IKKß deletion from CNS macrophages increases neuronal excitability and accelerates the onset of EAE, while from peripheral macrophages reduces disease severity.

BACKGROUND: Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease characterized by motor deficits and cognitive decline. Many immune aspects of the disease are understood through studies in the experimental autoimmune encephalomyelitis (EAE) model, including the contribution of the NF-κB transcription factor to neuroinflammation. However, the cell-specific roles of NF-κB to EAE and its cognitive comorbidities still needs further investigation. We have previously shown that the myeloid cell NF-κB plays a role in the healthy brain by exerting homeostatic regulation of neuronal excitability and synaptic plasticity and here we investigated its role in EAE. METHODS: We used constitutive MφIKKßΚΟ mice, in which depletion of IKKß, the main activating kinase of NF-κB, was global to CNS and peripheral macrophages, and ΜgΙΚΚßKO mice, in which depletion was inducible and specific to CNS macrophages by 28 days after tamoxifen administration. We subjected these mice to MOG35-55 induced EAE and cuprizone-induced demyelination. We measured pathology by immunohistochemistry, investigated molecular mechanisms by RNA sequencing analysis and studied neuronal functions by in vivo electrophysiology in awake animals. RESULTS: Global depletion of IKKß from myeloid cells in MφIKKßΚΟ mice accelerated the onset and significantly supressed chronic EAE. Knocking out IKKß only from CNS resident macrophages accelerated the onset and exacerbated chronic EAE, accompanied by earlier demyelination and immune cell infiltration but had no effect in cuprizone-induced demyelination. Peripheral T cell effector functions were not affected by myeloid cell deletion of IKKß, but CNS resident mechanisms, such as microglial activation and neuronal hyperexcitability were altered from early in EAE. Lastly, depletion of myeloid cell IKKß resulted in enhanced late long-term potentiation in EAE. CONCLUSIONS: IKKß-mediated activation of NF-κΒ in myeloid cells has opposing roles in EAE depending on the cell type and the disease stage. In CNS macrophages it is protective while in peripheral macrophages it is disease-promoting and acts mainly during chronic disease. Although clinically protective, CNS myeloid cell IKKß deletion dysregulates neuronal excitability and synaptic plasticity in EAE. These effects of IKKß on brain cognitive abilities deserve special consideration when therapeutic interventions that inhibit NF-κB are used in MS.

Novel cell-based analysis reveals region-dependent changes in microglial dynamics in grey matter in a cuprizone model of demyelination.

Microglia are key players in Multiple Sclerosis (MS), expressing many susceptibility genes for this disease. They constantly survey the brain microenvironment, but the precise functional relationships between microglia and pathological processes remain unknown. We performed a detailed assessment of microglial dynamics in three distinct grey matter regions in a cuprizone-induced demyelination model. We found that microglial activation preceded detectable demyelination and showed regional specificities, such as prominent phagocytic activity in cortical layer 5 and early hypertrophic morphology in hippocampal CA1. Demyelination happened earliest in cortical layer 5, although was more complete in CA1. In cortical layer 2/3, microglial activation and demyelination were less pronounced but microglia became hyper-ramified with slower process movement during remyelination, thereby maintaining local brain surveillance. Profiling of microglia using specific morphological and motility parameters revealed region-specific heterogeneity of microglial responses in the grey matter that might serve as sensitive indicators of progression in CNS demyelinating diseases.

Fundamentally different roles of neuronal TNF receptors in CNS pathology: TNFR1 and IKKß promote microglial responses and tissue injury in demyelination while TNFR2 protects against excitotoxicity in mice.

