Akhilesh Kumar

The TWEAK–Fn14 system is a critical regulator of denervation-induced skeletal muscle atrophy in mice

The TNF-related cytokine TWEAK promotes skeletal muscle atrophy that is associated with classical disuse syndromes.

Genomic Profiling of Messenger RNAs and MicroRNAs Reveals Potential Mechanisms of TWEAK-Induced Skeletal Muscle Wasting in Mice

Background Skeletal muscle wasting is a devastating complication of several physiological and pathophysiological conditions. Inflammatory cytokines play an important role in the loss of skeletal muscle mass in various chronic diseases. We have recently reported that proinflammatory cytokine TWEAK is a major muscle-wasting cytokine. Emerging evidence suggests that gene expression is regulated not only at transcriptional level but also at post-transcriptional level through the expression of specific non-coding microRNAs (miRs) which can affect the stability and/or translation of target mRNA. However, the role of miRs in skeletal muscle wasting is unknown. Methodology/Principal Findings To understand the mechanism of action of TWEAK in skeletal muscle, we performed mRNA and miRs expression profile of control and TWEAK-treated myotubes. TWEAK increased the expression of a number of genes involved in inflammatory response and fibrosis and reduced the expression of few cytoskeletal gene (e.g. Myh4, Ankrd2, and TCap) and metabolic enzymes (e.g. Pgam2). Low density miR array demonstrated that TWEAK inhibits the expression of several miRs including muscle-specific miR-1-1, miR-1-2, miR-133a, miR-133b and miR-206. The expression of a few miRs including miR-146a and miR-455 was found to be significantly increased in response to TWEAK treatment. Ingenuity pathway analysis showed that several genes affected by TWEAK are known/putative targets of miRs. Our cDNA microarray data are consistent with miRs profiling. The levels of specific mRNAs and miRs were also found to be similarly regulated in atrophying skeletal muscle of transgenic mice (Tg) mice expressing TWEAK. Conclusions/Significance Our results suggest that TWEAK affects the expression of several genes and microRNAs involved in inflammatory response, fibrosis, extracellular matrix remodeling, and proteolytic degradation which might be responsible for TWEAK-induced skeletal muscle loss.

Genetic Ablation of TWEAK Augments Regeneration and Post-Injury Growth of Skeletal Muscle in Mice

Impairment in the regeneration process is a critical determinant for skeletal muscle wasting in chronic diseases and degenerative muscle disorders. Inflammatory cytokines are known to cause significant muscle wasting, however, their role in myofiber regeneration is less clear. In this study we have investigated the role of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) in skeletal muscle regeneration in vivo. Our results show that expression levels of TWEAK and its receptor Fn14 are significantly increased in skeletal muscles of mice after injury. Genetic deletion of TWEAK increased the fiber cross-sectional area and levels of embryonic isoform of myosin heavy chain in regenerating tibial anterior muscle. Conversely, muscle-specific transgenic overexpression of TWEAK reduced the fiber cross-sectional area and levels of the embryonic myosin heavy chain in regenerating muscle. TWEAK induced the expression of several inflammatory molecules and increased interstitial fibrosis in regenerating muscle. Genetic ablation of TWEAK suppressed, whereas overexpression of TWEAK increased, the activation of nuclear factor-kappa B without affecting the activation of Akt or p38 kinase in regenerating myofibers. Primary myoblasts from TWEAK-null mice showed enhanced differentiation in vitro, whereas myoblasts from TWEAK-Tg mice showed reduced differentiation compared with wild-type mice. Collectively, our study suggests that TWEAK negatively regulates muscle regeneration and that TWEAK is a potential therapeutic target to enhance skeletal muscle regeneration in vivo.

