The 5HT2b Receptor in Alzheimer's Disease: Increased Levels in Patient Brains and Antagonist Attenuation of Amyloid and Tau Induced Dysfunction.

BACKGROUND: Background: Neurodegenerative diseases manifest behavioral dysfunction with disease progression. Intervention with neuropsychiatric drugs is part of most multi-drug treatment paradigms. However, only a fraction of patients responds to the treatments and those responding must deal with drug-drug interactions and tolerance issues generally attributed to off-target activities. Recent efforts have focused on the identification of underexplored targets and exploration of improved outcomes by treatment with selective molecular probes. Objective: As part of ongoing efforts to identify and validate additional targets amenable to therapeutic intervention, we examined levels of the serotonin 5-HT2b receptor (5-HT2bR) in Alzheimer's disease (AD) brains and the potential of a selective 5-HT2bR antagonist to counteract synaptic plasticity and memory damage induced by AD-related proteins, amyloid-ß, and tau. Methods: This work used a combination of biochemical, chemical biology, electrophysiological, and behavioral techniques. Biochemical methods included analysis of protein levels. Chemical biology methods included the use of an in vivo molecular probe MW071, a selective antagonist for the 5HT2bR. Electrophysiological methods included assessment of long-term potentiation (LTP), a type of synaptic plasticity thought to underlie memory formation. Behavioral studies investigated spatial memory and associative memory. Results: 5HT2bR levels are increased in brain specimens of AD patients compared to controls. 5HT2bR antagonist treatment rescued amyloid-ß and tau oligomer-induced impairment of synaptic plasticity and memory. Conclusions: The increased levels of 5HT-2bR in AD patient brains and the attenuation of disease-related synaptic and behavioral dysfunctions by MW071 treatment suggest that the 5HT-2bR is a molecular target worth pursuing as a potential therapeutic target.

PI3Kγ stimulates a high molecular weight form of myosin light chain kinase to promote myeloid cell adhesion and tumor inflammation.

Myeloid cells play key roles in cancer immune suppression and tumor progression. In response to tumor derived factors, circulating monocytes and granulocytes extravasate into the tumor parenchyma where they stimulate angiogenesis, immune suppression and tumor progression. Chemokines, cytokines and interleukins stimulate PI3Kγ-mediated Rap1 activation, leading to conformational changes in integrin α4ß1 that promote myeloid cell extravasation and tumor inflammation Here we show that PI3Kγ activates a high molecular weight form of myosin light chain kinase, MLCK210, that promotes myosin-dependent Rap1 GTP loading, leading to integrin α4ß1 activation. Genetic or pharmacological inhibition of MLCK210 suppresses integrin α4ß1 activation, as well as tumor inflammation and progression. These results demonstrate a critical role for myeloid cell MLCK210 in tumor inflammation and serve as basis for the development of alternative approaches to develop immune oncology therapeutics.

Therapeutic treatment with the anti-inflammatory drug candidate MW151 may partially reduce memory impairment and normalizes hippocampal metabolic markers in a mouse model of comorbid amyloid and vascular pathology.

Alzheimer's disease (AD) is the leading cause of dementia in the elderly, but therapeutic options are lacking. Despite long being able to effectively treat the ill-effects of pathology present in various rodent models of AD, translation of these strategies to the clinic has so far been disappointing. One potential contributor to this situation is the fact that the vast majority of AD patients have other dementia-contributing comorbid pathologies, the most common of which are vascular in nature. This situation is modeled relatively infrequently in basic AD research, and almost never in preclinical studies. As part of our efforts to develop small molecule, anti-inflammatory therapeutics for neurological injury and disease, we have recently been exploring potentially promising treatments in preclinical multi-morbidity contexts. In the present study, we generated a mouse model of mixed amyloid and hyperhomocysteinemia (HHcy) pathology in which to test the efficacy of one of our anti-inflammatory compounds, MW151. HHcy can cause cerebrovascular damage and is an independent risk factor for both AD dementia and vascular contributions to cognitive impairment and dementia. We found that MW151 was able to partially rescue hippocampal-dependent spatial memory and learning deficits in this comorbidity context, and further, that the benefit is associated with a normalization of hippocampal metabolites detectable via magnetic resonance spectroscopy. These findings provide evidence that MW151 in particular, and potentially anti-inflammatory treatment more generally, may be beneficial in AD patients with comorbid vascular pathology.