BACKGROUND: During inflammatory demyelination, TNF receptor 1 (TNFR1) mediates detrimental proinflammatory effects of soluble TNF (solTNF), whereas TNFR2 mediates beneficial effects of transmembrane TNF (tmTNF) through oligodendroglia, microglia, and possibly other cell types. This model supports the use of selective inhibitors of solTNF/TNFR1 as anti-inflammatory drugs for central nervous system (CNS) diseases. A potential obstacle is the neuroprotective effect of solTNF pretreatment described in cultured neurons, but the relevance in vivo is unknown. METHODS: To address this question, we generated mice with neuron-specific depletion of TNFR1, TNFR2, or inhibitor of NF-κB kinase subunit ß (IKKß), a main downstream mediator of TNFR signaling, and applied experimental models of inflammatory demyelination and acute and preconditioning glutamate excitotoxicity. We also investigated the molecular and cellular requirements of solTNF neuroprotection by generating astrocyte-neuron co-cultures with different combinations of wild-type (WT) and TNF and TNFR knockout cells and measuring N-methyl-D-aspartate (NMDA) excitotoxicity in vitro. RESULTS: Neither neuronal TNFR1 nor TNFR2 protected mice during inflammatory demyelination. In fact, both neuronal TNFR1 and neuronal IKKß promoted microglial responses and tissue injury, and TNFR1 was further required for oligodendrocyte loss and axonal damage in cuprizone-induced demyelination. In contrast, neuronal TNFR2 increased preconditioning protection in a kainic acid (KA) excitotoxicity model in mice and limited hippocampal neuron death. The protective effects of neuronal TNFR2 observed in vivo were further investigated in vitro. As previously described, pretreatment of astrocyte-neuron co-cultures with solTNF (and therefore TNFR1) protected them against NMDA excitotoxicity. However, protection was dependent on astrocyte, not neuronal TNFR1, on astrocyte tmTNF-neuronal TNFR2 interactions, and was reproduced by a TNFR2 agonist. CONCLUSIONS: These results demonstrate that neuronal TNF receptors perform fundamentally different roles in CNS pathology in vivo, with neuronal TNFR1 and IKKß promoting microglial inflammation and neurotoxicity in demyelination, and neuronal TNFR2 mediating neuroprotection in excitotoxicity. They also reveal that previously described neuroprotective effects of solTNF against glutamate excitotoxicity in vitro are indirect and mediated via astrocyte tmTNF-neuron TNFR2 interactions. These results consolidate the concept that selective inhibition of solTNF/TNFR1 with maintenance of TNFR2 function would have combined anti-inflammatory and neuroprotective properties required for safe treatment of CNS diseases.

Exploiting the Role of Hypoxia-Inducible Factor 1 and Pseudohypoxia in the Myelodysplastic Syndrome Pathophysiology.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem (HSCs) and/or progenitor cells disorders. The established dependence of MDS progenitors on the hypoxic bone marrow (BM) microenvironment turned scientific interests to the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 facilitates quiescence maintenance and regulates differentiation by manipulating HSCs metabolism, being thus an appealing research target. Therefore, we examine the aberrant HIF-1 stabilization in BMs from MDS patients and controls (CTRLs). Using a nitroimidazole-indocyanine conjugate, we show that HIF-1 aberrant expression and transcription activity is oxygen independent, establishing the phenomenon of pseudohypoxia in MDS BM. Next, we examine mitochondrial quality and quantity along with levels of autophagy in the differentiating myeloid lineage isolated from fresh BM MDS and CTRL aspirates given that both phenomena are HIF-1 dependent. We show that the mitophagy of abnormal mitochondria and autophagic death are prominently featured in the MDS myeloid lineage, their severity increasing with intra-BM blast counts. Finally, we use in vitro cultured CD34+ HSCs isolated from fresh human BM aspirates to manipulate HIF-1 expression and examine its potential as a therapeutic target. We find that despite being cultured under 21% FiO2, HIF-1 remained aberrantly stable in all MDS cultures. Inhibition of the HIF-1α subunit had a variable beneficial effect in all <5%-intra-BM blasts-MDS, while it had no effect in CTRLs or in ≥5%-intra-BM blasts-MDS that uniformly died within 3 days of culture. We conclude that HIF-1 and pseudohypoxia are prominently featured in MDS pathobiology, and their manipulation has some potential in the therapeutics of benign MDS.

Characterization of Asparagine Deamidation in Immunodominant Myelin Oligodendrocyte Glycoprotein Peptide Potential Immunotherapy for the Treatment of Multiple Sclerosis.

