JNK signaling provides a novel therapeutic target for Rett syndrome

Background Rett syndrome (RTT) is a monogenic X-linked neurodevelopmental disorder characterized by loss-of-function mutations in the MECP2 gene, which lead to structural and functional changes in synapse communication, and impairments of neural activity at the basis of cognitive deficits that progress from an early age. While the restoration of MECP2 in animal models has been shown to rescue some RTT symptoms, gene therapy intervention presents potential side effects, and with gene- and RNA-editing approaches still far from clinical application, strategies focusing on signaling pathways downstream of MeCP2 may provide alternatives for the development of more effective therapies in vivo. Here, we investigate the role of the c-Jun N-terminal kinase (JNK) stress pathway in the pathogenesis of RTT using different animal and cell models and evaluate JNK inhibition as a potential therapeutic approach. Results We discovered that the c-Jun N-terminal kinase (JNK) stress pathway is activated in Mecp2-knockout, Mecp2-heterozygous mice, and in human MECP2-mutated iPSC neurons. The specific JNK inhibitor, D-JNKI1, promotes recovery of body weight and locomotor impairments in two mouse models of RTT and rescues their dendritic spine alterations. Mecp2-knockout presents intermittent crises of apnea/hypopnea, one of the most invalidating RTT pathological symptoms, and D-JNKI1 powerfully reduces this breathing dysfunction. Importantly, we discovered that also neurons derived from hiPSC-MECP2 mut show JNK activation, high-phosphorylated c-Jun levels, and cell death, which is not observed in the isogenic control wt allele hiPSCs. Treatment with D-JNKI1 inhibits neuronal death induced by MECP2 mutation in hiPSCs mut neurons. Conclusions As a summary, we found altered JNK signaling in models of RTT and suggest that D-JNKI1 treatment prevents clinical symptoms, with coherent results at the cellular, molecular, and functional levels. This is the first proof of concept that JNK plays a key role in RTT and its specific inhibition offers a new and potential therapeutic tool to tackle RTT.

Noncanonical Wnt5a/JNK Signaling Contributes to the Development of D-Gal/LPS-Induced Acute Liver Failure

Acute liver failure (ALF) is a deadly clinical disorder with few effective treatments and unclear pathogenesis. In our previous study, we demonstrated that aberrant Wnt5a expression was involved in acute on chronic liver failure. However, the role of Wnt5a in ALF is unknown. We investigated the expression of Wnt5a and its downstream signaling of c-jun N-terminal kinase (JNK) in a mouse model of ALF established by co-injection of D-galactosamine (D-Gal) and lipopolysaccharide (LPS) in C57BL/6 mice. We also investigated the role of Box5, a Wnt5a antagonist in vivo. Moreover, the effect of Wnt5a/JNK signaling on downstream inflammatory cytokines expression, phagocytosis and migration in THP-1 macrophages was studied in vitro. Aberrant Wnt5a expression and JNK activation were detected in D-Gal/LPS-induced ALF mice. Box5 pretreatment reversed JNK activation, and eventually decreased the mortality rate of D-Gal/LPS-treated mice with reduced hepatic necrosis and apoptosis, serum ALT and AST levels, and liver inflammatory cytokines expression, although the last was not significant. We further demonstrated that recombined Wnt5a (rWnt5a) induced tumor necrosis α (TNF-α) and Interleukin-6 (IL-6) mRNA expression, and increased the phagocytosis ability of THP-1 macrophages in a JNK-dependent manner, which could be restored by Box5. In addition, rWnt5a-induced migration of THP-1 macrophages was also turned by Box5. Our findings suggested that Wnt5a/JNK signaling play important role in the development of ALF, and Box5 could have particular hepatoprotecive effects in ALF.

Nicotinamide riboside kinase-2 inhibits JNK pathway and limits dilated cardiomyopathy in mice with chronic pressure overload

Nicotinamide riboside kinase-2 (NRK-2) has recently emerged as a critical regulator of cardiac remodeling however, underlying molecular mechanisms is largely unknown. To explore the same, NRK2 knockout (KO) and littermate control mice were subjected to trans-aortic constriction (TAC) or sham surgeries and cardiac function was assessed by serial M-mode echocardiography. A mild cardiac contractile dysfunction was observed in the KOs at the early adaptive phase of remodeling followed by a significant deterioration during the maladaptive cardiac remodeling phase. Consistently, NRK2 KO hearts displayed increased cardiac hypertrophy and heart failure reflected by morphometric parameters as well as increased fetal genes ANP and BNP expressions. Histological assessment revealed an extensive left ventricular (LV) chamber dilatation accompanied by elevated cardiomyopathy and fibrosis in the KO hearts post-TAC. In a gain-of-function model, NRK-2 overexpressing in AC16 cardiomyocytes displayed significantly attenuated fetal genes ANP and BNP expression. Consistently, NRK-2 overexpression attenuated angiotensin II- induced cardiomyocyte death. Mechanistically, we identified NRK-2 as a critical regulator of JNK MAP kinase where NRK-2 overexpression in human cardiomyocytes markedly suppressed the angiotensin II- induced JNK activation. Thus, our results demonstrate that NRK-2 plays protective roles in pressure overload- induced dilatative cardiac remodeling and, genetic ablation exacerbates dilated cardiomyopathy, interstitial collagen deposition, and cardiac dysfunction post-TAC due, in part, to increased JNK activation.

