Deficiency of the Two-Pore Potassium Channel KCNK9 Impairs Intestinal Epithelial Cell Survival and Aggravates Dextran Sodium Sulfate-Induced Colitis.

BACKGROUND & AIMS: The 2-pore potassium channel subfamily K member 9 (KCNK9) regulates intracellular calcium concentration and thus modulates cell survival and inflammatory signaling pathways. It also was recognized as a risk allele for inflammatory bowel disease. However, it remains unclear whether KCNK9 modulates inflammatory bowel disease via its impact on immune cell function or whether its influence on calcium homeostasis also is relevant in intestinal epithelial cells. METHODS: Kcnk9-/- mice were challenged with 3% dextran sulfate sodium (DSS) to induce experimental acute colitis. Primary cultures of intestinal epithelial cells were generated, and expression of potassium channels as well as cytosolic calcium levels and susceptibility to apoptosis were evaluated. Furthermore, we evaluated whether KCNK9 deficiency was compensated by the closely related 2-pore potassium channel KCNK3 in vivo or in vitro. RESULTS: Compared with controls, KCNK9 deficiency or its pharmacologic blockade were associated with aggravated DSS-induced colitis compared with wild-type animals. In the absence of KCNK9, intestinal epithelial cells showed increased intracellular calcium levels and were more prone to mitochondrial damage and caspase-9-dependent apoptosis. We found that expression of KCNK3 was increased in Kcnk9-/- mice but did not prevent apoptosis after DSS exposure. Conversely, increased levels of KCNK9 in Kcnk3-/- mice were associated with an ameliorated course of DSS-induced colitis. CONCLUSIONS: KCNK9 enhances mitochondrial stability, reduces apoptosis, und thus supports epithelial cell survival after DSS exposure in vivo and in vitro. Conversely, its increased expression in Kcnk3-/- resulted in less mitochondrial damage and apoptosis and was associated with beneficial outcomes in DSS-induced colitis.

Glutaredoxin 2 promotes SP-1-dependent CSPG4 transcription and migration of wound healing NG2 glia and glioma cells: Enzymatic Taoism.

Redox regulation of specific cysteines via oxidoreductases of the thioredoxin family is increasingly being recognized as an important signaling pathway. Here, we demonstrate that the cytosolic isoform of the vertebrate-specific oxidoreductase Glutaredoxin 2 (Grx2c) regulates the redox state of the transcription factor SP-1 and thereby its binding affinity to both the promoter and an enhancer region of the CSPG4 gene encoding chondroitin sulfate proteoglycan nerve/glial antigen 2 (NG2). This leads to an increased number of NG2 glia during in vitro oligodendroglial differentiation and promotes migration of these wound healing cells. On the other hand, we found that the same mechanism also leads to increased invasion of glioma tumor cells. Using in vitro (human cell lines), ex vivo (mouse primary cells), and in vivo models (zebrafish), as well as glioblastoma patient tissue samples we provide experimental data highlighting the Yin and Yang of redox signaling in the central nervous system and the enzymatic Taoism of Grx2c.

Myelinating Co-Culture as a Model to Study Anti-NMDAR Neurotoxicity.

