Rhotekin regulates axon regeneration through the talin-Vinculin-Vinexin axis in Caenorhabditis elegans.

Axon regeneration requires actomyosin interaction, which generates contractile force and pulls the regenerating axon forward. In Caenorhabditis elegans, TLN-1/talin promotes axon regeneration through multiple down-stream events. One is the activation of the PAT-3/integrin-RHO-1/RhoA GTPase-LET-502/ROCK (Rho-associated coiled-coil kinase)-regulatory non-muscle myosin light-chain (MLC) phosphorylation signaling pathway, which is dependent on the MLC scaffolding protein ALP-1/ALP-Enigma. The other is mediated by the F-actin-binding protein DEB-1/vinculin and is independent of the MLC phosphorylation pathway. In this study, we identified the svh-7/rtkn-1 gene, encoding a homolog of the RhoA-binding protein Rhotekin, as a regulator of axon regeneration in motor neurons. However, we found that RTKN-1 does not function in the RhoA-ROCK-MLC phosphorylation pathway in the regulation of axon regeneration. We show that RTKN-1 interacts with ALP-1 and the vinculin-binding protein SORB-1/vinexin, and that SORB-1 acts with DEB-1 to promote axon regeneration. Thus, RTKN-1 links the DEB-1-SORB-1 complex to ALP-1 and physically connects phosphorylated MLC on ALP-1 to the actin cytoskeleton. These results suggest that TLN-1 signaling pathways coordinate MLC phosphorylation and recruitment of phosphorylated MLC to the actin cytoskeleton during axon regeneration.

ONO-1301 enhances post-transplantation survival of human induced pluripotent stem cell-derived cardiac tissue sheet by promoting angiogenesis.

BACKGROUND: Transplanting human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) tissue sheets effectively treat ischemic cardiomyopathy. Cardiac functional recovery relies on graft survival in which angiogenesis played an important part. ONO-1301 is a synthetic prostacyclin analog with proangiogenic effects. We hypothesized that transplantation of hiPSC-CM tissue sheets with slow-release ONO-1301 scaffold could promote hostgraft angiogenesis, enhance tissue survival and therapeutic effect. METHODS: We developed hiPSC-CM tissue sheets with ONO-1301 slow-release scaffold and evaluated their morphology, gene expression, and effects on angiogenesis. Three tissue sheet layers were transplanted into a rat myocardial infarction (MI) model. Left ventricular ejection fraction, gene expression in the MI border zone, and angiogenesis effects were investigated 4 weeks after transplantation. RESULTS: In vitro assessment confirmed the slow-release of ONO-1301, and its pro-angiogenesis effects. In addition, in vivo data demonstrated that ONO-1301 administration positively correlated with graft survival. Cardiac tissue as thick as ∼900 µm was retained in the ONO (+) treated group. Additionally, left ventricular ejection fraction of the ONO (+) group was significantly enhanced, compared to ONO (-) group. The ONO (+) group also showed significantly improved interstitial fibrosis, higher capillary density, increased number of mature blood vessels, along with an enhanced supply of oxygen, and nutrients. CONCLUSIONS: Slow-release ONO-1301 scaffold provided an efficient delivery method for thick hiPSC-CM tissue. ONO-1301 promotes angiogenesis between the host and graft and improves nutritional and oxygen supply, thereby enhancing the survival of transplanted cells, effectively improving ejection fraction, and therapeutic effects.

Histidine dephosphorylation of the Gß protein GPB-1 promotes axon regeneration in C. elegans.