Berberine Induces NSCLC Apoptosis Via Activation of ROS/ASK1/JNK Pathway in Vitro and Vivo

BackgroundNon-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. Berberine (BBR), as an isoquinoline alkaloid, is commonly utilized in traditional Chinese medicine. Previous studies have proven that BBR possesses potential anti-tumor effect. However, the mechanism of on mitochondrial function in anti-NSCLC are still unknown.MethodsCell Counting Kit-8 (CCK-8), flow cytometry and western blotting were utilized to characterize the roles and relationships among BBR, ROS, ASK1, JNK, coxIV,caspase-3, cytochrome c ,bcl-2 and bax in NSCLC. Immunohistochemical (IHC) analysis was built to examine their expression in vivo.ResultsIn this study, we found that BBR potently suppressed NSCLC cells (A549 and PC9) growth by inducing apoptosis in a dose- and time-dependent manner. BBR induced apoptosis in NSCLC as evidenced by caspase-3 cleavage, cytochrome c release, and mitochondrial membrane depolarization. Furthermore, BBR induced ROS generation and ASK1 and JNK activation. To explore whether such apoptosis was linked to ROS production and ASK1 and JNK activation, we treated cells with a JNK inhibitor (SP600125), which significantly suppressed BBR-induced apoptosis. We further found that treating these cells with the anti-oxidant N-acetyl cysteine (NAC) was sufficient to both suppress ASK1 and JNK activation and to disrupt apoptotic induction.ConclusionsTogether, these data suggest that BBR induces NSCLC cells apoptosis via ROS-mediated ASK1/JNK and mitochondrial pathway activation.

Huganbuzure Granule Attenuates Concanavalin-A-Induced Immune Liver Injury in Mice via Regulating the Balance of Th1/Th2/Th17/Treg Cells and Inhibiting Apoptosis

In Uygur medicine, Huganbuzure granule (HBG) is one of the classical prescriptions for liver protection. However, its role in immune liver injury remains unknown. This study evaluates the effect of HBG on concanavalin-A- (ConA-) induced immune liver injury and investigates its protective underlying mechanism. BALB/c mice were randomly divided into five groups (n = 24 mice per group): control, ConA, 1.6 g/kg HBG + ConA, 3.2 g/kg HBG + ConA, and 6 mg/kg prednisolone + ConA. HBG was intragastrically administrated once daily for ten consecutive days, prior to ConA (20 mg/kg) injection. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), superoxide dismutase (SOD), and malondialdehyde (MDA) in mouse serum were measured after ConA injection. Moreover, liver-related mRNA levels were evaluated by qPCR. The detection of liver-related proteins was assessed by immunohistochemistry and western blot analysis. Compared with the ConA group, HBG reduced the mRNA expression of IL-17A and IFN-γ and the protein expression of T-bet and ROR-γt. In addition, HBG increased the mRNA expression of IL-4 and TGF-β and protein expression of GATA3 and Foxp3, indicating that HBG regulated the balance of Th1/Th2 and Th17/Treg. Furthermore, HBG alleviated immune liver injury by reducing oxidative stress, inhibiting apoptosis, and decreasing the expression of p-JNK, p-ERK, p-p38, p-JAK1, p-STAT1, p-STAT3, and IRF1. Our data suggested that HBG attenuated ConA-induced immune liver injury by regulating the immune balance and inhibiting JAK1/STATs/IRF1 signaling, thereby reducing apoptosis induced by JNK activation. The findings indicate that HBG may be a promising drug for immune liver injury.

Abstract P326: Nicotinamide Riboside Kinase-2 Deficiency Promotes Pressure Overload- Induced Dilated Cardiomyopathy And Heart Failure