Anti-NMDA receptor (NMDAR) encephalitis is frequently associated with demyelinating disorders (e.g., multiple sclerosis (MS), neuromyelitis optica spectrum disorder (NMOSD), myelin oligodendrocyte glycoprotein-associated disease (MOGAD)) with regard to clinical presentation, neuropathological and cerebrospinal fluid findings. Indeed, autoantibodies (AABs) against the GluN1 (NR1) subunit of the NMDAR diminish glutamatergic transmission in both neurons and oligodendrocytes, leading to a state of NMDAR hypofunction. Considering the vital role of oligodendroglial NMDAR signaling in neuron-glia communication and, in particular, in tightly regulated trophic support to neurons, the influence of GluN1 targeting on the physiology of myelinated axon may be of importance. We applied a myelinating spinal cord cell culture model that contains all major CNS cell types, to evaluate the effects of a patient-derived GluN1-specific monoclonal antibody (SSM5) on neuronal and myelin integrity. A non-brain reactive (12D7) antibody was used as the corresponding isotype control. We show that in cultures at the late stage of myelination, prolonged treatment with SSM5, but not 12D7, leads to neuronal damage. This is characterized by neurite blebbing and fragmentation, and a reduction in the number of myelinated axons. However, this significant toxic effect of SSM5 was not observed in earlier cultures at the beginning of myelination. Anti-GluN1 AABs induce neurodegenerative changes and associated myelin loss in myelinated spinal cord cultures. These findings may point to the higher vulnerability of myelinated neurons towards interference in glutamatergic communication, and may refer to the disturbance of the NMDAR-mediated oligodendrocyte metabolic supply. Our work contributes to the understanding of the emerging association of NMDAR encephalitis with demyelinating disorders.

Regulation of sirtuin expression in autoimmune neuroinflammation: Induction of SIRT1 in oligodendrocyte progenitor cells.

In multiple sclerosis (MS) regeneration of oligodendrocytes following inflammatory demyelination is limited by the compromised ability of progenitors to repopulate lesioned areas and transition to functionally competent oligodendrocytes. Regarding underlying mechanisms, the involvement of epigenetic processes has been suggested, e.g. the contribution of histone deacetylases (HDAC) known to regulate oligodendrocyte progenitor cell (OPC) differentiation. However, their precise expression patterns, particular of redox-sensitive NAD+ HDACs, remains largely unknown. In this study, we determined the expression and activity of sirtuins, members of the HDAC class III family with a specific focus on SIRT1, previously associated with neurodegenerative, inflammatory and demyelinating disorders of the central nervous system (CNS). By investigating mouse experimental autoimmune encephalomyelitis (EAE), a model for MS, we found that transcription of SIRT1, SIRT2 and SIRT6 was significantly increased in the CNS during chronic disease stages. We confirmed this finding for SIRT1 protein expression and were able to localize upregulated SIRT1 in nuclei of NG2+ or PDGFRα+ OPCs in demyelinated brain lesions. In cultured mouse A2B5+ OPCs blockade of SIRT1 activity by the small molecule compound Ex527 enhanced mitotic activity but did not affect the capacity to differentiate. A similar pattern was detectable in OPCs derived from SIRT1-deficient animals. Taken together, our data suggest that SIRT1 inhibition may help to expand the endogenous pool of OPCs without affecting their differentiation.

Nimodipine confers clinical improvement in two models of experimental autoimmune encephalomyelitis.

Multiple sclerosis is characterised by inflammatory neurodegeneration, with axonal injury and neuronal cell death occurring in parallel to demyelination. Regarding the molecular mechanisms responsible for demyelination and axonopathy, energy failure, aberrant expression of ion channels and excitotoxicity have been suggested to lead to Ca2+ overload and subsequent activation of calcium-dependent damage pathways. Thus, the inhibition of Ca2+ influx by pharmacological modulation of Ca2+ channels may represent a novel neuroprotective strategy in the treatment of secondary axonopathy. We therefore investigated the effects of the L-type voltage-gated calcium channel blocker nimodipine in two different models of mouse experimental autoimmune encephalomyelitis (EAE), an established experimental paradigm for multiple sclerosis. We show that preventive application of nimodipine (10 mg/kg per day) starting on the day of induction had ameliorating effects on EAE in SJL/J mice immunised with encephalitic myelin peptide PLP139-151 , specifically in late-stage disease. Furthermore, supporting these data, administration of nimodipine to MOG35-55 -immunised C57BL/6 mice starting at the peak of pre-established disease, also led to a significant decrease in disease score, indicating a protective effect on secondary CNS damage. Histological analysis confirmed that nimodipine attenuated demyelination, axonal loss and pathological axonal ß-amyloid precursor protein accumulation in the cerebellum and spinal cord in the chronic phase of disease. Of note, we observed no effects of nimodipine on the peripheral immune response in EAE mice with regard to distribution, antigen-specific proliferation or activation patterns of lymphocytes. Taken together, our data suggest a CNS-specific effect of L-type voltage-gated calcium channel blockade to inflammation-induced neurodegeneration.

