JNK MAP Kinase Research Articles

The Helicobacter pylori type IV secretion system upregulates epithelial cortactin expression by a CagA- and JNK-dependent pathway.

Cortactin represents an important actin-binding factor, which controls actin-cytoskeletal remodelling in host cells. In this way, cortactin has been shown to exhibit crucial functions both for cell movement and tumour cell invasion. In addition, the cortactin gene cttn is amplified in various cancer types of humans. Helicobacter pylori is the causative agent of multiple gastric diseases and represents a significant risk factor for the development of gastric adenocarcinoma. It has been repeatedly shown that H. pylori manipulates cancer-related signal transduction events in infected gastric epithelial cells such as the phosphorylation status of cortactin. In fact, H. pylori modifies the activity of cortactin's binding partners to stimulate changes in the actin-cytoskeleton, cell adhesion and motility. Here we show that H. pylori infection of cultured AGS and Caco-2 cells for 24-48 hr leads to the overexpression of cortactin by 2-3 fold at the protein level. We demonstrate that this activity requires the integrity of the type IV secretion system (T4SS) encoded by the cag pathogenicity island (cagPAI) as well as the translocated effector protein CagA. We further show that ectopic expression of CagA is sufficient to stimulate cortactin overexpression. Furthermore, phosphorylation of CagA at the EPIYA-repeat region is not required, suggesting that this CagA activity proceeds in a phosphorylation-independent fashion. Inhibitor studies further demonstrate that the involved signalling pathway comprises the mitogen-activated protein kinase JNK (c-Jun N-terminal kinase), but not ERK1/2 or p38. Taken together, using H. pylori as a model system, this study discovered a previously unrecognised cortactin activation cascade by a microbial pathogen. We suggest that H. pylori targets cortactin to manipulate the cellular architecture and epithelial barrier functions that can impact gastric cancer development. TAKE AWAYS: Helicobacter pylori infection induces overexpression of cortactin at the protein level Cortactin upregulation requires the T4SS and effector protein CagA Ectopic expression of CagA is sufficient to stimulate cortactin overexpression Overexpression of cortactin proceeds CagA phosphorylation-independent The involved host cell signalling pathway comprises the MAP kinase JNK.

A signalling cascade for Ral

ABSTRACT Ras is the most mutated oncoprotein in cancer. Among the three oncogenic effectors of Ras – Raf, PI3 Kinase and RalGEF>Ral – signalling through RalGEF>Ral (Ras-like) is by far the least well understood. A variety of signals and binding partners have been defined for Ral, yet we know little of how Ral functions in vivo. This review focuses on previous research in Drosophila that defined a function for Ral in apoptosis and established indirect relationships among Ral, the CNH-domain MAP4 Kinase misshapen, and the JNK MAP kinase basket. Most of the described signalling components are not essential in C. elegans, facilitating subsequent analysis using developmental patterning of the C. elegans vulval precursor cells (VPCs). The functions of two paralogous CNH-domain MAP4 Kinases were defined relative to Ras>Raf, Notch and Ras>RalGEF>Ral signalling in VPCs. MIG-15, the nematode ortholog of misshapen, antagonizes both the Ral-dependent and Ras>Raf-dependent developmental outcomes. In contrast, paralogous GCK-2, the C. elegans ortholog of Drosophila happyhour, propagates the 2°-promoting signal of Ral. Manipulations via CRISPR of Ral signalling through GCK-2 coupled with genetic epistasis delineated a Ras>RalGEF>Ral>Exo84>GCK-2>MAP3KMLK−1> p38PMK−1 cascade. Thus, genetic analysis using invertebrate experimental organisms defined a cascade from Ras to p38 MAP kinase.

Cell-cell and cell-substratum contacts in the regulation of MAPK and Akt signalling: Importance in therapy, biopharmacy and bioproduction