Mannan (polysaccharide) conjugated with a myelin oligodendrocyte glycoprotein (MOG) peptide, namely (KG)5MOG35-55, represents a potent and promising new approach for the immunotherapy of Multiple Sclerosis (MS). The MOG35-55 epitope conjugated with the oxidized form of mannan (poly-mannose) via a (KG)5 linker was found to inhibit the symptoms of MOG35-55-induced experimental autoimmune encephalomyelitis (EAE) in mice using prophylactic and therapeutic vaccinated protocols. Deamidation is a common modification in peptide and protein sequences, especially for Gln and Asn residues. In this study, the structural solution motif of deaminated peptides and their functional effects in an animal model for MS were explored. Several peptides based on the MOG35-55 epitope have been synthesized in which the Asn53 was replaced with Ala, Asp, or isoAsp. Our results demonstrate that the synthesized MOG peptides were formed to the deaminated products in basic conditions, and the Asn53 was mainly modified to Asp. Moreover, both peptides (wild type and deaminated derivative) conjugated with mannan (from Saccharomyces cerevisiae) independently inhibited the development of neurological symptoms and inflammatory demyelinating spinal cord lesions in MOG35-55-induced EAE. To conclude, mannan conjugated with a deamidated product did not affect the efficacy of the parent peptide.

Mesenchymal Stem Cell Protection of Neurons against Glutamate Excitotoxicity Involves Reduction of NMDA-Triggered Calcium Responses and Surface GluR1, and Is Partly Mediated by TNF.

Mesenchymal stem cells (MSC) provide therapeutic effects in experimental CNS disease models and show promise as cell-based therapies for humans, but their modes of action are not well understood. We previously show that MSC protect rodent neurons against glutamate excitotoxicity in vitro, and in vivo in an epilepsy model. Neuroprotection is associated with reduced NMDA glutamate receptor (NMDAR) subunit expression and neuronal glutamate-induced calcium (Ca2+) responses, and increased expression of stem cell-associated genes. Here, to investigate whether MSC-secreted factors modulate neuronal AMPA glutamate receptors (AMPAR) and gene expression, we performed longitudinal studies of enriched mouse cortical neurons treated with MSC conditioned medium (CM). MSC CM did not alter total levels of GluR1 AMPAR subunit in neurons, but its distribution, reducing cell surface levels compared to non-treated neurons. Proportions of NeuN-positive neurons, and of GFAP- and NG2-positive glia, were equal in untreated and MSC CM-treated cultures over time suggesting that neurons, rather than differentially-expanded glia, account for the immature gene profile previously reported in MSC CM-treated cultures. Lastly, MSC CM contained measurable amounts of tumor necrosis factor (TNF) bioactivity and pre-treatment of MSC CM with the TNF inhibitor etanercept reduced its ability to protect neurons. Together these results indicate that MSC-mediated neuroprotection against glutamate excitotoxicity involves reduced NMDAR and GluR1-containing AMPAR function, and TNF-mediated neuroprotection.

A Cyclic Altered Peptide Analogue Based on Myelin Basic Protein 87-99 Provides Lasting Prophylactic and Therapeutic Protection Against Acute Experimental Autoimmune Encephalomyelitis.

In this report, amide-linked cyclic peptide analogues of the 87-99 myelin basic protein (MBP) epitope, a candidate autoantigen in multiple sclerosis (MS), are tested for therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE). Cyclic altered peptide analogues of MBP87-99 with substitutions at positions 91 and/or 96 were tested for protective effects when administered using prophylactic or early therapeutic protocols in MBP72-85-induced EAE in Lewis rats. The Lys91 and Pro96 of MBP87-99 are crucial T-cell receptor (TCR) anchors and participate in the formation of trimolecular complex between the TCR-antigen (peptide)-MHC (major histocompability complex) for the stimulation of encephalitogenic T cells that are necessary for EAE induction and are implicated in MS. The cyclic peptides were synthesized using Solid Phase Peptide Synthesis (SPPS) applied on the 9-fluorenylmethyloxycarboxyl/tert-butyl Fmoc/tBu methodology and combined with the 2-chlorotrityl chloride resin (CLTR-Cl). Cyclo(91-99)[Ala96]MBP87-99, cyclo(87-99)[Ala91,96]MBP87-99 and cyclo(87-99)[Arg91, Ala96]MBP87-99, but not wild-type linear MBP87-99, strongly inhibited MBP72-85-induced EAE in Lewis rats when administered using prophylactic and early therapeutic vaccination protocols. In particular, cyclo(87-99)[Arg91, Ala96]MBP87-99 was highly effective in preventing the onset and development of clinical symptoms and spinal cord pathology and providing lasting protection against EAE induction.

