RESUMO
BACKGROUND: Multiple sclerosis (MS) is a progressive neurodegenerative disease of the central nervous system characterized by inflammation-driven synaptic abnormalities. Interleukin-9 (IL-9) is emerging as a pleiotropic cytokine involved in MS pathophysiology. METHODS: Through biochemical, immunohistochemical, and electrophysiological experiments, we investigated the effects of both peripheral and central administration of IL-9 on C57/BL6 female mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. RESULTS: We demonstrated that both systemic and local administration of IL-9 significantly improved clinical disability, reduced neuroinflammation, and mitigated synaptic damage in EAE. The results unveil an unrecognized central effect of IL-9 against microglia- and TNF-mediated neuronal excitotoxicity. Two main mechanisms emerged: first, IL-9 modulated microglial inflammatory activity by enhancing the expression of the triggering receptor expressed on myeloid cells-2 (TREM2) and reducing TNF release. Second, IL-9 suppressed neuronal TNF signaling, thereby blocking its synaptotoxic effects. CONCLUSIONS: The data presented in this work highlight IL-9 as a critical neuroprotective molecule capable of interfering with inflammatory synaptopathy in EAE. These findings open new avenues for treatments targeting the neurodegenerative damage associated with MS, as well as other inflammatory and neurodegenerative disorders of the central nervous system.
Assuntos
Encefalomielite Autoimune Experimental , Interleucina-9 , Camundongos Endogâmicos C57BL , Microglia , Sinapses , Fator de Necrose Tumoral alfa , Animais , Camundongos , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/induzido quimicamente , Interleucina-9/metabolismo , Interleucina-9/farmacologia , Glicoproteínas de Membrana/metabolismo , Microglia/metabolismo , Microglia/efeitos dos fármacos , Microglia/patologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Sinapses/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
MiR-142-3p has recently emerged as key factor in tailoring personalized treatments for multiple sclerosis (MS), a chronic autoimmune demyelinating disease of the central nervous system (CNS) with heterogeneous pathophysiology and an unpredictable course. With its involvement in a detrimental regulatory axis with interleukin-1beta (IL1ß), miR-142-3p orchestrates excitotoxic synaptic alterations that significantly impact both MS progression and therapeutic outcomes. In this study, we investigated for the first time the influence of individual genetic variability on the miR-142-3p excitotoxic effect in MS. We specifically focused on the single-nucleotide polymorphism Val66Met (rs6265) of the brain-derived neurotrophic factor (BDNF) gene, known for its crucial role in CNS functioning. We assessed the levels of miR-142-3p and IL1ß in cerebrospinal fluid (CSF) obtained from a cohort of 114 patients with MS upon diagnosis. By stratifying patients according to their genetic background, statistical correlations with clinical parameters were performed. Notably, in Met-carrier patients, we observed a decoupling of miR-142-3p levels from IL1ß levels in the CSF, as well as from of disease severity (Expanded Disability Status Score, EDSS; Multiple Sclerosis Severity Score, MSSS; Age-Related Multiple Sclerosis Severity Score, ARMSS) and progression (Progression Index, PI). Our discovery of the interference between BDNF Val66Met polymorphism and the synaptotoxic IL1ß-miR-142-3p axis, therefore hampering miR-142-3p action on MS course, provides valuable insights for further development of personalized medicine in the field.