Phosphorylation-dependent subcellular redistribution of small myosin light chain kinase.

BACKGROUND: Myosin light chain kinase (MLCK) is a Ca2+-calmodulin-dependent enzyme dedicated to phosphorylate and activate myosin II to provide force for various motile processes. In smooth muscle cells and many other cells, small MLCK (S-MLCK) is a major isoform. S-MLCK is an actomyosin-binding protein firmly attached to contractile machinery in smooth muscle cells. Still, it can leave this location and contribute to other cellular processes. However, molecular mechanisms for switching the S-MLCK subcellular localization have not been described. METHODS: Site-directed mutagenesis and in vitro protein phosphorylation were used to study functional roles of discrete in-vivo phosphorylated residues within the S-MLCK actin-binding domain. In vitro co-sedimentation analysis was applied to study the interaction of recombinant S-MLCK actin-binding fragment with filamentous actin. Subcellular distribution of phosphomimicking S-MLCK mutants was studied by fluorescent microscopy and differential cell extraction. RESULTS: Phosphorylation of S-MLCK actin-binding domain at Ser25 and/or Thr56 by proline-directed protein kinases or phosphomimicking these posttranslational modifications alters S-MLCK binding to actin filaments both in vitro and in cells, and induces S-MLCK subcellular translocation with no effect on the enzyme catalytic properties. CONCLUSIONS: Phosphorylation of the amino terminal actin-binding domain of S-MLCK renders differential subcellular targeting of the enzyme and may, thereby, contribute to a variety of context-dependent responses of S-MLCK to cellular and tissue stimuli. GENERAL SIGNIFICANCE: S-MLCK physiological function can potentially be modulated via phosphorylation of its actin recognition domain, a regulation distinct from the catalytic and calmodulin regulatory domains.

First-in-Human Studies of MW01-6-189WH, a Brain-Penetrant, Antineuroinflammatory Small-Molecule Drug Candidate: Phase 1 Safety, Tolerability, Pharmacokinetic, and Pharmacodynamic Studies in Healthy Adult Volunteers.

MW01-6-189WH (MW189) is a novel central nervous system-penetrant small-molecule drug candidate that selectively attenuates stressor-induced proinflammatory cytokine overproduction and is efficacious in intracerebral hemorrhage and traumatic brain injury animal models. We report first-in-human, randomized, double-blind, placebo-controlled phase 1 studies to evaluate the safety, tolerability, and pharmacokinetics (PK) of single and multiple ascending intravenous doses of MW189 in healthy adult volunteers. MW189 was safe and well tolerated in single and multiple doses up to 0.25 mg/kg, with no clinically significant concerns. The most common drug-related treatment-emergent adverse event was infusion-site reactions, likely related to drug solution acidity. No clinically concerning changes were seen in vital signs, electrocardiograms, physical or neurological examinations, or safety laboratory results. PK analysis showed dose-proportional increases in plasma concentrations of MW189 after single or multiple doses, with approximately linear kinetics and no significant drug accumulation. Steady state was achieved by dose 3 for all dosing cohorts. A pilot pharmacodynamic study administering low-dose endotoxin to induce a systemic inflammatory response was done to evaluate the effects of a single intravenous dose of MW189 on plasma cytokine levels. MW189 treatment resulted in lower levels of the proinflammatory cytokine TNF-α and higher levels of the anti-inflammatory cytokine IL-10 compared with placebo treatment. The outcomes are consistent with the pharmacological mechanism of MW189. Overall, the safety profile, PK properties, and pharmacodynamic effect support further development of MW189 for patients with acute brain injury.