Altered expression of oligodendrocyte and neuronal marker genes predicts the clinical onset of autoimmune encephalomyelitis and indicates the effectiveness of multiple sclerosis-directed therapeutics.

Experimental autoimmune encephalomyelitis (EAE) is a valuable model for studying immunopathology in multiple sclerosis (MS) and for exploring the interface between autoimmune responses and CNS tissue that ultimately leads to lesion development. In this study, we measured gene expression in mouse spinal cord during myelin oligodendrocyte gp35-55 peptide-induced EAE, using quantitative RT-PCR, to identify gene markers that monitor individual hallmark pathological processes. We defined a small panel of genes whose longitudinal expression patterns provided insight into the timing, interrelationships, and mechanisms of individual disease processes and the efficacy of therapeutics for the treatment of MS. Earliest transcriptional changes were upregulation of Il17a and sharp downregulation of neuronal and oligodendrocyte marker genes preceding clinical disease onset, whereas neuroinflammatory markers progressively increased as symptoms and tissue lesions developed. EAE-induced gene-expression changes were not altered in mice deficient in IKKß in cells of the myeloid lineage compared with controls, but the administration of a selective inhibitor of soluble TNF to mice from the day of immunization delayed changes in the expression of innate inflammation, myelin, and neuron markers from the presymptomatic phase. Proof of principle that the gene panel shows drug screening potential was obtained using a well-established MS therapeutic, glatiramer acetate. Prophylactic treatment of mice with glatiramer acetate normalized gene marker expression, and this correlated with the level of therapeutic success. These results show that neurons and oligodendrocytes are highly sensitive to CNS-directed autoimmunity before the development of clinical symptoms and immunopathology and reveal a role for soluble TNF in mediating the earliest changes in gene expression.

Differential contributions of microglial and neuronal IKKß to synaptic plasticity and associative learning in alert behaving mice.

Microglia are CNS resident immune cells and a rich source of neuroactive mediators, but their contribution to physiological brain processes such as synaptic plasticity, learning, and memory is not fully understood. In this study, we used mice with partial depletion of IκB kinase ß, the main activating kinase in the inducible NF-κB pathway, selectively in myeloid lineage cells (mIKKßKO) or excitatory neurons (nIKKßKO) to measure synaptic strength at hippocampal Schaffer collaterals during long-term potentiation (LTP) and instrumental conditioning in alert behaving individuals. Resting microglial cells in mIKKßKO mice showed less Iba1-immunoreactivity, and brain IL-1ß mRNA levels were selectively reduced compared with controls. Measurement of field excitatory postsynaptic potentials (fEPSPs) evoked by stimulation of the CA3-CA1 synapse in mIKKßKO mice showed higher facilitation in response to paired pulses and enhanced LTP following high frequency stimulation. In contrast, nIKKßKO mice showed normal basic synaptic transmission and LTP induction but impairments in late LTP. To understand the consequences of such impairments in synaptic plasticity for learning and memory, we measured CA1 fEPSPs in behaving mice during instrumental conditioning. IKKß was not necessary in either microglia or neurons for mice to learn lever-pressing (appetitive behavior) to obtain food (consummatory behavior) but was required in both for modification of their hippocampus-dependent appetitive, not consummatory behavior. Our results show that microglia, through IKKß and therefore NF-κB activity, regulate hippocampal synaptic plasticity and that both microglia and neurons, through IKKß, are necessary for animals to modify hippocampus-driven behavior during associative learning.

Oxidative tissue injury in multiple sclerosis is only partly reflected in experimental disease models.