The use of cultured cells as a tool for research, precision medicine, biopharmacy, and biomanufacturing is constantly increasing. In parallel, the role of cell-cell and cell-substratum contacts in cell functioning is increasingly validated. Adhesion signalling plays a key role here. The activity of cell fate-regulating signalling molecules is an important factor in determining cell behaviour, as well as their response to treatment, depending on cell phenotypic status and location in the body. Three cellular state models (adherent, single cells in suspension, and aggregated cells) were compared for cell signalling, including focal adhesion (FAK), mitogen-activated (MAPK), as well as Akt protein kinases, and transcription factor cJun, by using lung adenocarcinoma A549, muscle-derived stem Myo, as well as primary lung cancer cell lines. Survival of both A549 and Myo cells was dependent on kinases Akt and ERK in detached conditions. Intercellular contacts in aggregates promoted activation of Akt and ERK, and cell survival. Loss of contacts with the substrate increased phosphorylation of MAP kinases JNK and p38, while decreased Akt phosphorylation by processes involving FAK. Unexpectedly, detachment increased phosphorylation of antiapoptotic kinase ERK in A549, while in Myo stem cells ERK phosphorylation was downregulated. JNK target transcription factor cJun protein level was markedly diminished by contacts between cells possibly involving mechanism of proteasomal degradation. Furthermore, studies revealed the opposite dependence of molecules of the same signalling pathway – phospho-cJun and phospho-JNK – on cell culture density. Differences in ERK activation under detachment conditions indicate that targeting of prosurvival kinases during anoikis should be different in different cells. Moreover, the outcome of JNK activation in cells may depend on the amount of cJun, which is determined by cell-cell contacts.

Nutrition and PI3K/Akt signaling are required for p38-dependent regeneration.

Regeneration after damage requires early signals to trigger the tissue repair machinery. Reactive oxygen species (ROS) act as early signals that are sensed by the MAP3 kinase Ask1, which in turn activates by phosphorylation the MAP kinases p38 and JNK. The sustained or high activation of these kinases can result in apoptosis, whereas short or low activation can promote regeneration. Using the Ask1-dependent regeneration program, we demonstrate in Drosophila wing that PI3K/Akt signaling is necessary for Ask1 to activate p38, but not JNK. In addition, nutrient restriction or mutations that target Ser83 of the Drosophila Ask1 protein, a PI3K/Akt-sensitive residue, block regeneration. However, these effects can be reversed by the ectopic activation of p38, but not of JNK. Our results demonstrate that Ask1 controls the activation of p38 through Ser83, and that the phosphorylation of p38 during regeneration is nutrient sensitive. This mechanism is important for discriminating between p38 and JNK in the cells involved in tissue repair and regenerative growth.

Cefoxitin treatment of MRSA leads to a shift in the IL-12/IL-23 production pattern in dendritic cells by a mechanism involving changes in the MAPK signaling

Methicillin resistant Staphylococcus aureus (MRSA) constitute a serious health care problem worldwide. This study addresses the effect of β-lactam treatment on the ability of clinically relevant MRSA strains to induce IL-12 and IL-23. MRSA strains induced a dose-dependent IL-12 response in murine bone-marrow-derived dendritic cells that was dependent on endocytosis and acidic degradation. Facilitated induction of IL-12 (but not of IL-23) called for activation of the MAP kinase JNK, and was suppressed by p38. Compromised peptidoglycan structure in cefoxitin-treated bacteria – as denoted by increased sensitivity to mutanolysin –caused a shift from IL-12 towards IL-23. Moreover, cefoxitin treatment of MRSA led to a p38 MAPK-dependent early up-regulation of Dual Specificity Phosphatase (DUSP)-1. Compared to common MRSA, characteristics associated with a persister phenotype increased intracellular survival and upon cefoxitin treatment, the peptidoglycan was not equally compromised and the cytokine induction still required phagosomal acidification. Together, these data demonstrate that β-lactam treatment changes the MRSA-induced IL-12/IL-23 pattern determined by the activation of JNK and p38. We suggest that accelerated endosomal degradation of the peptidoglycan in cefoxitin-treated MRSA leads to an early expression of DUSP-1 and accordingly, a reduction in the IL-12/IL-23 ratio in dendritic cells. This may influence the clearance of S. aureus.

Impaired protein adduct removal following repeat administration of subtoxic doses of acetaminophen enhances liver injury in fed mice.

Acetaminophen (APAP) is a widely used analgesic and is safe at therapeutic doses. However, an overdose of APAP is hepatotoxic and accidental overdoses are increasingly common due to the presence of APAP in several combination medications. Formation of protein adducts (APAP-CYS) is central to APAP-induced liver injury and their removal by autophagy is an essential adaptive response after an acute overdose. Since the typical treatment for conditions such as chronic pain involves multiple doses of APAP over time, this study investigated APAP-induced liver injury after multiple subtoxic doses and examined the role of autophagy in responding to this regimen. Fed male C57BL/6J mice were administered repeated doses (75mg/kg and 150mg/kg) of APAP, followed by measurement of adducts within the liver, mitochondria, and in plasma, activation of the MAP kinase JNK, and markers of liver injury. The role of autophagy was investigated by treatment of mice with the autophagy inhibitor, leupeptin. Our data show that multiple treatments at the 150mg/kg dose of APAP resulted in protein adduct formation in the liver and mitochondria, activation of JNK, and hepatocyte cell death, which was significantly exacerbated by inhibition of autophagy. While repeated dosing with the milder 75mg/kg dose did not cause mitochondrial protein adduct formation, JNK activation, or liver injury, autophagy inhibition resulted in hepatocyte death even at this lower dose. These data illustrate the importance of adaptive responses such as autophagy in removing protein adducts and preventing liver injury, especially in clinically relevant situations involving repeated dosing with APAP.