Histidine phosphorylation is an emerging noncanonical protein phosphorylation in animals, yet its physiological role remains largely unexplored. The protein histidine phosphatase (PHPT1) was recently identified for the first time in mammals. Here, we report that PHIP-1, an ortholog of PHPT1 in Caenorhabditis elegans, promotes axon regeneration by dephosphorylating GPB-1 Gß at His-266 and inactivating GOA-1 Goα signaling, a negative regulator of axon regeneration. Overexpression of the histidine kinase NDK-1 also inhibits axon regeneration via GPB-1 His-266 phosphorylation. Thus, His-phosphorylation plays an antiregenerative role in C. elegans. Furthermore, we identify a conserved UNC-51/ULK kinase that functions in autophagy as a PHIP-1-binding protein. We demonstrate that UNC-51 phosphorylates PHIP-1 at Ser-112 and activates its catalytic activity and that this phosphorylation is required for PHIP-1-mediated axon regeneration. This study reveals a molecular link from ULK to protein histidine phosphatase, which facilitates axon regeneration by inhibiting trimeric G protein signaling.

Chemical Signaling Regulates Axon Regeneration via the GPCR-Gqα Pathway in Caenorhabditis elegans.

Chemical communication controls a wide range of behaviors via conserved signaling networks. Axon regeneration in response to injury is determined by the interaction between the extracellular environment and intrinsic growth potential. In this study, we investigated the role of chemical signaling in axon regeneration in Caenorhabditis elegans We find that the enzymes involved in ascaroside pheromone biosynthesis, ACOX-1.1, ACOX-1.2, and DAF-22, participate in axon regeneration by producing a dauer-inducing ascaroside, ascr#5. We demonstrate that the chemoreceptor genes, srg-36 and srg-37, which encode G-protein-coupled receptors for ascr#5, are required for adult-specific axon regeneration. Furthermore, the activating mutation in egl-30 encoding Gqα suppresses axon regeneration defective phenotype in acox-1.1 and srg-36 srg-37 mutants. Therefore, the ascaroside signaling system provides a unique example of a signaling molecule that regulates the regenerative pathway in the nervous system.SIGNIFICANCE STATEMENT In Caenorhabditis elegans, axon regeneration is positively regulated by the EGL-30 Gqα-JNK MAP kinase cascade. However, it remains unclear what signals activate the EGL-30 pathway in axon regeneration. Here, we show that SRG-36 and SRG-37 act as upstream G-protein-coupled receptors (GPCRs) that activate EGL-30. C. elegans secretes a family of small-molecule pheromones called ascarosides, which serve various functions in chemical signaling. SRG-36 and SRG-37 are GPCRs for the dauer-inducing ascaroside ascr#5. Consistent with this, we found that ascr#5 activates the axon regeneration pathway via SRG-36/SRG-37 and EGL-30. Thus, ascaroside signaling promotes axon regeneration by activating the GPCR-Gqα pathway.

Combined administration of laminin-221 and prostacyclin agonist enhances endogenous cardiac repair in an acute infarct rat heart.

Although endogenous cardiac repair by recruitment of stem cells may serve as a therapeutic approach to healing a damaged heart, how to effectively enhance the migration of stem cells to the damaged heart is unclear. Here, we examined whether the combined administration of prostacyclin agonist (ONO1301), a multiple-cytokine inducer, and stem cell niche laminin-221 (LM221), enhances regeneration through endogenous cardiac repair. We administered ONO1301- and LM221-immersed sheets, LM221-immersed sheets, ONO1301-immersed sheets, and PBS-immersed sheets (control) to an acute infarction rat model. Four weeks later, cardiac function, histology, and cytokine expression were analysed. The combined administration of LM221 and ONO1301 upregulated angiogenic and chemotactic factors in the myocardium after 4 weeks and enhanced the accumulation of ILB4 positive cells, SMA positive cells, and platelet-derived growth factor receptor alpha (PDGFRα) and CD90 double-positive cells, leading to the generation of mature microvascular networks. Interstitial fibrosis reduced and functional recovery was prominent in LM221- and ONO1301-administrated hearts as compared with those in ONO1301-administrated or control hearts. LM221 and ONO1301 combination enhanced recruitment of PDGFRα and CD90 double-positive cells, maturation of vessels, and functional recovery in rat acute myocardial infarction hearts, highlighting a new promising acellular approach for the failed heart.

CDK14 Promotes Axon Regeneration by Regulating the Noncanonical Wnt Signaling Pathway in a Kinase-Independent Manner.