Recent data suggest that oxidative injury may play an important role in demyelination and neurodegeneration in multiple sclerosis (MS). We compared the extent of oxidative injury in MS lesions with that in experimental models driven by different inflammatory mechanisms. It was only in a model of coronavirus-induced demyelinating encephalomyelitis that we detected an accumulation of oxidised phospholipids, which was comparable in extent to that in MS. In both, MS and coronavirus-induced encephalomyelitis, this was associated with massive microglial and macrophage activation, accompanied by the expression of the NADPH oxidase subunit p22phox but only sparse expression of inducible nitric oxide synthase (iNOS). Acute and chronic CD4(+) T cell-mediated experimental autoimmune encephalomyelitis lesions showed transient expression of p22phox and iNOS associated with inflammation. Macrophages in chronic lesions of antibody-mediated demyelinating encephalomyelitis showed lysosomal activity but very little p22phox or iNOS expressions. Active inflammatory demyelinating lesions induced by CD8(+) T cells or by innate immunity showed macrophage and microglial activation together with the expression of p22phox, but low or absent iNOS reactivity. We corroborated the differences between acute CD4(+) T cell-mediated experimental autoimmune encephalomyelitis and acute MS lesions via gene expression studies. Furthermore, age-dependent iron accumulation and lesion-associated iron liberation, as occurring in the human brain, were only minor in rodent brains. Our study shows that oxidative injury and its triggering mechanisms diverge in different models of rodent central nervous system inflammation. The amplification of oxidative injury, which has been suggested in MS, is only reflected to a limited degree in the studied rodent models.

Spontaneous human CD8 T cell and autoimmune encephalomyelitis-induced CD4/CD8 T cell lesions in the brain and spinal cord of HLA-DRB1*15-positive multiple sclerosis humanized immune system mice.

Autoimmune diseases of the central nervous system (CNS) such as multiple sclerosis (MS) are only partially represented in current experimental models and the development of humanized immune mice is crucial for better understanding of immunopathogenesis and testing of therapeutics. We describe a humanized mouse model with several key features of MS. Severely immunodeficient B2m-NOG mice were transplanted with peripheral blood mononuclear cells (PBMCs) from HLA-DRB1-typed MS and healthy (HI) donors and showed rapid engraftment by human T and B lymphocytes. Mice receiving cells from MS patients with recent/ongoing Epstein-Barr virus reactivation showed high B cell engraftment capacity. Both HLA-DRB1*15 (DR15) MS and DR15 HI mice, not HLA-DRB1*13 MS mice, developed human T cell infiltration of CNS borders and parenchyma. DR15 MS mice uniquely developed inflammatory lesions in brain and spinal cord gray matter, with spontaneous, hCD8 T cell lesions, and mixed hCD8/hCD4 T cell lesions in EAE immunized mice, with variation in localization and severity between different patient donors. Main limitations of this model for further development are poor monocyte engraftment and lack of demyelination, lymph node organization, and IgG responses. These results show that PBMC humanized mice represent promising research tools for investigating MS immunopathology in a patient-specific approach.

Transmembrane tumour necrosis factor is neuroprotective and regulates experimental autoimmune encephalomyelitis via neuronal nuclear factor-kappaB.

Tumour necrosis factor mediates chronic inflammatory pathologies including those affecting the central nervous system, but non-selective tumour necrosis factor inhibitors exacerbate multiple sclerosis. In addition, TNF receptor SF1A, which encodes one of the tumour necrosis factor receptors, has recently been identified as a multiple sclerosis susceptibility locus in genome-wide association studies in large patient cohorts. These clinical data have emphasized the need for a better understanding of the beneficial effects of tumour necrosis factor during central nervous system inflammation. In this study, we present evidence that the soluble and transmembrane forms of tumour necrosis factor exert opposing deleterious and beneficial effects, respectively, in a multiple sclerosis model. We compared the effects, in experimental autoimmune encephalomyelitis, of selectively inhibiting soluble tumour necrosis factor, and of both soluble and transmembrane tumour necrosis factor. Blocking the action of soluble tumour necrosis factor, but not of soluble tumour necrosis factor and transmembrane tumour necrosis factor, protected mice against the clinical symptoms of experimental autoimmune encephalomyelitis. Therapeutic benefit was independent of changes in antigen-specific immune responses and focal inflammatory spinal cord lesions, but was associated with reduced overall central nervous system immunoreactivity, increased expression of neuroprotective molecules, and was dependent upon the activity of neuronal nuclear factor-κB, a downstream mediator of neuroprotective tumour necrosis factor/tumour necrosis factor receptor signalling, because mice lacking IκB kinase ß in glutamatergic neurons were not protected by soluble tumour necrosis factor blockade. Furthermore, blocking the action of soluble tumour necrosis factor, but not of soluble tumour necrosis factor and transmembrane tumour necrosis factor, protected neurons in astrocyte-neuron co-cultures against glucose deprivation, an in vitro neurodegeneration model relevant for multiple sclerosis, and this was dependent upon contact between the two cell types. Our results show that soluble tumour necrosis factor promotes central nervous system inflammation, while transmembrane tumour necrosis factor is neuroprotective, and suggest that selective inhibition of soluble tumour necrosis factor may provide a new way forward for the treatment of multiple sclerosis and possibly other inflammatory central nervous system disorders.