Endothelial Protease Activated Receptor 1 (PAR1) Signalling Is Required for Lymphocyte Transmigration across Brain Microvascular Endothelial Cells.

Lymphocyte transendothelial migration (TEM) relies on ICAM-1 engagement on the luminal surface of the endothelial cells (ECs). In blood–brain barrier (BBB) ECs, ICAM-1 triggers TEM signalling, including through JNK MAP kinase and AMP-activated protein kinase (AMPK), which lead to the phosphorylation and internalisation of the adherens junction protein VE-cadherin. In addition to ICAM-1, G protein-coupled receptors (GPCRs) are also required for lymphocytes TEM across BBB ECs. Here, we investigated the role of protease activated GPCRs (PARs) and found a specific role for PAR1 in support of lymphocyte TEM across BBB ECs in vitro. PAR1 requirement for TEM was confirmed using protease inhibitors, specific small molecule and peptide antagonists, function blocking antibodies and siRNA-mediated knockdown. In BBB ECs, PAR1 stimulation led to activation of signalling pathways essential to TEM; notably involving JNK and endothelial nitric oxide synthase (eNOS), with the latter downstream of AMPK. In turn, nitric oxide production through eNOS was essential for TEM by modulating VE-cadherin on Y731. Collectively, our data showed that non-canonical PAR1 activation by a lymphocyte-released serine protease is required for lymphocyte TEM across the BBB in vitro, and that this feeds into previously established ICAM-1-mediated endothelial TEM signalling pathways.

Mitogen-activated protein kinase p38 modulates pacemaker ion channels differentiation in P19-derived pluripotent cells

Signal regulators during early cardiogenetic differentiation for the cellular automaticity are largely unknown. Our investigations were designed to clarify the role of transcription factors and their modulators in P19-derived cardiomyocytes to the expression of cardiac pacemaker ion channels. Transcription factors Csx/Nkx2.5 and GATA4 but not MEF2C were markedly inhibited by p38 MAP kinase inhibition in a distinct manner; expression but not phosphorylation of GATA4 was reduced by inhibition of p38 MAP kinase actions. In the presence of an ERK1/2,5 inhibitor PD98059 or a JNK MAP kinase inhibitor SP600125, P19 cells successfully differentiated into cardiomyocytes displaying spontaneous beatings with expression of three types of pacemaker ion channels. We demonstrate that acquisition of cellular automaticity and the expression of pacemaker ion channels are regulated by the transcription factors, Csx/Nkx2.5 and GATA4, through intracellular signals including p38 MAP kinase in the process of P19-derived pluripotent cells differentiation into cardiomyocytes.

Microglial-specific depletion of TAK1 is neuroprotective in the acute phase after ischemic stroke