The postinjury regenerative capacity of neurons is known to be mediated by a complex interaction of intrinsic regenerative pathways and external cues. In Caenorhabditis elegans, the initiation of axon regeneration is regulated by the nonmuscle myosin light chain-4 (MLC-4) phosphorylation signaling pathway. In this study, we have identified svh-16/cdk-14, a mammalian CDK14 homolog, as a positive regulator of axon regeneration in motor neurons. We then isolated the CDK-14-binding protein MIG-5/Disheveled (Dsh) and found that EGL-20/Wnt and the MIG-1/Frizzled receptor (Fz) are required for efficient axon regeneration. Further, we demonstrate that CDK-14 activates EPHX-1, the C. elegans homolog of the mammalian ephexin Rho-type GTPase guanine nucleotide exchange factor (GEF), in a kinase-independent manner. EPHX-1 functions as a GEF for the CDC-42 GTPase, inhibiting myosin phosphatase, which maintains MLC-4 phosphorylation. These results suggest that CDK14 activates the RhoGEF-CDC42-MLC phosphorylation axis in a noncanonical Wnt signaling pathway that promotes axon regeneration.SIGNIFICANCE STATEMENT Noncanonical Wnt signaling is mediated by Frizzled receptor (Fz), Disheveled (Dsh), Rho-type GTPase, and nonmuscle myosin light chain (MLC) phosphorylation. This study identified svh-16/cdk-14, which encodes a mammalian CDK14 homolog, as a regulator of axon regeneration in Caenorhabditis elegans motor neurons. We show that CDK-14 binds to MIG-5/Dsh, and that EGL-20/Wnt, MIG-1/Fz, and EPHX-1/RhoGEF are required for axon regeneration. The phosphorylation-mimetic MLC-4 suppressed axon regeneration defects in mig-1, cdk-14, and ephx-1 mutants. CDK-14 mediates kinase-independent activation of EPHX-1, which functions as a guanine nucleotide exchange factor for CDC-42 GTPase. Activated CDC-42 inactivates myosin phosphatase and thereby maintains MLC phosphorylation. Thus, the noncanonical Wnt signaling pathway controls axon regeneration via the CDK-14-EPHX-1-CDC-42-MLC phosphorylation axis.

Cardiac fibrosis models using human induced pluripotent stem cell-derived cardiac tissues allow anti-fibrotic drug screening in vitro.

Drug efficacy assessment without using animals is important for development of cardiac fibrosis treatment. In this study, potential anti-fibrotic drugs were screened in a model of diseased myocardium using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and non-CM in in vitro and in vivo heart failure models. Cardiomyogenic differentiation was induced in hiPSC to generate cardiac tissue, including both iPSC-CM and non-CM expressing fibroblast markers. Stimulation with TGF-ß significantly increased cardiac fibrotic extracellular matrix (ECM) gene expression, and decreased cardiac contractile/relaxation velocity. Anti-fibrotic HGF significantly decreased fibrotic changes induced by TGF-ß. A prostacyclin agonist, ONO-1301 (ONO), camostat mesilate (Cs), and pirfenidone (Pf) significantly decreased fibrotic ECM expression, and improved contraction/relaxation in the model stimulated with TGF-ß. Consistent with the in vitro assay, the administration of ONO, Cs, or Pf for 8 weeks in J2N-k hamsters preserved the left ventricular ejection fraction and decreased cardiac fibrosis compared with the controls. The in vitro model simulating fibrotic cardiac tissue showed precise screening of anti-fibrotic drugs which indicated the expected therapeutic response in an in vivo heart failure model, suggesting that the in vitro model presented in this study is a useful tool for the screening of anti-fibrotic drugs.

A serine protease inhibitor camostat mesilate prevents podocyte apoptosis and attenuates podocyte injury in metabolic syndrome model rats.