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure with a poor prognosis. Nicotinamide riboside kinase-2 (NRK-2), a muscle-specific β1 integrin-binding protein, is predominantly expressed in skeletal muscle and upregulates in several heart failure models. Emerging pieces of evidence suggest that NRK-2 plays a key role in cardiac pathogenesis, however, its role in chronic pressure overload (PO)-induced cardiac remodeling is largely unknown. To investigate the potential role of NRK-2 in PO-induced DCM and delineate the underlying molecular mechanisms, NRK2 knockout (KO) and littermate control mice were subjected to trans-aortic constriction (TAC) or sham surgeries and cardiac function was assessed by serial M-mode echocardiography. A mild cardiac contractile dysfunction was observed in the KOs at the early adaptive phase of remodeling followed by a significant LV chamber dilatation [LVIDd(mm); 5.32±0.36 vs. 4.75±0.39, P =0.001] and functional deterioration [LVEF(%); 20.53±8.81vs. 31.2±7.0, P =0.003] during the maladaptive cardiac remodeling phase (6 week). Consistently, NRK2 KO hearts displayed increased cardiac hypertrophy and heart failure reflected by morphometric parameters as well as increased fetal genes ANP and BNP expressions. Histological assessment revealed an extensive left ventricular (LV) chamber dilatation accompanied by elevated cardiomyopathy and fibrosis in the KO hearts post-TAC. In a gain-of-function model, NRK-2 overexpressing AC16 cardiomyocytes displayed significantly attenuated fetal genes expression and, NRK-2 further suppressed the fetal gene expression when challenged with angiotensin II. Consistently, NRK-2 overexpression attenuated angiotensin II-induced cardiomyocyte death. Mechanistically, NRK-2 was identified as a critical regulator of JNK MAP kinase and NRK-2 overexpression markedly suppressed the angiotensin II-induced JNK activation. Overall, our results demonstrate that NRK-2 regulates dilatative cardiac remodeling and, genetic ablation exacerbates dilated cardiomyopathy, interstitial collagen deposition, and cardiac dysfunction post-TAC due, in part, to increased JNK activation.

Mitogen Kinase Kinase (MKK7) Controls Cytokine Production In Vitro and In Vivo in Mice

Mitogen kinase kinase 4 (MKK4) and mitogen kinase kinase 7 (MKK7) are members of the MAP2K family that can activate downstream mitogen-activated protein kinases (MAPKs). MKK4 has been implicated in the activation of both c-Jun N-terminal kinase (JNK) and p38 MAPK, while MKK7 has been reported to activate only JNK in response to different stimuli. The stimuli, as well as the cell type determine which MAP2K member will mediate a given response. In various cell types, MKK7 contributes to the activation of downstream MAPKs, JNK, which is known to regulate essential cellular processes, such as cell death, differentiation, stress response, and cytokine secretion. Previous studies have also implicated the role of MKK7 in stress signaling pathways and cytokine production. However, little is known about the degree to which MKK4 and MKK7 contribute to innate immune responses in macrophages or during inflammation in vivo. To address this question and to elucidate the role of MKK4 and MKK7 in macrophage and in vivo, we developed MKK4- and MKK7-deficient mouse models with tamoxifen-inducible Rosa26 CreERT. This study reports that MKK7 is required for JNK activation both in vitro and in vivo. Additionally, we demonstrated that MKK7 in macrophages is necessary for lipopolysaccharide (LPS)-induced cytokine production, M1 polarization, and migration, which appear to be a major contributor to the inflammatory response in vivo. Conversely, MKK4 plays a significant, but minor role in cytokine production in vivo.

JNK activation in TA and EDL muscle is load-dependent in rats receiving identical excitation patterns

As the excitation–contraction coupling is inseparable during voluntary exercise, the relative contribution of the mechanical and neural input on hypertrophy-related molecular signalling is still poorly understood. Herein, we use a rat in-vivo strength exercise model with an electrically-induced standardized excitation pattern, previously shown to induce a load-dependent increase in myonuclear number and hypertrophy, to study acute effects of load on molecular signalling. We assessed protein abundance and specific phosphorylation of the four protein kinases FAK, mTOR, p70S6K and JNK after 2, 10 and 28 min of a low- or high-load contraction, in order to assess the effects of load, exercise duration and muscle-type on their response to exercise. Specific phosphorylation of mTOR, p70S6K and JNK was increased after 28 min of exercise under the low- and high-load protocol. Elevated phosphorylation of mTOR and JNK was detectable already after 2 and 10 min of exercise, respectively, but greatest after 28 min of exercise, and JNK phosphorylation was highly load-dependent. The abundance of all four kinases was higher in TA compared to EDL muscle, p70S6K abundance was increased after exercise in a load-independent manner, and FAK and JNK abundance was reduced after 28 min of exercise in both the exercised and control muscles. In conclusion, the current study shows that JNK activation after a single resistance exercise is load-specific, resembling the previously reported degree of myonuclear accrual and muscle hypertrophy with repetition of the exercise stimulus.

Cytoskeleton and Associated Proteins: Pleiotropic JNK Substrates and Regulators

This review extensively reports data from the literature concerning the complex relationships between the stress-induced c-Jun N-terminal kinases (JNKs) and the four main cytoskeleton elements, which are actin filaments, microtubules, intermediate filaments, and septins. To a lesser extent, we also focused on the two membrane-associated cytoskeletons spectrin and ESCRT-III. We gather the mechanisms controlling cytoskeleton-associated JNK activation and the known cytoskeleton-related substrates directly phosphorylated by JNK. We also point out specific locations of the JNK upstream regulators at cytoskeletal components. We finally compile available techniques and tools that could allow a better characterization of the interplay between the different types of cytoskeleton filaments upon JNK-mediated stress and during development. This overview may bring new important information for applied medical research.