Transforming growth factor-β-activated kinase 1 (TAK1) is upregulated after cerebral ischemia and contributes to an aggravation of brain injury. TAK1 acts as a key regulator of NF-ΚB and the MAP kinases JNK and p38 and modulates post-ischemic neuroinflammation and apoptosis. Microglia are the main TAK1-expressing immunocompetent cells of the brain. However, little is known about the function and regulation of microglial TAK1 after cerebral ischemia. Tamoxifen-dependent conditional depletion of TAK1 in microglial cells was induced in Cx3cr1creER-Tak1fl/fl mice. The creER-negative Tak1fl/fl mice and vehicle-treated (corn oil) mice served as control groups. A transient intraluminal middle cerebral artery occlusion of 30 min followed by 6 h and 72 h of reperfusion was performed in male mice. Oxygen-glucose-deprivation (OGD) was performed with primary cortical glial cell cultures to examine the effect of microglial-specific and general (5Z-7-Oxozeaenol) TAK1 inhibition after different reperfusion times (1 h, 6 h, and 72 h). Cx3cr1creER-Tak1fl/fl mice showed reduced infarct sizes and improved neurological outcomes compared to the control group. The mRNA and protein levels of pro-inflammatory Il1b/IL-1β and Tnf/TNF-α in the peri-infarct zones of microglial-specific TAK1-depleted mice were significantly reduced. Furthermore, TAK1 depletion in vitro led to reduced cell death rates after OGD. Moreover, hypoxia-mediated activation of TAK1 and its downstream signalling proteins, JNK and p38, were dampened by microglial TAK1 depletion. In contrast, 5Z-7-Oxozeaenol-induced pharmacological inhibition of TAK1 completely diminished MAPK-signalling including the kinases JNK and p38 in all cells. Microglial TAK1 depletion abrogates post-ischemic neuroinflammation and apoptosis in the acute phase, hence might be considered as a potential target in the treatment of cerebral hypoxia.Key messagesTAK1 is activated after cerebral ischemia and induces MAP kinases p38 and JNK.Activated TAK1 increases apoptosis rate and the level pro-inflammatory cytokines IL-1β and TNF-α.Microglial cells seem to be the main source of TAK1-mediated post-ischemic neuroinflammation.Microglial-specific TAK1-depletion mediates sustainable neuroprotective effects, which might be superior to global TAK1 inhibition.

A MAP kinase‐driven mechanical stress response mediates the hypertrophic response in skeletal muscle

When cells are exposed to unfavorable conditions they cope by activating cellular stress responses. Such signaling depends on activation of the p38 and JNK MAP kinases and is crucial for both general tissue homeostasis and a range of physiological reactions. However, the mechanisms linking diverse stress stimuli to MAP kinase activation remain poorly understood.Here we show how a specific MAP3 kinase mediates activation of p38 and JNK upon hyperosmotic‐ and mechanical stress. Employing fluorescence microscopy techniques, we show that this MAP3 kinase translocates from the cytoplasm to the nuclear lamina and other detergent‐resistant structures in the nucleus upon osmotic stress induction. Here, it co‐localizes with structures rich in DNA damage response factors such as 53BP1, but void of DNA strand breaks, which were shown to constitute nuclear sensor domains for mechanical stress.Mechanical stress is experienced in skeletal muscle due to the repeated contraction of muscle fibers upon physical exercise. The signaling from these events underlie the hypertrophic response, where muscles respond to exercise by growing larger and stronger. We show that the above signaling pathway operates during exercise‐associated stimulus of C2C12 myotubes, where it is responsible for the production of a range of inflammatory transcripts and myokines (such as IL6). Consequently, in a mouse model deficient for the MAP3 kinase, we see clear signs of pathology associated with decreased muscle repair and adaptation to exercise.In conclusion, our work provides novel insight into the mechanisms by which cells sense and react to mechanical stress stimuli and identify skeletal muscle as a major target organ upon deficiency in these responses.

Bergamot Polyphenols Improve Dyslipidemia and Pathophysiological Features in a Mouse Model of Non-Alcoholic Fatty Liver Disease

There is a need for continued drug development for nonalcoholic steatohepatitis (NASH). Bergamot is a plant whose fruit juice is enriched with flavonoids and phenolic compounds which improves dyslipidemia and markers of systemic inflammation in patients with Metabolic Syndrome. The aim of this study was to perform a preclinical “proof of concept” study of Bergamot polyphenolic formulation (BPF99) for the treatment of NASH. A disease reversal study was performed in the diet-induced animal model of NAFLD (DIAMOND). Groups of 8 weeks old mice were randomly assigned to receive chow diet, high fat diet with sugar in drinking water (Western diet- WD). Mice on WD were further randomized to continue on WD gavaged with vehicle or continue on WD with additional gavage of BPF99 (50 mg/kg) after 16 weeks of diet. Mice were euthanized after 11 additional weeks. The primary endpoint was resolution of NASH. Secondary endpoints included changes in individual histological features, body weight, liver enzymes, dyslipidemia, markers of oxidative stress and molecular markers of disease activity and fibrosis. The results showed that BPF99 reduced ALT (mean 71.6 vs 44.6 IU/l, p < 0.01), triglycerides (38.8 vs 28.1 mg/dl, p < 0.05), LDL-C (39.2 vs 23.7 mg/dl, p < 0.001). It significantly improved NASH resolution (p < 0.001) and the SAF scores (p < 0.05) while the NAS improvement approached significance. BPF99 reduced markers of oxidative stress, along with reduced JNK and p38 MAP kinase activity. BPF99 did not reduce the number of mice with fibrosis but improved collagen proportional area (p < 0.04) and procollagen I and III expression. Collectively our results showed that BPF99 resolves NASH and ameliorates key histological and pathophysiological features of NASH along with improvement in ALT and dyslipidemia in the DIAMOND mice.