Stasimon Contributes to the Loss of Sensory Synapses and Motor Neuron Death in a Mouse Model of Spinal Muscular Atrophy.

Reduced expression of the survival motor neuron (SMN) protein causes the neurodegenerative disease spinal muscular atrophy (SMA). Here, we show that adeno-associated virus serotype 9 (AAV9)-mediated delivery of Stasimon-a gene encoding an endoplasmic reticulum (ER)-resident transmembrane protein regulated by SMN-improves motor function in a mouse model of SMA through multiple mechanisms. In proprioceptive neurons, Stasimon overexpression prevents the loss of afferent synapses on motor neurons and enhances sensory-motor neurotransmission. In motor neurons, Stasimon suppresses neurodegeneration by reducing phosphorylation of the tumor suppressor p53. Moreover, Stasimon deficiency converges on SMA-related mechanisms of p53 upregulation to induce phosphorylation of p53 through activation of p38 mitogen-activated protein kinase (MAPK), and pharmacological inhibition of this kinase prevents motor neuron death in SMA mice. These findings identify Stasimon dysfunction induced by SMN deficiency as an upstream driver of distinct cellular cascades that lead to synaptic loss and motor neuron degeneration, revealing a dual contribution of Stasimon to motor circuit pathology in SMA.

Genetic knockout of myosin light chain kinase (MLCK210) prevents cerebral microhemorrhages and attenuates neuroinflammation in a mouse model of vascular cognitive impairment and dementia.

The blood-brain barrier (BBB) is critical in maintenance of brain homeostasis, and loss of its functional integrity is a key feature across a broad range of neurological insults. This includes both acute injuries such as traumatic brain injury and stroke, as well as more chronic pathologies associated with aging, such as vascular cognitive impairment and dementia (VCID). A specific form of myosin light chain kinase (MLCK210) is a major regulator of barrier integrity in general, including the BBB. Studies have demonstrated the potential of MLCK210 as a therapeutic target for peripheral disorders involving tissue barrier dysfunction, but less is known about its potential as a target for chronic neurologic disorders. We report here that genetic knockout (KO) of MLCK210 protects against cerebral microhemorrhages and neuroinflammation induced by chronic dietary hyperhomocysteinemia. Overall, the results are consistent with an accumulating body of evidence supporting MLCK210 as a potential therapeutic target for tissue barrier dysfunction and specifically implicate it in BBB dysfunction and neuroinflammation in a model of VCID.

A Selective and Brain Penetrant p38αMAPK Inhibitor Candidate for Neurologic and Neuropsychiatric Disorders That Attenuates Neuroinflammation and Cognitive Dysfunction.

The p38αMAPK is a serine/threonine protein kinase and a key node in the intracellular signaling networks that transduce and amplify stress signals into physiological changes. A preponderance of preclinical data and clinical observations established p38αMAPK as a brain drug discovery target involved in neuroinflammatory responses and synaptic dysfunction in multiple degenerative and neuropsychiatric brain disorders. We summarize the discovery of highly selective, brain-penetrant, small molecule p38αMAPK inhibitors that are efficacious in diverse animal models of neurologic disorders. A crystallography and pharmacoinformatic approach to fragment expansion enabled the discovery of an efficacious hit. The addition of secondary pharmacology screens to refinement delivered lead compounds with improved selectivity, appropriate pharmacodynamics, and efficacy. Safety considerations and additional secondary pharmacology screens drove optimization that delivered the drug candidate MW01-18-150SRM (MW150), currently in early stage clinical trials.

p38α MAPK signaling drives pharmacologically reversible brain and gastrointestinal phenotypes in the SERT Ala56 mouse.