Metabolic syndrome (MetS) is associated with chronic kidney disease and proteinuria. Previously, we reported that a synthetic serine protease inhibitor, camostat mesilate (CM), mitigated hypertension and proteinuria in rodent disease models. The present study evaluated the anti-hypertensive and anti-proteinuric effects of CM in MetS model rats (SHR/ND mcr-cp). Rats were divided into normal salt-fed (NS), high salt-fed (HS), HS and CM-treated (CM), and HS and hydralazine-treated (Hyd) groups. Rats were sacrificed after four weeks of treatment. Severe hypertension and proteinuria were observed in the HS group. Although CM and Hyd equally alleviated hypertension, CM suppressed proteinuria and glomerular sclerosis more efficiently than Hyd. The HS group revealed a decrease in podocyte number and podocyte-specific molecules, together with an increase in glomerular apoptotic cells and apoptosis-related proteins in the kidney. These changes were significantly attenuated by CM, but not by Hyd. Furthermore, CM ameliorated the apoptotic signals in murine cultured podocytes stimulated with the high glucose and aldosterone medium. In conclusion, CM could exert renoprotective effects in MetS model rats, together with the inhibition of podocyte apoptosis. Our study suggests that serine protease inhibition may become a new therapeutic strategy against MetS-related hypertension and renal injuries.

The Integrin Signaling Network Promotes Axon Regeneration via the Src-Ephexin-RhoA GTPase Signaling Axis.

Axon regeneration is an evolutionarily conserved process essential for restoring the function of damaged neurons. In Caenorhabditis elegans hermaphrodites, initiation of axon regeneration is regulated by the RhoA GTPase-ROCK (Rho-associated coiled-coil kinase)-regulatory nonmuscle myosin light-chain phosphorylation signaling pathway. However, the upstream mechanism that activates the RhoA pathway remains unknown. Here, we show that axon injury activates TLN-1/talin via the cAMP-Epac (exchange protein directly activated by cAMP)-Rap GTPase cascade and that TLN-1 induces multiple downstream events, one of which is integrin inside-out activation, leading to the activation of the RhoA-ROCK signaling pathway. We found that the nonreceptor tyrosine kinase Src, a key mediator of integrin signaling, activates the Rho guanine nucleotide exchange factor EPHX-1/ephexin by phosphorylating the Tyr-568 residue in the autoinhibitory domain. Our results suggest that the C. elegans integrin signaling network regulates axon regeneration via the Src-RhoGEF-RhoA axis.SIGNIFICANCE STATEMENT The ability of axons to regenerate after injury is governed by cell-intrinsic regeneration pathways. We have previously demonstrated that the Caenorhabditis elegans RhoA GTPase-ROCK (Rho-associated coiled-coil kinase) pathway promotes axon regeneration by inducing MLC-4 phosphorylation. In this study, we found that axon injury activates TLN-1/talin through the cAMP-Epac (exchange protein directly activated by cAMP)-Rap GTPase cascade, leading to integrin inside-out activation, which promotes axonal regeneration by activating the RhoA signaling pathway. In this pathway, SRC-1/Src acts downstream of integrin activation and subsequently activates EPHX-1/ephexin RhoGEF by phosphorylating the Tyr-568 residue in the autoinhibitory domain. Our results suggest that the C. elegans integrin signaling network regulates axon regeneration via the Src-RhoGEF-RhoA axis.

Innovative therapeutic strategy using prostaglandin I2 agonist (ONO1301) combined with nano drug delivery system for pulmonary arterial hypertension.

Clinical outcomes of pulmonary arterial hypertension (PAH) may be improved using targeted delivery system. We investigated the efficacy of ONO1301 (prostacyclin agonist) nanospheres (ONONS) in Sugen5416/hypoxia rat models of PAH. The rats were injected with saline (control) or ONONS (n = 10, each) on days 21 and 28, respectively. Hepatocyte growth factor (HGF)-expressing fibroblasts and inflammatory cytokines were measured. Cardiac performance was assessed and targeted delivery was monitored in vivo, using Texas red-labeled nanoparticles. Compared with control, HGF-expressing fibroblasts and HGF expression levels were significantly higher in the ONONS group, while the levels of interleukin-6, interleukin-1ß, transforming growth factor-ß, and platelet-derived growth factor were lower. Histological assessment revealed significant amelioration of the percent medial wall thickness in pulmonary vasculature of rats in the ONONS group. Rats in the ONONS group showed decreased proliferating cell nuclear antigen-positive smooth muscle cells and improved right ventricle pressure/left ventricle pressure. No difference was seen in the accumulation of Texas red-labeled nanoparticles in the brain, heart, liver, and spleen between PAH and normal rats. However, a significant area of nanoparticles was detected in the lungs of PAH rats. ONONS effectively ameliorated PAH, with selective delivery to the damaged lung.