Maturation of circulating Ly6ChiCCR2+ monocytes by mannan-MOG induces antigen-specific tolerance and reverses autoimmune encephalomyelitis.

Autoimmune diseases affecting the CNS not only overcome immune privilege mechanisms that protect neural tissues but also peripheral immune tolerance mechanisms towards self. Together with antigen-specific T cells, myeloid cells are main effector cells in CNS autoimmune diseases such as multiple sclerosis, but the relative contributions of blood-derived monocytes and the tissue resident macrophages to pathology and repair is incompletely understood. Through the study of oxidized mannan-conjugated myelin oligodendrocyte glycoprotein 35-55 (OM-MOG), we show that peripheral maturation of Ly6ChiCCR2+ monocytes to Ly6ChiMHCII+PD-L1+ cells is sufficient to reverse spinal cord inflammation and demyelination in MOG-induced autoimmune encephalomyelitis. Soluble intradermal OM-MOG drains directly to the skin draining lymph node to be sequestered by subcapsular sinus macrophages, activates Ly6ChiCCR2+ monocytes to produce MHC class II and PD-L1, prevents immune cell trafficking to spinal cord, and reverses established lesions. We previously showed that protection by OM-peptides is antigen specific. Here, using a neutralizing anti-PD-L1 antibody in vivo and dendritic cell-specific Pdl1 knockout mice, we further demonstrate that PD-L1 in non-dendritic cells is essential for the therapeutic effects of OM-MOG. These results show that maturation of circulating Ly6ChiCCR2+ monocytes by OM-myelin peptides represents a novel mechanism of immune tolerance that reverses autoimmune encephalomyelitis.

Neuronal I kappa B kinase beta protects mice from autoimmune encephalomyelitis by mediating neuroprotective and immunosuppressive effects in the central nervous system.

Some aspects of CNS-directed autoimmunity in multiple sclerosis are modeled in mice by immunization with myelin Ags where tissue damage is driven by myelin-reactive Th1 and Th17 effector lymphocytes. Whether the CNS plays an active role in controlling such autoimmune diseases is unknown. We used mice in which IkappaB kinase beta was deleted from Ca(2+)/calmodulin-dependent kinase IIalpha-expressing neurons (nIKKbetaKO) to investigate the contribution of neuronal NF-kappaB to the development of myelin oligodendrocyte glycoprotein 35-55-induced experimental autoimmune encephalomyelitis. We show that nIKKbetaKO mice developed a severe, nonresolving disease with increased axon loss compared with controls and this was associated with significantly reduced CNS production of neuroprotective factors (vascular endothelial growth factor, CSF1-R, and FLIP) and increased production of proinflammatory cytokines (IL-6, TNF, IL-12, IL-17, and CD30L) and chemokines. The isolation of CNS-infiltrating monocytes revealed greater numbers of CD4(+) T cells, reduced numbers of NK1.1(+) cells, and a selective accumulation of Th1 cells in nIKKbetaKO CNS from early in the disease. Our results show that neurons play an important role in determining the quality and outcome of CNS immune responses, specifically that neuronal IkappaB kinase beta is required for neuroprotection, suppression of inflammation, limitation of Th1 lymphocyte accumulation, and enhancement of NK cell recruitment in experimental autoimmune encephalomyelitis-affected CNS and stress the importance of neuroprotective strategies for the treatment of multiple sclerosis.

TNF receptor I sensitizes neurons to erythropoietin- and VEGF-mediated neuroprotection after ischemic and excitotoxic injury.