Autism spectrum disorder (ASD) is a common neurobehavioral disorder with limited treatment options. Activation of p38 MAPK signaling networks has been identified in ASD, and p38 MAPK signaling elevates serotonin (5-HT) transporter (SERT) activity, effects mimicked by multiple, hyperfunctional SERT coding variants identified in ASD subjects. Mice expressing the most common of these variants (SERT Ala56) exhibit hyperserotonemia, a biomarker observed in ASD subjects, as well as p38 MAPK-dependent SERT hyperphosphorylation, elevated hippocampal 5-HT clearance, hypersensitivity of CNS 5-HT1A and 5-HT2A/2C receptors, and behavioral and gastrointestinal perturbations reminiscent of ASD. As the α-isoform of p38 MAPK drives SERT activation, we tested the hypothesis that CNS-penetrant, α-isoform-specific p38 MAPK inhibitors might normalize SERT Ala56 phenotypes. Strikingly, 1-week treatment of adult SERT Ala56 mice with MW150, a selective p38α MAPK inhibitor, normalized hippocampal 5-HT clearance, CNS 5-HT1A and 5-HT2A/2C receptor sensitivities, social interactions, and colonic motility. Conditional elimination of p38α MAPK in 5-HT neurons of SERT Ala56 mice restored 5-HT1A and 5-HT2A/2C receptor sensitivities as well as social interactions, mirroring effects of MW150. Our findings support ongoing p38α MAPK activity as an important determinant of the physiological and behavioral perturbations of SERT Ala56 mice and, more broadly, supports consideration of p38α MAPK inhibition as a potential treatment for core and comorbid phenotypes present in ASD subjects.

An isoform-selective p38α mitogen-activated protein kinase inhibitor rescues early entorhinal cortex dysfunctions in a mouse model of Alzheimer's disease.

Neuroinflammation is a fundamental mechanism in Alzheimer's disease (AD) progression. The stress-induced activation of the p38α mitogen-activated protein kinase (MAPK) leads to increased production of proinflammatory cytokines and neurodegeneration. We investigated the effects of an isoform selective p38α MAPK inhibitor, MW01-18-150SRM (MW150), administered at 2.5 mg/kg/d (i.p.; 14 days) on early entorhinal cortex (EC) alterations in an AD mouse model carrying human mutations of the amyloid precursor protein (mhAPP). We used electrophysiological analyses with long-term potentiation induction in EC-containing brain slices and EC-relevant associative memory tasks. We found that MW150 was capable of rescuing long-term potentiation in 2-month old mhAPP mice. Acute delivery of MW150 to brain slices was similarly effective in rescuing long-term potentiation, with a comparable efficacy to that of the widely used multikinase inhibitor SB203580. MW150-treated mhAPP mice demonstrated improved ability to discriminate novel associations between objects and their position/context. Our findings suggest that the selective inhibition of the stress-activated p38α MAPK with MW150 can attenuate the EC dysfunctions associated with neuroinflammation in an early stage of AD progression.

Retention of normal glia function by an isoform-selective protein kinase inhibitor drug candidate that modulates cytokine production and cognitive outcomes.