New regional drug delivery system by direct epicardial placement of slow-release prostacyclin agonist promise therapeutic angiogenesis in a porcine chronic myocardial infarction.

Although prostacyclin is an endogenous factor for the protection and regeneration of damaged tissue, the use of clinically available prostacyclin analogues for treating chronic pathological conditions is limited owing to their short half-lives. A new reagent, ONO-1301SR, which is a unique synthetic prostacyclin agonist polymerized with lactic and glycolic acid, has been demonstrated to constitutively release prostacyclin analogues to adjacent tissues, suggesting its therapeutic potential via slow-release delivery into a specific organ. In this study, we investigated the regenerative effect of direct epicardial delivery of the ONO-1301SR on a heart with a chronic myocardial infarct. An ameroid constrictor was placed on the left anterior descending coronary artery of Göttingen minipigs for 4 weeks to induce ischemic cardiomyopathy; this was followed by direct epicardial placement of ONO-1301SR-immersed gelatinous sheet, or only a gelatinous sheet on the anterolateral surface of the heart. Epicardial placement of ONO-1301SR resulted in significant recovery of global cardiac functions and regional wall motion of the lateral wall. Importantly, after epicardial placement of ONO-1301SR for 4 weeks, the myocardial blood flow significantly increased in the lateral region as assessed by 13N-ammonia positron emission tomography; this finding was consistent with significantly increased capillary density in the peri-infarct area with up-regulated angiogenic cytokine expression. Conclusion: Use of the slow-release drug delivery system of prostacyclin agonist yielded regenerative angiogenesis, including increased regional blood perfusion and systolic function in a porcine model of chronic myocardial infarction.

BRCA1-BARD1 Regulates Axon Regeneration in Concert with the Gqα-DAG Signaling Network.

The breast cancer susceptibility protein BRCA1 and its partner BRCA1-associated RING domain protein 1 (BARD1) form an E3-ubiquitin (Ub) ligase complex that acts as a tumor suppressor in mitotic cells. However, the roles of BRCA1-BARD1 in postmitotic cells, such as neurons, remain poorly defined. Here, we report that BRC-1 and BRD-1, the Caenorhabditis elegans orthologs of BRCA1 and BARD1, are required for adult-specific axon regeneration, which is positively regulated by the EGL-30 Gqα-diacylglycerol (DAG) signaling pathway. This pathway is downregulated by DAG kinase (DGK), which converts DAG to phosphatidic acid (PA). We demonstrate that inactivation of DGK-3 suppresses the brc-1 brd-1 defect in axon regeneration, suggesting that BRC-1-BRD-1 inhibits DGK-3 function. Indeed, we show that BRC-1-BRD-1 poly-ubiquitylates DGK-3 in a manner dependent on its E3 ligase activity, causing DGK-3 degradation. Furthermore, we find that axon injury causes the translocation of BRC-1 from the nucleus to the cytoplasm, where DGK-3 is localized. These results suggest that the BRC-1-BRD-1 complex regulates axon regeneration in concert with the Gqα-DAG signaling network. Thus, this study describes a new role for breast cancer proteins in fully differentiated neurons and the molecular mechanism underlying the regulation of axon regeneration in response to nerve injury.SIGNIFICANCE STATEMENT BRCA1-BRCA1-associated RING domain protein 1 (BARD1) is an E3-ubiquitin (Ub) ligase complex acting as a tumor suppressor in mitotic cells. The roles of BRCA1-BARD1 in postmitotic cells, such as neurons, remain poorly defined. We show here that Caenorhabditis elegans BRC-1/BRCA1 and BRD-1/BARD1 are required for adult-specific axon regeneration, a process that requires high diacylglycerol (DAG) levels in injured neurons. The DAG kinase (DGK)-3 inhibits axon regeneration by reducing DAG levels. We find that BRC-1-BRD-1 poly-ubiquitylates and degrades DGK-3, thereby keeping DAG levels elevated and promoting axon regeneration. Furthermore, we demonstrate that axon injury causes the translocation of BRC-1 from the nucleus to the cytoplasm, where DGK-3 is localized. Thus, this study describes a new role for BRCA1-BARD1 in fully-differentiated neurons.