CNS neurons use robust cytoprotective mechanisms to ensure survival and functioning under conditions of injury. These involve pathways induced by endogenous neuroprotective cytokines such as erythropoietin (EPO). Recently, in contrast to its well known deleterious roles, TNF has also been shown to exhibit neuroprotective properties. In the present study, we investigated the molecular mechanisms by which TNF receptor (TNFR)I mediates neuroprotection by comparing the gene expression profiles of lesioned cortex from WT and TNFRI KO mice after permanent middle cerebral artery occlusion. Several known neuroprotective molecules were identified as TNFRI targets, notably members of the Bcl-2 family, DNA repair machinery and cell cycle, developmental, and differentiation factors, neurotransmitters and growth factors, as well as their receptors, including EPO receptor (EPOR), VEGF, colony-stimulating factor receptor 1, insulin-like growth factor (IGF), and nerve growth factor (NGF). Further analysis showed that induction of EPOR and VEGF expression in primary cortical neurons after glucose deprivation (GD) largely depended on TNFRI and was further up-regulated by TNF. Also, EPO- and VEGF-induced neuroprotection against GD, oxygen-glucose deprivation, and NMDA excitotoxicity depended significantly on TNFRI presence. Finally, EPO prevented neuronal damage induced by kainic acid in WT but not TNFRI KO mice. Our results identify cross-talk between tissue protective cytokines, specifically that TNFRI is necessary for constitutive and GD-induced expression of EPOR and VEGF and for EPO-mediated neuroprotection.

Comparative gene expression analysis in mouse models for multiple sclerosis, Alzheimer's disease and stroke for identifying commonly regulated and disease-specific gene changes.

The brain responds to injury and infection by activating innate defense and tissue repair mechanisms. Working upon the hypothesis that the brain defense response involves common genes and pathways across diverse pathologies, we analysed global gene expression in brain from mouse models representing three major central nervous system disorders, cerebral stroke, multiple sclerosis and Alzheimer's disease compared to normal brain using DNA microarray expression profiling. A comparison of dysregulated genes across disease models revealed common genes and pathways including key components of estrogen and TGF-beta signaling pathways that have been associated with neuroprotection as well as a neurodegeneration mediator, TRPM7. Further, for each disease model, we discovered collections of differentially expressed genes that provide novel insight into the individual pathology and its associated mechanisms. Our data provide a resource for exploring the complex molecular mechanisms that underlie brain neurodegeneration and a new approach for identifying generic and disease-specific targets for therapy.

A National Representative, Cross-Sectional Study by the Hellenic Academy of NeuroImmunology (HEL.A.NI.) on COVID-19 and Multiple Sclerosis: Overall Impact and Willingness Toward Vaccination.

Background: In the context of the coronavirus disease 2019 (COVID-19) pandemic, the constant needs of people with multiple sclerosis (PwMS) and their caregivers were urgently highlighted. Aim: The present study aims to capture the effects of the COVID-19 pandemic in several aspects of the quality of life of PwMS, in perception and behavior to COVID-19 and multiple sclerosis (MS), as well as concerning healthcare, working conditions, and the willingness toward COVID-19 vaccination. Methods: This study is an initiative of the Hellenic Academy of Neuroimmunology (HEL.A.NI.) and it has been included in the MS Data Alliance (MSDA) Catalog, which can be accessed after creating an account on https://msda.emif-catalogue.eu/login. Two online questionnaires were administered: (i) impact of the COVID-19 pandemic on the quality of life, behavior, and healthcare of PwMS (Questionnaire A) and (ii) vaccination against COVID-19 (Questionnaire B). People with MS were invited to participate by the Hellenic Federation of Persons with Multiple Sclerosis (HFoPwMS). Results: Three-hundred-ninety PwMS responded to Questionnaire A, whereas 176 PwMS provided answers for Questionnaire B. Older age, longer disease duration, and higher MS-related disability were associated with the increased perceived sensitivity toward severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as well as the increased perceived severity of COVID-19 upon potential infection. A significant proportion of PwMS experienced restricted access to MS-related health professionals, disease-modifying therapy (DMT) prescription, and/or to MS-related laboratory examination due to the pandemic. Subgroups of PwMS reported exacerbated symptoms (i.e., chronic MS-related symptoms, fatigue and/or worsening of pre-existing fatigue, and sexual dysfunction and or/worsening of pre-existing sexual dysfunction). Overall, the majority of the participants reported either a strong willingness to get vaccinated against COVID-19 or a likeliness to undergo vaccination. Being aware of the HEL.A.NI. recommendations regarding COVID-19 vaccination for PwMS were reported to increase the willingness of the participants to receive the vaccine. Conclusions: Our results highlight the necessity of scientific and patient organizations in taking joint action to increase awareness on health-related issues during the pandemic and to provide accurate and up-to-date guidance for PwMS. Online information and communications technology (ICT) tools for polling public belief and behavior may prove valuable as means of retaining active routes of communication between stakeholders.