BACKGROUND: Brain p38α mitogen-activated protein kinase (MAPK), a potential therapeutic target for cognitive dysfunction based on the neuroinflammation-synaptic dysfunction cycle of pathophysiology progression, offers an innovative pharmacological strategy via inhibiting the same activated target in both glia and neurons, thereby enhancing the possibility for efficacy. The highly selective, brain-penetrant p38αMAPK inhibitor MW150 attenuates cognitive dysfunction in two distinct Alzheimer's disease (AD)-relevant models and avoids the problems encountered with previous mixed-kinase inhibitor drug candidates. Therefore, it is essential that the glial effects of this CNS-active kinase inhibitor be addressed in order to anticipate future use in clinical investigations. METHODS: We explored the effects of MW150 on glial biology in the AD-relevant APP/PS1 knock-in (KI) mouse model where we previously showed efficacy in suppression of hippocampal-dependent associative and spatial memory deficits. MW150 (2.5 mg/kg/day) was administered daily to 11-12-month-old KI mice for 14 days, and levels of proinflammatory cytokines IL-1ß, TNFα, and IL-6 measured in homogenates of mouse cortex using ELISA. Glial markers IBA1, CD45, CD68, and GFAP were assessed by immunohistochemistry. Microglia and amyloid plaques were quantified by immunofluorescence staining followed by confocal imaging. Levels of soluble and insoluble of Aß40 and Aß42 were measured by ELISA. The studies of in vivo pharmacodynamic effects on markers of neuroinflammation were complemented by mechanistic studies in the murine microglia BV2 cell line, using live cell imaging techniques to monitor proliferation, migration, and phagocytosis activities. RESULTS: Intervention with MW150 in KI mice during the established therapeutic time window attenuated the increased levels of IL-1ß and TNFα but not IL-6. MW150 treatment also increased the IBA1+ microglia within a 15 µm radius of the amyloid plaques, without significantly affecting overall microglia or plaque volume. Levels of IBA1, CD45, CD68, GFAP, and Aß40 and Aß42 were not affected by MW150 treatment. MW150 did not significantly alter microglial migration, proliferation, or phagocytosis in BV2 cells. CONCLUSIONS: Our results demonstrate that MW150 at an efficacious dose can selectively modulate neuroinflammatory responses associated with pathology progression without pan-suppression of normal physiological functions of microglia.

Phylogenetic Diversity of Koala Retrovirus within a Wild Koala Population.

Koala populations are in serious decline across many areas of mainland Australia, with infectious disease a contributing factor. Koala retrovirus (KoRV) is a gammaretrovirus present in most wild koala populations and captive colonies. Five subtypes of KoRV (A to E) have been identified based on amino acid sequence divergence in a hypervariable region of the receptor binding domain of the envelope protein. However, analysis of viral genetic diversity has been conducted primarily on KoRV in captive koalas housed in zoos in Japan, the United States, and Germany. Wild koalas within Australia have not been comparably assessed. Here we report a detailed analysis of KoRV genetic diversity in samples collected from 18 wild koalas from southeast Queensland. By employing deep sequencing we identified 108 novel KoRV envelope sequences and determined their phylogenetic diversity. Genetic diversity in KoRV was abundant and fell into three major groups; two comprised the previously identified subtypes A and B, while the third contained the remaining hypervariable region subtypes (C, D, and E) as well as four hypervariable region subtypes that we newly define here (F, G, H, and I). In addition to the ubiquitous presence of KoRV-A, which may represent an exclusively endogenous variant, subtypes B, D, and F were found to be at high prevalence, while subtypes G, H, and I were present in a smaller number of animals. IMPORTANCE: Koala retrovirus (KoRV) is thought to be a significant contributor to koala disease and population decline across mainland Australia. This study is the first to determine KoRV subtype prevalence among a wild koala population, and it significantly expands the total number of KoRV sequences available, providing a more precise picture of genetic diversity. This understanding of KoRV subtype prevalence and genetic diversity will be important for conservation efforts attempting to limit the spread of KoRV. Furthermore, KoRV is one of the only retroviruses shown to exist in both endogenous (transmitted vertically to offspring in the germ line DNA) and exogenous (horizontally transmitted between infected individuals) forms, a division of fundamental evolutionary importance.

Selective suppression of the α isoform of p38 MAPK rescues late-stage tau pathology.