Caenorhabditis elegans F-Box Protein Promotes Axon Regeneration by Inducing Degradation of the Mad Transcription Factor.

In Caenorhabditis elegans, axon regeneration is activated by a signaling cascade through the receptor tyrosine kinase (RTK) SVH-2. Axonal injury induces svh-2 gene expression by degradation of the Mad-like transcription factor MDL-1. In this study, we identify the svh-24/sdz-33 gene encoding a protein containing F-box and F-box-associated domains as a regulator of axon regeneration in motor neurons. We find that sdz-33 is required for axon injury-induced svh-2 expression. SDZ-33 targets MDL-1 for poly-ubiquitylation and degradation. Furthermore, we demonstrate that SDZ-33 promotes axotomy-induced nuclear degradation of MDL-1, resulting in the activation of svh-2 expression in animals. These results suggest that the F-box protein is required for RTK signaling in the control of axon regeneration.SIGNIFICANCE STATEMENT In Caenorhabditis elegans, axon regeneration is positively regulated by the growth factor SVH-1 and its receptor tyrosine kinase SVH-2. Expression of the svh-2 gene is induced by axonal injury via the Ets-like transcription factor ETS-4, whose transcriptional activity is inhibited by the Mad-like transcription factor MDL-1. Axon injury leads to the degradation of MDL-1, and this is linked to the activation of ETS-4 transcriptional activity. In this study, we identify the sdz-33 gene encoding a protein containing an F-box domain as a regulator of axon regeneration. We demonstrate that MDL-1 is poly-ubiquitylated and degraded through the SDZ-33-mediated 26S proteasome pathway. These results reveal that an F-box protein promotes axon regeneration by degrading the Mad transcription factor.

Synthetic Prostacyclin Agonist Attenuates Pressure-Overloaded Cardiac Fibrosis by Inhibiting FMT.

Fibroblast-to-myofibroblast transition (FMT) is the primary inducer of cardiac fibrosis. ONO-1301, a synthetic prostacyclin agonist, reportedly promotes tissue fibrosis repair by enhancing anti-fibrotic cytokine production. We hypothesized that ONO-1301 attenuates pressure-overloaded cardiac fibrosis by modulating FMT and generated a pressure-overloaded murine model via transverse aortic constriction (TAC) to evaluate the in vivo effects of ONO-1301. Cardiac fibrosis, left ventricular dilatation, and systolic dysfunction were established 4 weeks after TAC; however, ONO-1301 treatment initiated 2 weeks after TAC significantly attenuated those effects. Furthermore, ONO-1301 treatment significantly upregulated expression levels of cardioprotective cytokines such as vascular endothelial growth factor and hepatocyte growth factor in TAC hearts, whereas FMT-related factors, including transforming growth factor (TGF)-ß1 and connective tissue growth factor, were significantly downregulated. The number of α-smooth muscle actin (α-SMA)- and vimentin-positive cells, representing fibroblast-originated cells transitioned into myofibroblasts, was significantly reduced in ONO-1301-treated TAC hearts. We isolated cardiac fibroblasts (CFs) from the left ventricles of adult male mice and assessed the effects of ONO-1301 on CFs stimulated by TGF-ß. Results showed that ONO-1301 co-incubation significantly suppressed TGF-ß-induced α-SMA expression and collagen synthesis, and significantly inhibited TGF-ß-induced CF proliferation and migration. Our findings suggest that ONO-1301 ameliorates pressure overloaded cardiac fibrosis by inhibiting TGF-ß-induced FMT.