TNFRI is a positive T-cell costimulatory molecule important for the timing of cytokine responses.

Tumor necrosis factor (TNF)- and TNF receptor I (TNFRI)-deficient mice are resistant to initiation and show delayed resolution of disease in paradigms of autoimmune disease, but the contribution of TNF/TNFRI signaling to T-cell activation and effector responses has not been determined. In this study, we investigated the role of TNFRI in T-cell receptor (TCR)-mediated T-cell activation in vitro and in vivo using CD3(+)-enriched primary T cells and mice deficient in TNFRI. Following TCR engagement, TNFRI knockout (KO) T cells showed significantly delayed proliferation, cell division, upregulation of interleukin 2 (IL-2) and IL-2 receptor alpha chain (CD25) mRNA and cell-surface expression of CD25 compared with wild-type (WT) cells. Thus, WT and TNFRI KO cells showed equivalent proliferation peaks at 48 and 72 h, respectively. TNFRI KO mice also developed a defective primary T-cell response to ovalbumin and an acute contact hypersensitivity response to oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one). However, TNFRI KO splenocytes that were stimulated by TCR engagement in vitro for 96 h produced significantly higher intracellular levels of interferon-gamma (IFN-gamma), IL-2 and TNF-alpha, but not IL-17, compared with WT cells, in correlation with their relatively higher proliferation rate at this time point. Further, TCR-stimulated CD3(+)-enriched TNFRI KO T cells showed similarly higher production and secretion of IFN-gamma and IL-2 compared with WT, suggesting that TNFRI-mediated cytokine regulation might involve a T-cell autonomous effect. Our results show a novel role for TNFRI as a positive T-cell costimulatory molecule that is important for timely T-cell activation and effector cytokine production and the development of primary immune responses in mice.

Gene expression profile associated with oncogenic ras-induced senescence, cell death, and transforming properties in human cells.

We developed inducible and constitutive expression systems of Ha-RasV12 in HEK 293 cells to examine early oncogenic RasV12 signaling. Inducible expression of oncogenic Ras-triggered growth arrest, early senescence, and later apoptosis. Gene expression profile analysis revealed early Ras proliferation and cell cycle genes like c-fos, cyclin E, cdk2, cell-cell contact, and signaling like integrin a6, MEK5, and free radical signaling genes, like proline oxidase. Therefore, Ras-mediated signaling is a fine regulated process both positively and negatively influencing cell cycle, senescence, and apoptosis pathways. Novel early RAS-target genes could be potentially exploited in cancer diagnostics and therapeutics.

Inflammation and Oxidative Stress in Multiple Sclerosis: Consequences for Therapy Development.

CNS inflammation is a major driver of MS pathology. Differential immune responses, including the adaptive and the innate immune system, are observed at various stages of MS and drive disease development and progression. Next to these immune-mediated mechanisms, other mediators contribute to MS pathology. These include immune-independent cell death of oligodendrocytes and neurons as well as oxidative stress-induced tissue damage. In particular, the complex influence of oxidative stress on inflammation and vice versa makes therapeutic interference complex. All approved MS therapeutics work by modulating the autoimmune response. However, despite substantial developments in the treatment of the relapsing-remitting form of MS, approved therapies for the progressive forms of MS as well as for MS-associated concomitants are limited and much needed. Here, we summarize the contribution of inflammation and oxidative stress to MS pathology and discuss consequences for MS therapy development.