Matrix Metalloproteinase Inhibitor Batimastat Alleviates Pathology and Improves Skeletal Muscle Function in Dystrophin-Deficient mdx Mice

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, involves severe muscle degeneration, inflammation, fibrosis, and early death in afflicted boys. Matrix metalloproteinases (MMPs) are extracellular proteases that cause tissue degradation in several disease states. In this study, we tested the hypothesis that the expression levels of various MMPs are abnormally increased and that their inhibition will ameliorate muscle pathogenesis in animal models of DMD. Our results show that the transcript levels of several MMPs are significantly up-regulated, whereas tissue inhibitors of MMPs are down-regulated, in dystrophic muscle of mdx mice. Chronic administration of batimastat (BB-94), a broad spectrum peptide inhibitor of MMPs, reduced necrosis, infiltration of macrophages, centronucleated fibers, and the expression of embryonic myosin heavy chain in skeletal muscle of mdx mice. Batimastat also reduced the expression of several inflammatory molecules and augmented the levels of sarcolemmal protein beta-dystroglycan and neuronal nitric oxide in mdx mice. In addition, muscle force production in isometric contraction was increased in batimastat-treated mdx mice compared with those treated with vehicle alone. Furthermore, inhibition of MMPs using batimastat reduced the activation of mitogen-activated protein kinases and activator protein-1 in myofibers of mdx mice. Our study provides the novel evidence that the expression of MMPs is atypically increased in DMD, that their inhibition ameliorates pathogenesis, and that batimastat could prove to be a significant candidate for DMD therapy.

GSK3β and the control of infectious bacterial diseases

Glycogen synthase kinase 3β (GSK3β) has been shown to be a crucial mediator of the intensity and direction of the innate immune system response to bacterial stimuli. This review focuses on: (i) the central role of GSK3β in the regulation of pathogen-induced inflammatory responses through the regulation of pro- and anti-inflammatory cytokine production, (ii) the extensive ongoing efforts to exploit GSK3β for its therapeutic potential in the control of infectious diseases, and (iii) the increasing evidence that specific pathogens target GSK3β-related pathways for immune evasion. A better understanding of complex bacteria-GSK3β interactions is likely to lead to more effective anti-inflammatory interventions and novel targets to circumvent pathogen colonization and survival.

Effects of palmitoylethanolamide on aqueous humor outflow.

PURPOSE To study the effects of palmitoylethanolamide (PEA), a fatty acid ethanolamide, on aqueous humor outflow facility. METHODS The effects of PEA on outflow facility were measured using a porcine anterior segment-perfused organ culture model. The involvements of different receptors in PEA-induced changes were investigated using receptor antagonists and adenovirus delivered small hairpin RNAs (shRNAs). PEA-induced activation of p42/44 mitogen-activated protein kinase (MAPK) was determined by Western blot analysis using an antiphospho p42/44 MAPK antibody. RESULTS PEA caused a concentration-dependent enhancement of outflow facility, with the maximum effect (151.08 ± 11.12% of basal outflow facility) achieved at 30 nM of PEA. Pretreatment of anterior segments with 1 μM cannabinoid receptor 2 antagonist SR144528 and 1 μM PPARα antagonist GW6471, but not 1 μM cannabinoid receptor 1 antagonist SR141716A, produced a partial antagonism on the PEA-induced increase of outflow facility. Treatment of TM cells with PEA for 10 minutes activated phosphorylation of p42/44 MAPK, which was blocked by pretreatment with SR1444528 and GW6471, but not SR141716A. Knocking down the expression of either GPR55 or PPARα receptors with specific shRNAs for these receptors partially blocked PEA-induced increase in outflow facility and abolished PEA-induced phosphorylation of p42/44 MAPK. PD98059, an inhibitor of the p42/44 MAPK pathway, blocked both PEA-induced enhancement of aqueous humor outflow facility and PEA-induced phosphorylation of p42/44 MAPK. CONCLUSIONS Our results demonstrate that PEA increases aqueous humor outflow through the TM pathway and these effects are mediated by GPR55 and PPARα receptors through activation of p42/44 MAPK.

Structure-activity relationships of fatty acid amide ligands in activating and desensitizing G protein-coupled receptor 119

The purpose of the current study was to apply a high throughput assay to investigate the structure-activity relationships of fatty acid amides for activating and desensitizing G protein-coupled receptor 119, a promising therapeutic target for both type 2 diabetes and obesity. A cell-based, homogenous time resolved fluorescence (HTRF) method for measuring G protein-coupled receptor 119-mediated increase of cyclic adenosine monophosphate (cAMP) levels was validated and applied in this study. Using novel fatty acid amides and detailed potency and efficacy analyses, we have demonstrated that degree of saturation in acyl chain and charged head groups of fatty acid amides have profound effects on the ability of these compounds to activate G protein-coupled receptor 119. In addition, we have demonstrated for the first time that pretreatments with G protein-coupled receptor 119 agonists desensitize the receptor and the degrees of desensitization caused by fatty acid amides correlate well with their structure-activity relationships in activating the receptor.