Administration of Slow-Release Synthetic Prostacyclin Agonist Promoted Angiogenesis and Skeletal Muscle Regeneration for Limb Ischemia.

Gene or cell therapy is currently not fully efficacious for arteriosclerosis obliterans (ASO). In this study, we determined whether YS-1402, a slow-release synthetic prostacyclin agonist, promoted neovascularization and skeletal muscle regeneration in a mouse model of critical limb ischemia (CLI). We ligated the femoral artery and its branches to obtain the CLI mouse model, administered saline (S group) or YS-1402 (YS group) to the thigh adductor 1 week after femoral artery occlusion, and evaluated tissue blood flow after surgery. After treatment, the leg muscle was obtained for histological, gene expression, and protein analyses to assess angiogenesis and skeletal muscle regeneration. Tissue blood flow improved in the YS group compared with that in the S group, and the number of CD31+/α-smooth muscle actin (αSMA)+ arterioles increased in the YS group. Prostacyclin receptor (IPR), stromal cell-derived factor-1, hepatocyte growth factor, and neural cell adhesion molecule expression levels were higher in the YS than in the S group. Skeletal muscle regeneration was detected based on PAX7- and Ki-67-positive satellite cells in the YS group. Myogenin and MyoD expression was higher in the YS than in the S group. Therefore, YS-1402 promoted functional angiogenesis and skeletal muscle regeneration in the CLI mouse model, suggesting a new therapy for ASO.

TDP2 negatively regulates axon regeneration by inducing SUMOylation of an Ets transcription factor.

In Caenorhabditis elegans, the JNK MAP kinase (MAPK) pathway is important for axon regeneration. The JNK pathway is activated by a signaling cascade consisting of the growth factor SVH-1 and its receptor tyrosine kinase SVH-2. Expression of the svh-2 gene is induced by axonal injury in a process involving the transcription factors ETS-4 and CEBP-1. Here, we find that svh-14/mxl-1, a gene encoding a Max-like transcription factor, is required for activation of svh-2 expression in response to axonal injury. We show that MXL-1 binds to and inhibits the function of TDPT-1, a C. elegans homolog of mammalian tyrosyl-DNA phosphodiesterase 2 [TDP2; also called Ets1-associated protein II (EAPII)]. Deletion of tdpt-1 suppresses the mxl-1 defect, but not the ets-4 defect, in axon regeneration. TDPT-1 induces SUMOylation of ETS-4, which inhibits ETS-4 transcriptional activity, and MXL-1 counteracts this effect. Thus, TDPT-1 interacts with two different transcription factors in axon regeneration.

N-Glycosylation of the Discoidin Domain Receptor Is Required for Axon Regeneration in Caenorhabditis elegans.

Axon regeneration following neuronal injury is an important repair mechanism that is not well understood at present. In Caenorhabditis elegans, axon regeneration is regulated by DDR-2, a receptor tyrosine kinase (RTK) that contains a discoidin domain and modulates the Met-like SVH-2 RTK-JNK MAP kinase signaling pathway. Here, we describe the svh-10/sqv-3 and svh-11 genes, which encode components of a conserved glycosylation pathway, and show that they modulate axon regeneration in C. elegans Overexpression of svh-2, but not of ddr-2, can suppress the axon regeneration defect observed in svh-11 mutants, suggesting that SVH-11 functions between DDR-2 and SVH-2 in this glycosylation pathway. Furthermore, we found that DDR-2 is N-glycosylated at the Asn-141 residue located in its discoidin domain, and mutation of this residue caused an axon regeneration defect. These findings indicate that N-linked glycosylation plays an important role in axon regeneration in C. elegans.

Prostacyclin Analogue-Loaded Nanoparticles Attenuate Myocardial Ischemia/Reperfusion Injury in Rats.