Activation of Wnt signaling promotes hippocampal neurogenesis in experimental autoimmune encephalomyelitis.

BACKGROUND: Disease progression in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), as one of its animal models, is characterized by demyelination and neuronal damage in white and gray matter structures, including the hippocampus. It is thought that dysfunction of the hippocampus, a primary locus of learning and memory consolidation, may contribute to cognitive impairment in MS patients. Previously, we reported an increased generation of hippocampal neuronal progenitors in the acute stage of EAE, whereas the microenvironmental signals triggering this process remained uninvestigated. RESULTS: In the present study, we used the Wnt signaling reporter mouse Axin2(LacZ), to elucidate the molecular mechanisms underlying the activation of the hippocampal neurogenic niche upon autoimmune neuroinflammation. Histological and enzymatic examinations of ß-gal during the disease course of EAE, allowed us to survey hippocampal Wnt/ß-catenin activity, one of the key signaling pathways of adult neurogenesis. We found that Wnt signaling is transiently upregulated in the acute stage of disease, consistent with a timely induction of canonical Wnt ligands. The enhancement of signaling coincided with hippocampal neuronal damage and local expression of immune cytokines such as TNFα and IFNγ, implicating the role of the inflammatory milieu in activation of the Wnt/ß-catenin pathway. Supporting this finding, we show that transient exposure to pro-inflammatory cytokine TNFα triggers Wnt signaling in hippocampal organotypic slice cultures. Importantly, inflammation-mediated activation of the Wnt/ß-catenin pathway was associated with enhanced neurogenesis in vitro and in vivo, indicating its potential role in hippocampal tissue regeneration and repair. CONCLUSIONS: This study raises the possibility that enhancement of Wnt signaling may support neurogenic processes to cope with neuronal deficits upon immune-mediated neuroinflammation.

ERK1 as a Therapeutic Target for Dendritic Cell Vaccination against High-Grade Gliomas.

Glioma regression requires the recruitment of potent antitumor immune cells into the tumor microenvironment. Dendritic cells (DC) play a role in immune responses to these tumors. The fact that DC vaccines do not effectively combat high-grade gliomas, however, suggests that DCs need to be genetically modified specifically to promote their migration to tumor relevant sites. Previously, we identified extracellular signal-regulated kinase (ERK1) as a regulator of DC immunogenicity and brain autoimmunity. In the current study, we made use of modern magnetic resonance methods to study the role of ERK1 in regulating DC migration and tumor progression in a model of high-grade glioma. We found that ERK1-deficient mice are more resistant to the development of gliomas, and tumor growth in these mice is accompanied by a higher infiltration of leukocytes. ERK1-deficient DCs exhibit an increase in migration that is associated with sustained Cdc42 activation and increased expression of actin-associated cytoskeleton-organizing proteins. We also demonstrated that ERK1 deletion potentiates DC vaccination and provides a survival advantage in high-grade gliomas. Considering the therapeutic significance of these results, we propose ERK1-deleted DC vaccines as an additional means of eradicating resilient tumor cells and preventing tumor recurrence. Mol Cancer Ther; 15(8); 1975-87. ©2016 AACR.

Natalizumab restores aberrant miRNA expression profile in multiple sclerosis and reveals a critical role for miR-20b.