BACKGROUND: Hyperphosphorylation and aggregation of tau protein are the pathological hallmarks of Alzheimer's disease and related tauopathies. We previously demonstrated that the microglial activation induces tau hyperphosphorylation and cognitive impairment via activation of p38 mitogen-activated protein kinase (p38 MAPK) in the hTau mouse model of tauopathy that was deficient for microglial fractalkine receptor CX3CR1. METHOD: We report an isoform-selective, brain-permeable, and orally bioavailable small molecule inhibitor of p38α MAPK (MW181) and its effects on tau phosphorylation in vitro and in hTau mice. RESULTS: First, pretreatment of mouse primary cortical neurons with MW181 completely blocked inflammation-induced p38α MAPK activation and AT8 (pS199/pS202) site tau phosphorylation, with the maximum effect peaking at 60-90 min after stimulation. Second, treatment of old (~20 months of age) hTau mice with MW181 (1 mg/kg body weight; 14 days via oral gavage) significantly reduced p38α MAPK activation compared with vehicle-administered hTau mice. This also resulted in a significant reduction in AT180 (pT231) site tau phosphorylation and Sarkosyl-insoluble tau aggregates. Third, MW181 treatment significantly increased synaptophysin protein expression and resulted in improved working memory. Fourth, MW181 administration reduced phosphorylated MAPK-activated protein kinase 2 (pMK2) and phosphorylated activating transcription factor 2 (pATF2), which are known substrates of p38α MAPK. Finally, MW181 reduced the expression of interferon-γ and interleukin-1ß. CONCLUSIONS: Taken together, these studies support p38α MAPK as a valid therapeutic target for the treatment of tauopathies.

Product release is rate-limiting for catalytic processing by the Dengue virus protease.

Dengue Virus (DENV) is the most prevalent global arbovirus, yet despite an increasing burden to health care there are currently no therapeutics available to treat infection. A potential target for antiviral drugs is the two-component viral protease NS2B-NS3pro, which is essential for viral replication. Interactions between the two components have been investigated here by probing the effect on the rate of enzyme catalysis of key mutations in a mobile loop within NS2B that is located at the interface of the two components. Steady-state kinetic assays indicated that the mutations greatly affect catalytic turnover. However, single turnover and fluorescence experiments have revealed that the mutations predominantly affect product release rather than substrate binding. Fluorescence analysis also indicated that the addition of substrate triggers a near-irreversible change in the enzyme conformation that activates the catalytic centre. Based on this mechanistic insight, we propose that residues within the mobile loop of NS2B control product release and present a new target for design of potent Dengue NS2B-NS3 protease inhibitors.

Riboflavin and ultraviolet light: impact on dengue virus infectivity.

BACKGROUND: Dengue viruses (DENV 1-4) are emerging across the world, and these viruses pose a risk to transfusion safety. Pathogen inactivation may be an alternative approach for managing the risk of DENV transfusion transmission. This study aimed to investigate the ability of riboflavin and UV light to inactivate DENV 1-4 in platelet concentrates. MATERIALS AND METHODS: DENV 1-4 were spiked into buffy coat-derived platelet concentrates in additive solution (SSP+) before being treated with riboflavin and UV light. Infectious virus was quantified pre- and posttreatment, and the reduction in viral infectivity was calculated. RESULTS: All four DENV serotypes were modestly reduced after treatment. The greatest amount of reduction in infectivity was observed for DENV-4 (1·81 log reduction) followed by DENV-3 (1·71 log reduction), DENV-2 (1·45 log reduction) and then DENV-1 (1·28 log reduction). CONCLUSION: Our study demonstrates that DENV 1-4 titres are modestly reduced following treatment with riboflavin and UV light. With the increasing number of transfusion-transmitted cases of DENV around the globe, and the increasing incidence and geographical distribution of DENV, additional approaches for maintaining blood safety may be required in the future.

MW151 Inhibited IL-1ß Levels after Traumatic Brain Injury with No Effect on Microglia Physiological Responses.

A prevailing neuroinflammation hypothesis is that increased production of proinflammatory cytokines contributes to progressive neuropathology, secondary to the primary damage caused by a traumatic brain injury (TBI). In support of the hypothesis, post-injury interventions that inhibit the proinflammatory cytokine surge can attenuate the progressive pathology. However, other post-injury neuroinflammatory responses are key to endogenous recovery responses. Therefore, it is critical that pharmacological attenuation of detrimental or dysregulated neuroinflammatory processes avoid pan-suppression of inflammation. MW151 is a CNS-penetrant, small molecule experimental therapeutic that restores injury- or disease-induced overproduction of proinflammatory cytokines towards homeostasis without immunosuppression. Post-injury administration of MW151 in a closed head injury model of mild TBI suppressed acute cytokine up-regulation and downstream cognitive impairment. Here, we report results from a diffuse brain injury model in mice using midline fluid percussion. Low dose (0.5-5.0 mg/kg) administration of MW151 suppresses interleukin-1 beta (IL-1ß) levels in the cortex while sparing reactive microglia and astrocyte responses. To probe molecular mechanisms, we used live cell imaging of the BV-2 microglia cell line to demonstrate that MW151 does not affect proliferation, migration, or phagocytosis of the cells. Our results provide insight into the roles of glial responses to brain injury and indicate the feasibility of using appropriate dosing for selective therapeutic modulation of injurious IL-1ß increases while sparing other glial responses to injury.