A General Method for Targeted Quantitative Cross-Linking Mass Spectrometry.

Chemical cross-linking mass spectrometry (XL-MS) provides protein structural information by identifying covalently linked proximal amino acid residues on protein surfaces. The information gained by this technique is complementary to other structural biology methods such as x-ray crystallography, NMR and cryo-electron microscopy[1]. The extension of traditional quantitative proteomics methods with chemical cross-linking can provide information on the structural dynamics of protein structures and protein complexes. The identification and quantitation of cross-linked peptides remains challenging for the general community, requiring specialized expertise ultimately limiting more widespread adoption of the technique. We describe a general method for targeted quantitative mass spectrometric analysis of cross-linked peptide pairs. We report the adaptation of the widely used, open source software package Skyline, for the analysis of quantitative XL-MS data as a means for data analysis and sharing of methods. We demonstrate the utility and robustness of the method with a cross-laboratory study and present data that is supported by and validates previously published data on quantified cross-linked peptide pairs. This advance provides an easy to use resource so that any lab with access to a LC-MS system capable of performing targeted quantitative analysis can quickly and accurately measure dynamic changes in protein structure and protein interactions.

Involvement of a non-CB1/CB2 cannabinoid receptor in the aqueous humor outflow-enhancing effects of abnormal-cannabidiol

The purpose of this study was to investigate the effects of abnormal-cannabidiol (abn-cbd), a non-psychoactive cannabinoid agonist, on aqueous humor outflow via the trabecular meshwork (TM) of porcine eye, and to examine the involvement of a non-CB1/CB2 cannabinoid receptor and the p42/44 mitogen-activated protein kinase (p42/44 MAPK) pathway. The effects of abn-cbd on aqueous humor outflow were measured using a porcine anterior segment perfused organ culture model. The activation of p42/44 MAPK by abn-cbd was determined in cultured TM cells with western blot analysis using an anti-phospho-p42/44 MAPK antibody. Administration of abn-cbd caused a concentration-dependent enhancement of aqueous humor outflow facility with a maximum effect (155.0 ± 11.7% of basal outflow facility) after administration of 30 nM abn-cbd. Pretreatment with 1 μM of O-1918, a cannabidiol analog that acts as a selective antagonist at the non-CB1/CB2 receptor, produced a full antagonism of 30 nM abn-cbd induced increase of aqueous humor outflow facility. Pretreatment with 1 μM of CB1 antagonist SR141716A partially blocked, whereas pretreatment with either 1 μM of CB1 antagonist AM251 or 1 μM of CB2 antagonist SR144528 had no effect on abn-cbd induced enhancement of outflow facility. Treatment of TM cells with 30 nM of abn-cbd activated p42/44 MAPK, which was blocked completely by pretreatment with O-1918, and partially by pretreatment with SR141716A, but not by either AM251 or SR144528. In addition, PD98059, an inhibitor of p42/44 MAPK pathway, blocked completely the abn-cbd induced p42/44 MAPK activation and blocked partially the abn-cbd induced enhancement of outflow facility. In conclusion, the results from this study demonstrate that abn-cbd increases aqueous humor outflow through the TM pathway of the eye, and this effect is mediated by a non-CB1/CB2 cannabinoid receptor, with an involvement of p42/44 MAPK signaling pathway.

Nicotinic augmentation of anti-inflammatory GSK3b signaling

Background Glycogen synthesis kinase 3b (GSK3b) has been shown to be a critical mediator of the intensity and direction of the innate immune system responding to bacterial stimuli. Stimulation of the anti-cholinergic anti-inflammatory system by tobacco alkaloids (nicotine; cotinine) leads to phosphorylation and inactivation of GSK3b and, subsequently, to immune suppression. This presentation will review the tobacco-induced dysregulation of GSK3b signaling and provide insight into the increased susceptibility of smokers to multiple bacterial diseases, including those caused by Mycobacterium tuberculosis, Legionella pneumophila, and Neisseria meningitidis. The extensive ongoing efforts to exploit GSK3b for its therapeutic potential in the control of infectious diseases will also be reviewed.