OBJECTIVE: To identify microRNAs (miRNAs) regulated by anti-α4 integrin monoclonal antibody therapy (natalizumab) in the peripheral blood of patients with relapsing-remitting (RR) multiple sclerosis (MS) and to confirm their role in experimental settings in vivo. METHODS: In a longitudinal study of 17 RR-MS patients, we investigated blood miRNA expression profiles at baseline and after 1 year of natalizumab therapy by microarray technique and quantitative PCR validation. We compared the baseline expression profiles of these patients to those of 18 age- and sex-matched healthy controls. We confirmed the contribution of resulting candidate miRNAs in an animal model of MS, experimental autoimmune encephalomyelitis (EAE) induced by adoptive transfer of proteolipid protein (PLP)139-151-activated lymphocytes in SJL/J mice or by active immunization of miR-106a∼363-deficient C57BL/6 mice (or wildtype litter mates) with myelin oligodendrocyte glycoprotein (MOG)35-55. RESULTS: Our longitudinal analysis revealed that miR-18a, miR-20b, miR-29a, and miR-103 were upregulated and predominantly expressed by CD4(+) T cells, whereas miR-326 was downregulated upon natalizumab treatment. A comparison of untreated RR-MS patients at baseline with healthy controls revealed that the four natalizumab-upregulated targets were initially downregulated in MS. All confirmed targets showed disease-dependent expression in splenocytes of mice suffering from EAE. Genetic deletion of the miRNA cluster miR-106a∼363 (containing natalizumab-regulated miR-20b) resulted in a more severe EAE course and an in vivo upregulation of the miR-20b target genes rorgt, stat3, and vegfa. INTERPRETATION: Our study indicates that natalizumab restores dysregulated miRNA patterns in MS and reveals the contribution of miR-20b in autoimmune demyelination in vivo.

Redox-regulated fate of neural stem progenitor cells.

BACKGROUND: Accumulated data indicate that self-renewal, multipotency, and differentiation of neural stem cells are under an intrinsic control mediated by alterations in the redox homeostasis. These dynamic redox changes not only reflect and support the ongoing metabolic and energetic processes, but also serve to coordinate redox-signaling cascades. Controlling particular redox couples seems to have a relevant impact on cell fate decision during development, adult neurogenesis and regeneration. SCOPE OF REVIEW: Our own research provided initial evidence for the importance of NAD+-dependent enzymes in neural stem cell fate decision. In this review, we summarize recent knowledge on the active role of reactive oxygen species, redox couples and redox-signaling mechanisms on plasticity and function of neural stem and progenitor cells focusing on NAD(P)+/NAD(P)H-mediated processes. MAJOR CONCLUSIONS: The compartmentalized subcellular sources and availability of oxidizing/reducing molecules in particular microenvironment define the specificity of redox regulation in modulating the delicate balance between stemness and differentiation of neural progenitors. The generalization of "reactive oxygen species" as well as the ambiguity of their origin might explain the diametrically-opposed findings in the field of redox-dependent cell fate reflected by the literature. GENERAL SIGNIFICANCE: Increasing knowledge of temporary and spatially defined redox regulation is of high relevance for the development of novel approaches in the field of cell-based regeneration of nervous tissue in various pathological states. This article is part of a special issue entitled Redox regulation of differentiation and de-differentiation.

Critical residues for structure and catalysis in short-chain dehydrogenases/reductases.

Short-chain dehydrogenases/reductases form a large, evolutionarily old family of NAD(P)(H)-dependent enzymes with over 60 genes found in the human genome. Despite low levels of sequence identity (often 10-30%), the three-dimensional structures display a highly similar alpha/beta folding pattern. We have analyzed the role of several conserved residues regarding folding, stability, steady-state kinetics, and coenzyme binding using bacterial 3beta/17beta-hydroxysteroid dehydrogenase and selected mutants. Structure determination of the wild-type enzyme at 1.2-A resolution by x-ray crystallography and docking analysis was used to interpret the biochemical data. Enzyme kinetic data from mutagenetic replacements emphasize the critical role of residues Thr-12, Asp-60, Asn-86, Asn-87, and Ala-88 in coenzyme binding and catalysis. The data also demonstrate essential interactions of Asn-111 with active site residues. A general role of its side chain interactions for maintenance of the active site configuration to build up a proton relay system is proposed. This extends the previously recognized catalytic triad of Ser-Tyr-Lys residues to form a tetrad of Asn-Ser-Tyr-Lys in the majority of characterized short-chain dehydrogenases/reductase enzymes.