Inhibition of USP9X Downregulates JAK2-V617F and Induces Apoptosis Synergistically with BH3 Mimetics Preferentially in Ruxolitinib-Persistent JAK2-V617F-Positive Leukemic Cells.

JAK2-V617F plays a key role in the pathogenesis of myeloproliferative neoplasm. However, its inhibitor ruxolitinib has shown limited clinical efficacies because of the ruxolitinib-persistent proliferation of JAK2-V617F-positive cells. We here demonstrate that the USP9X inhibitor WP1130 or EOAI3402143 (G9) inhibited proliferation and induced apoptosis more efficiently in cells dependent on JAK2-V617F than on cytokine-activated JAK2. WP1130 preferentially downregulated activated and autophosphorylated JAK2-V617F by enhancing its K63-linked polyubiquitination and inducing its aggresomal translocation to block downstream signaling. Furthermore, JAK2-V617F associated physically with USP9X in leukemic HEL cells. Induction of apoptosis by inhibition of USP9X was mediated through the intrinsic mitochondria-mediated pathway, synergistically enhanced by BH3 mimetics, prevented by overexpression of Bcl-xL, and required oxidative stress to activate stress-related MAP kinases p38 and JNK as well as DNA damage responses in HEL cells. Although autophosphorylated JAK2-V617F was resistant to WP1130 in the ruxolitinib-persistent HEL-R cells, these cells expressed Bcl-2 and Bcl-xL at lower levels and showed an increased sensitivity to WP1130 as well as BH3 mimetics as compared with ruxolitinib-naive HEL cells. Thus, USP9X represents a promising target along with anti-apoptotic Bcl-2 family members for novel therapeutic strategies against JAK2-V617F-positive myeloproliferative neoplasms, particularly under the ruxolitinib persistence conditions.

Acute Heat Exposure Alters Autophagy Signaling in C2C12 Myotubes.

Autophagy is a major intracellular degradation process that is essential for the clearance of unnecessary proteins/organelles and the maintenance of cellular homeostasis. The inhibition of autophagy results in cellular consequences associated with many skeletal muscle pathologies, and therapies designed to elevate autophagic activity may provide protection from such pathologies. Acute exposure to low levels of heat has therapeutic effects; however, the impact of heat on skeletal muscle autophagy remains unclear. In the present study, C2C12 myotubes were maintained at 37°C thermoneutral (TN) or heated at 40°C heat treatment (HT) for 1 h. Myotubes were harvested immediately after heating, or returned to 37°C for recovery of 2 or 24 h. HT resulted in an elevation in pAMPK (T172), Beclin-1, and LC3 II, a marker for autophagosome formation, but no change in p62. In the context of autophagy inhibition with Bafilomycin A1, HT resulted in lower LC3 II compared to TN. The applied heat load induced the heat shock response, as evidenced by immediate upregulation of HSF1 and Hsp70. Hsp70 continued to increase during recovery, whereas pHsp27 was downregulated acutely in response to HT, but retuned to TN levels by 2 h of recovery. HT also reduced the phosphorylation of the MAP-kinases p38 and JNK. These findings suggest that an acute, short bout of mild heat may be beneficial to skeletal muscle by increasing AMPK activity, markers of autophagasome formation, and the heat shock response.

Multitasking: Dual Leucine Zipper-Bearing Kinases in Neuronal Development and Stress Management.

The dual leucine zipper-bearing kinase (DLK) and leucine zipper-bearing kinase (LZK) are evolutionarily conserved MAPKKKs of the mixed-lineage kinase family. Acting upstream of stress-responsive JNK and p38 MAP kinases, DLK and LZK have emerged as central players in neuronal responses to a variety of acute and traumatic injuries. Recent studies also implicate their function in astrocytes, microglia, and other nonneuronal cells, reflecting their expanding roles in the multicellular response to injury and in disease. Of particular note is the potential link of these kinases to neurodegenerative diseases and cancer. It is thus critical to understand the physiological contexts under which these kinases are activated, as well as the signal transduction mechanisms that mediate specific functional outcomes. In this review we first provide a historical overview of the biochemical and functional dissection of these kinases. We then discuss recent findings on regulating their activity to enhance cellular protection following injury and in disease, focusing on but not limited to the nervous system.

