rho kinase inhibition evidenced by increased phosphorylation of RhoA and decreased phosphorylation of downstream factors including cofilin. Administration of PPT, a specific ERα agonist, or a membrane impermeable estrogen-albumin construct also inhibited cofilin phosphorylation, suggesting that cofilin phosphorylation is achieved through the recruitment of a membrane ERα. The ERβ-selective agonist, DPN, did not alter cofilin phosphorylation levels, but the ER antagonist, ICI 182,780 increased cofilin phosphorylation levels. G-15 (an inhibitor of G-protein coupled estrogen receptor) did not affect the phosphorylation as compared with estradiol alone. The inhibitor of Rho kinase, Y-27632, attenuated cofilin phosphorylation levels, similar to the effect produced by estradiol. These results indicate that chronic use of estradiol after infarction attenuates cardiac fibrosis by inhibiting RhoA/ROCK/cofilin pathway, which is exerted through membrane ERα-mediated receptor mechanism. Postinfarction was associated with increased cardiac fibrosis. Myocardial fibrosis was significantly reduced after administering estradiol (E2). Administration of the membrane ERα agonist PPT or E2-BSA inhibited cofilin phosphorylation. Combination of E2 and Y-27632 did not enhance the attenuation of cofilin phosphorylation. Chronic use of E2 attenuated cardiac fibrosis by inhibiting the RhoA/ROCK/cofilin pathway via ERα.

Adiponectin Increases LPL Activity via RhoA/ROCK-Mediated Actin Remodelling in Adult Rat Cardiomyocytes

Cardiomyocyte substrate utilization is important in maintaining optimal cardiac function. Adiponectin has been shown to confer cardioprotective effects in part via regulating glucose and fatty acid uptake and oxidation in cardiomyocytes. Here we investigated mechanisms whereby adiponectin mediates a particular metabolic effect by focusing on lipoprotein lipase (LPL), an enzyme that increases free fatty acid availability to the heart by breakdown of chylomicrons and very-low-density lipoproteins in circulation. We used primary adult rat cardiomyocytes and demonstrate that adiponectin increased LPL translocation to the cell surface where it could be released at least partly in its active form, as evidenced by measuring basal and heparin-releasable LPL activity. Furthermore, these effects of adiponectin were mediated via remodeling of the actin cytoskeleton. We quantitatively assessed the filamentous to globular actin ratio and show that increased stress fiber formation, visualized by rhodamine-phalloidin immunofluorescence, in response to adiponectin, is achieved via stimulating Ras homolog gene family A (RhoA) activity, determined using G-LISA RhoA activation assay kit. We also demonstrate that adiponectin induces phosphorylation and inhibition of cofilin, leading to a reduction in actin treadmilling. Increased cofilin phosphorylation and stress fiber formation in response to adiponectin were prevented by inhibition of either RhoA or its downstream kinase Rho-associated protein kinase. Importantly, inhibition of cytoskeletal remodeling prevented adiponectin-stimulated plasma membrane LPL content detected by immunofluorescence and also subsequent LPL activity. In summary, we show that adiponectin mediates actin cytoskeleton remodeling to translocate LPL and allow subsequent activation.