Phosphorylation Regulates Interaction of 210-kDa Myosin Light Chain Kinase N-terminal Domain with Actin Cytoskeleton.

High molecular weight myosin light chain kinase (MLCK210) is a multifunctional protein involved in myosin II activation and integration of cytoskeletal components in cells. MLCK210 possesses actin-binding regions both in the central part of the molecule and in its N-terminal tail domain. In HeLa cells, mitotic protein kinase Aurora B was suggested to phosphorylate MLCK210 N-terminal tail at serine residues (Dulyaninova, N. G., and Bresnick, A. R. (2004) Exp. Cell Res., 299, 303-314), but the functional significance of the phosphorylation was not established. We report here that in vitro, the N-terminal actin-binding domain of MLCK210 is located within residues 27-157 (N27-157, avian MLCK210 sequence) and is phosphorylated by cAMP-dependent protein kinase (PKA) and Aurora B at serine residues 140/149 leading to a decrease in N27-157 binding to actin. The same residues are phosphorylated in a PKA-dependent manner in transfected HeLa cells. Further, in transfected cells, phosphomimetic mutants of N27-157 showed reduced association with the detergent-stable cytoskeleton, whereas in vitro, the single S149D mutation reduced N27-157 association with F-actin to a similar extent as that achieved by N27-157 phosphorylation. Altogether, our results indicate that phosphorylation of MLCK210 at distinct serine residues, mainly at S149, attenuates the interaction of MLCK210 N-terminus with the actin cytoskeleton and might serve to regulate MLCK210 microfilament cross-linking activity in cells.

Matrix metalloproteinase 9-induced increase in intestinal epithelial tight junction permeability contributes to the severity of experimental DSS colitis.

Recent studies have implicated a pathogenic role for matrix metalloproteinases 9 (MMP-9) in inflammatory bowel disease. Although loss of epithelial barrier function has been shown to be a key pathogenic factor for the development of intestinal inflammation, the role of MMP-9 in intestinal barrier function remains unclear. The aim of this study was to investigate the role of MMP-9 in intestinal barrier function and intestinal inflammation. Wild-type (WT) and MMP-9(-/-) mice were subjected to experimental dextran sodium sulfate (DSS) colitis by administration of 3% DSS in drinking water for 7 days. The mouse colonic permeability was measured in vivo by recycling perfusion of the entire colon using fluorescently labeled dextran. The DSS-induced increase in the colonic permeability was accompanied by an increase in intestinal epithelial cell MMP-9 expression in WT mice. The DSS-induced increase in intestinal permeability and the severity of DSS colitis was found to be attenuated in MMP-9(-/-) mice. The colonic protein expression of myosin light chain kinase (MLCK) and phospho-MLC was found to be significantly increased after DSS administration in WT mice but not in MMP-9(-/-) mice. The DSS-induced increase in colonic permeability and colonic inflammation was attenuated in MLCK(-/-) mice and MLCK inhibitor ML-7-treated WT mice. The DSS-induced increase in colonic surface epithelial cell MLCK mRNA was abolished in MMP-9(-/-) mice. Lastly, increased MMP-9 protein expression was detected within the colonic surface epithelial cells in ulcerative colitis cases. These data suggest a role of MMP-9 in modulation of colonic epithelial permeability and inflammation via MLCK.