P3111Empagliflozin, a SGLT2 inhibitor, attenuates endothelial dysfunction and atherogenesis by inhibiting inflammatory responses in the vasculature and adipose tissue in diabetic apolipoprotein E-deficient

Abstract Background Inflammation and oxidative stress associated with hyperglycemia are major causes of vascular dysfunction and cardiovascular complications in diabetes. Recent studies reported that cardioprotective effects of sodium glucose co-transporter 2 (SGLT2) inhibitors, however underlying mechanisms are still obscure. Purpose The aim of this study was to investigate whether empagliflozin attenuates atherogenesis and endothelial dysfunction in diabetic apolipoprotein E-deficient (ApoE−/−) mice and investigated underlying mechanisms. Methods ApoE−/− mice were injected with streptozotocin (75 mg/kg) for 3 consecutive days. One week after last injection, a western type diet and administration of empagliflozin (20 mg/kg/day) or vehicle via oral gavage were started. Atherosclerotic plaque area was examined by en face Sudan IV staining. Lipid deposition and inflammatory features of atherosclerotic plaques was examined on lesions in the aortic root by immunohistochemical analysis. Vascular function was assessed by isometric tension recording. mRNA or protein expression level was examined by quantitative RT-PCR (qPCR) or western blot analysis, respectively. In in vitro experiments, murine macrophage cell line, RAW264.7, was used. Results Treatment with empagliflozin for 12 weeks significantly decreased atherosclerotic plaque size in the aortic arch compared with untreated group (p&lt;0.01). Empagliflozin reduced blood glucose (p&lt;0.001) and plasma lipid levels. Results of histological analyses revealed that empagliflozin decreased lipid deposition, macrophage accumulation, and the expression of inflammatory molecules in the aortic root. Empagliflozin treatment for 8 weeks significantly attenuated endothelial dysfunction as determined by vascular response to acetylcholine. qPCR results demonstrated that empagliflozin reduced the expression of inflammatory molecules such as MCP-1 (p&lt;0.05), ICAM-1 (p&lt;0.05) and Nox-2 (p&lt;0.05), a major NADPH oxidase subunit, in the aorta compared with the untreated group. Furthermore, empagliflozin significantly mitigated the expression of these inflammatory molecules in fat tissues around the aortic arch as determined by qPCR. In in vitro studies, methylglyoxal (MGO), a precursor of AGEs, increased the expression of inflammatory molecules (e.g., MCP-1, IL-1b and TNF-a (p&lt;0.05, respectively)) in RAW264.7 cells. MGO also significantly induced activation of JNK and p38 MAP kinase (p&lt;0.001, respectively) in this cell-type. Conclusions Empagliflozin attenuated endothelial dysfunction and atherogenesis in diabetic ApoE−/− mice. Reduction of inflammation in the vasculature and peri-vascular adipose tissues may have a role as underlying mechanisms at least partially.

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.

Regulation of apoptosis in the ischemic penumbra in the first day post-stroke.

Regulation of apoptosis in the ischemic penumbra in the first day post-stroke.

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.

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 β 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 β 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.

Transcriptomic Analysis of MAPK Signaling in NSC-34 Motor Neurons Treated with Vitamin E.

Vitamin E family is composed of different tocopherols and tocotrienols that are well-known as antioxidants but that exert also non-antioxidant effects. Oxidative stress may be involved in the progression of neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), characterized by motor neuron death. The aim of the study was the evaluation of the changes induced in the transcriptional profile of NSC-34 motor neurons treated with α-tocopherol. In particular, cells were treated for 24 h with 10 µM α-tocopherol, RNA was extracted and transcriptomic analysis was performed using Next Generation Sequencing. Vitamin E treatment modulated MAPK signaling pathway. The evaluation revealed that 34 and 12 genes, respectively belonging to “Classical MAP kinase pathway” and “JNK and p38 MAP kinase pathway”, were involved. In particular, a downregulation of the genes encoding for p38 (Log2 fold change −0.87 and −0.67) and JNK (Log2 fold change −0.16) was found. On the contrary, the gene encoding for ERK showed a higher expression in cells treated with vitamin E (Log2 fold change 0.30). Since p38 and JNK seem more involved in cell death, while ERK in cell survival, the data suggested that vitamin E treatment may exert a protective role in NSC-34 motor neurons. Moreover, Vitamin E treatment reduced the expression of the genes which encode proteins involved in mitophagy. These results indicate that vitamin E may be an efficacious therapy in preventing motor neuron death, opening new strategies for those diseases that involve motor neurons, including ALS.