Forgetting to remember in epilepsy

Amyloid-β (Aβ) drives the synaptic impairment and dendritic spine loss characteristic of Alzheimer9s disease (AD), but how Aβ affects the actin cytoskeleton remains unknown and contentious. The actin-binding protein, cofilin-1 (cof1), is a major regulator of actin dynamics in dendritic spines, and is subject to phospho-regulation by multiple pathways, including the Rho-associated protein kinase (ROCK) pathway. While cof1 is implicated as a driver of the synaptotoxicity characteristic of the early phases of AD pathophysiology, questions remain about the molecular mechanisms involved. Cofilin-actin rods are observed in neurons exposed to Aβ oligomers (Aβo) and in tissue from AD patients, and others have described an increased cofilin phosphorylation (p-cof1) in AD patients. Here, we report elevated p-cof1 of the postsynaptic enriched fraction of synaptosomes from cortical samples of male APP/PS1 mice and human AD cases of either sex. In primary cortical neurons, Aβo induced rapid actin stabilization and increased p-cof1 in the postsynaptic compartment of excitatory synapses within 30 min. Fluorescence recovery after photobleaching of actin-GFP and calcium imaging in live neurons expressing active or inactive cof1 mutants suggest that cof1 phosphorylation is necessary and sufficient for Aβo-induced synaptic impairment via actin stabilization before the reported formation of cofilin-actin rods. Moreover, the clinically available and well-tolerated ROCK inhibitor, fasudil, prevented Aβo-induced actin stabilization, synaptic impairment, and synaptic loss by blocking cofilin phosphorylation. Aβo also blocked the LTP-induced insertion of the AMPAR subunit, GluA1, at the postsynaptic density, in a fasudil-sensitive manner. These data support an important role for ROCKs and cofilin in mediating Aβ-induced synaptic impairment. <b>SIGNIFICANCE STATEMENT</b> We report that amyloid-β oligomers rapidly induce aberrant stabilization of F-actin within dendritic spines, which impairs synaptic strength and plasticity. Activation of the Rho-associated protein kinase (ROCK) pathway results in phosphorylation of cof1 and is sufficient to mediate Aβo-induced actin stabilization synaptic impairment and synaptic loss. Further, the ROCK inhibitor, fasudil, prevents cofilin phosphorylation, acute synaptic disruption, and synaptotoxicity in primary cortical neurons. Together, the herein presented data provide strong support for further study of the ROCK pathway as a therapeutic target for the cognitive decline and synaptotoxicity in Alzheimer9s disease.

Mechanisms of Vascular Smooth Muscle NADPH Oxidase 1 (Nox1) Contribution to Injury-Induced Neointimal Formation

Vascular NADPH oxidases (Noxes) have been implicated in cardiovascular diseases; however, the importance of individual Nox homologues remains unclear. Here, the role of the vascular smooth muscle cell (VSMC) Nox1 in neointima formation was studied using genetically modified animal models. Wire injury-induced neointima formation in the femoral artery, along with proliferation and apoptosis, was reduced in Nox1(y/-) mice, but there was little difference in Tg(SMCnox1) mice compared with wild-type (WT) mice. Proliferation and migration were reduced in cultured Nox1(y/-) VSMCs and increased in Tg(SMCnox1) cells. Tg(SMCnox1) cells exhibited increased fibronectin secretion, but neither collagen I production nor cell adhesion was affected by alteration of Nox1. Using antibody microarray and Western blotting analysis, increased cofilin phosphorylation and mDia1 expression and decreased PAK1 expression were detected in Nox1(y/-) cells. Overexpression of S3A, a constitutively active cofilin mutant, partially recovered reduced migration of Nox1(y/-) cells, suggesting that reduction in cofilin activity contributes to impaired migration of Nox1(y/-) VSMCs. These results indicate that Nox1 plays a critical role in neointima formation by mediating VSMC migration, proliferation, and extracellular matrix production, and that cofilin is a major effector of Nox1-mediated migration. Inhibition of Nox1 may be an efficient strategy to suppress neointimal formation.

Hyperosmotic stress induces Rho‐Rho kinase‐LIM kinase‐mediated cofilin phosphorylation

Cytoskeleton remodeling is a major adaptive response in osmotic stress, however the underlying mechanisms are incompletely understood. Our previous studies have shown that hypertonicity regulates various Rho GTPases and triggers de novo actin polymerization and myosin phosphorylation. Here we investigated whether osmotic shock impacts on actin-severing pathways as well. Hyperosmolarity induced threonine phosphorylation of LIM kinase and its target, the actin-severing protein cofilin in kidney tubular cells. Cofilin phosphorylation was rapid (< 2 min) sustained (>20 min), proportional with the osmotic concentration, and reversible upon restoration of isotonicity. Since both the Rho-Rho kinase (ROK) and the Rac/Cdc42-PAK pathways can induce LIMK-mediated cofilin phosphorylation, we asked which (if any) of these was responsible for the hypertonic cofilin response. Although hypertonicity activated PAK, neither dominant negative (DN) Rac nor DN-PAK1 prevented cofilin phosphorylation. In contrast, both DN-Rho and DN-ROK inhibited this effect. Moreover, the ROK inhibitor Y-27632 abolished the hypertonic LIMK and cofilin phosphorylation. Consistent with increased cofilin phosphorylation, hyperosmolarity appeared to reduce in situ actin severing and this effect was prevented by cofilin downregulation. These results suggest that Rho-ROK-LIMK-mediated cofilin phosphorylation contributes to osmotic stress-induced cytoskeleton remodeling.