Intravenously injected ONO-1301-containing nanoparticles (ONO-1301NPs), unlike an ONO-1301 solution, selectively accumulated in the ischemia/reperfusion (I/R)-injured myocardium of rats and contributed to the prolonged retention of ONO-1301 in the targeted myocardial tissue. In the ischemic area, proangiogenic cytokines were up-regulated and inflammatory cytokines were down-regulated upon ONO-1301NP administration. Consequently, ONO-1301NP-injected rats exhibited a smaller infarct size, better-preserved capillary networks, and a better-preserved myocardial blood flow at 24 h after I/R injury, compared with those in vehicle-injected or ONO-1301 solution-injected rats. ONO-1301NPs attenuate the myocardial I/R injury via proangiogenic and anti-inflammatory effects of the drug.

C. elegans Tensin Promotes Axon Regeneration by Linking the Met-like SVH-2 and Integrin Signaling Pathways.

Axon regeneration is a conserved mechanism induced by axon injury that initiates a neuronal response leading to regrowth of the axon. In Caenorhabditis elegans, the initiation of axon regeneration is regulated by the JNK MAP kinase (MAPK) pathway. We have previously identified a number of genes affecting the JNK pathway using an RNAi-based screen. Analysis of these genes, called the svh genes, has shed new light on the regulation of axon regeneration, revealing the involvement of a signaling cascade consisting of a growth factor SVH-1 and its receptor, the tyrosine kinase SVH-2. Here, we characterize the svh-6/tns-1 gene, which is a homolog of mammalian tensin, and show that it is a positive regulator of axon regeneration in motor neurons. We demonstrate that TNS-1 interacts with tyrosine-autophosphorylated SVH-2 and the integrin ß subunit PAT-3 via its SH2 and PTB domains, respectively, to promote axon regeneration. These results suggest that TNS-1 acts as an adaptor to link the SVH-2 and integrin signaling pathways.SIGNIFICANCE STATEMENT The Caenorhabditis elegans JNK MAPK pathway regulates the initiation of axon regeneration. Previously, we showed that a signaling cascade consisting of the HGF-like growth factor SVH-1 and its Met-like receptor tyrosine kinase SVH-2 promotes axon regeneration through activation of the JNK pathway. In this study, we show that the C. elegans tensin, TNS-1, is required for efficient regeneration after axon injury. Phosphorylation of SVH-2 on tyrosine mediates its interaction with the SH2 domain of TNS-1 to positively regulate axon regeneration. Furthermore, TNS-1 interacts via its PTB domain with the integrin ß subunit PAT-3. These results suggest that TNS-1 plays a critical role in the regulation of axon regeneration by linking the SVH-2 and integrin signaling pathways.

Single injection of sustained-release prostacyclin analog ONO-1301-MS ameliorates hypoxic toxicity in the murine model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by several pathologies including oxidative stress, apoptosis, neuroinflammation, and glutamate toxicity. Although multiple reports suggest that ischemia and hypoxia in the spinal cord plays a pivotal role in the pathogenesis of ALS, the precise role of hypoxia in disease progression remains unknown. In this study, we detected higher expression levels of Hypoxia-inducible factor 1-alpha (HIF-1α), a key regulator of cellular responses to hypoxia, in the spinal cord of ALS patients and in the transgenic mice overexpressing the familial ALS-associated G93A SOD1 mutation (mSOD1G93A mice) compared to controls. Single subcutaneous administration of sustained-release prostacyclin analog ONO-1301-MS to mSOD1G93A mice abrogated the expression of HIF-1α in their spinal cords, as well as erythropoietin (EPO) and vascular endothelial growth factor (VEGF), both of which are downstream to HIF-1α. Furthermore, ONO-1301-MS increased the level of mature brain-derived neurotrophic factor (BDNF) and ATP production in the spinal cords of mSOD1G93A mice. At late disease stages, the motor function and the survival of motor neurons of ONO-1301-MS-treated mSOD1G93A mice was significantly improved compared to vehicle-treated mSOD1G93A mice. Our data suggest that vasodilator therapy modulating local blood flow in the spinal cord has beneficial effects against ALS disease progression.