Assuntos
Fator Neurotrófico Derivado do Encéfalo , Interleucina-1beta , MicroRNAs , Esclerose Múltipla , Polimorfismo de Nucleotídeo Único , Humanos , Fator Neurotrófico Derivado do Encéfalo/genética , MicroRNAs/genética , Feminino , Masculino , Adulto , Esclerose Múltipla/genética , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/patologia , Pessoa de Meia-Idade , Interleucina-1beta/genética , Interleucina-1beta/líquido cefalorraquidiano , Índice de Gravidade de Doença , Predisposição Genética para DoençaRESUMO
BACKGROUND: Elevated levels of specific proinflammatory molecules in the cerebrospinal fluid (CSF) have been associated with disability progression, enhanced neurodegeneration and higher incidence of mood disorders in people with multiple sclerosis (MS). Studies in animal models of MS suggest that preventive exercise may play an immunomodulatory activity, with beneficial effects on both motor deficits and behavioral alterations. Here we explored the impact of lifestyle physical activity on clinical presentation and associated central inflammation in a large group of newly diagnosed patients with MS. Furthermore, we addressed the causal link between exercise-mediated immunomodulation and mood symptoms in the animal setting. METHODS: A cross-sectional study was conducted on 235 relapsing-remitting MS patients at the time of the diagnosis. Patients were divided into 3 groups ("sedentary", "lifestyle physical activity" and "exercise") according to the level of physical activity in the six months preceding the evaluation. Patients underwent clinical, neuropsychological and psychiatric evaluation, magnetic resonance imaging and lumbar puncture for diagnostic purposes. The CSF levels of proinflammatory and anti-inflammatory cytokines were analyzed and compared with a group of 80 individuals with non-inflammatory and non-degenerative diseases. Behavioral and electrophysiological studies were carried out in control mice receiving intracerebral injection of IL-2 or vehicle. Behavior was also assessed in mice with experimental autoimmune encephalomyelitis (EAE), animal model of MS, reared in standard (sedentary group) or running wheel-equipped (exercise group) cages. RESULTS: In exercising MS patients, depression and anxiety were reduced compared to sedentary patients. The CSF levels of the interleukin-2 and 6 (IL-2, IL-6) were increased in MS patients compared with control individuals. In MS subjects exercise was associated with normalized CSF levels of IL-2. In EAE mice exercise started before disease onset reduced both behavioral alterations and striatal IL-2 expression. Notably, a causal role of IL-2 in mood disorders was shown. IL-2 administration in control healthy mice induced anxious- and depressive-like behaviors and impaired type-1 cannabinoid (CB1) receptor-mediated neurotransmission at GABAergic synapses, mimicking EAE-induced synaptic dysfunction. CONCLUSIONS: Our results indicate an immunomodulatory effect of exercise in MS patients, associated with reduced CSF expression of IL-2, which might result in reduced mood disorders. These data suggest that exercise in the early stages may act as a disease-modifying therapy in MS although further longitudinal studies are needed to clarify this issue.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Animais , Estudos Transversais , Encefalomielite Autoimune Experimental/patologia , Humanos , Interleucina-2/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Transtornos do Humor/etiologiaRESUMO
AIM: We recently proposed miR-142-3p as a molecular player in inflammatory synaptopathy, a new pathogenic hallmark of multiple sclerosis (MS) and of its mouse model experimental autoimmune encephalomyelitis (EAE), that leads to neuronal loss independently of demyelination. MiR-142-3p seems to be unique among potential biomarker candidates in MS, since it is an inflammatory miRNA playing a dual role in the immune and central nervous systems. Here, we aimed to verify the impact of miR-142-3p circulating in the cerebrospinal fluid (CSF) of MS patients on clinical parameters, neuronal excitability and its potential interaction with disease modifying therapies (DMTs). METHODS AND RESULTS: In a cohort of 151 MS patients, we found positive correlations between CSF miR-142-3p levels and clinical progression, IL-1ß signalling as well as synaptic excitability measured by transcranial magnetic stimulation. Furthermore, therapy response of patients with 'low miR-142-3p' to dimethyl fumarate (DMF), an established disease-modifying treatment (DMT), was superior to that of patients with 'high miR-142-3p' levels. Accordingly, the EAE clinical course of heterozygous miR-142 mice was ameliorated by peripheral DMF treatment with a greater impact relative to their wild type littermates. In addition, a central protective effect of this drug was observed following intracerebroventricular and ex vivo acute treatments of EAE wild type mice, showing a rescue of miR-142-3p-dependent glutamatergic alterations. By means of electrophysiology, molecular and biochemical analysis, we suggest miR-142-3p as a molecular target of DMF. CONCLUSION: MiR-142-3p is a novel and potential negative prognostic CSF marker of MS and a promising tool for identifying personalised therapies.
Assuntos
Encefalomielite Autoimune Experimental/líquido cefalorraquidiano , MicroRNAs/líquido cefalorraquidiano , Esclerose Múltipla/líquido cefalorraquidiano , Transdução de Sinais/fisiologia , Adulto , Animais , Progressão da Doença , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Interleucina-1beta/metabolismo , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/genética , Pessoa de Meia-Idade , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Estudos ProspectivosRESUMO
Exercise is increasingly recommended as a supportive therapy for people with Multiple Sclerosis (pwMS). While clinical research has still not disclosed the real benefits of exercise on MS disease, animal studies suggest a substantial beneficial effect on motor disability and pathological hallmarks such as central and peripheral dysregulated immune response. The hippocampus, a core area for memory formation and learning, is a brain region involved in MS pathophysiology. Human and rodent studies suggest that the hippocampus is highly sensitive to the effects of exercise, the impact of which on MS hippocampal damage is still elusive. Here we addressed the effects of chronic voluntary exercise on hippocampal function and damage in experimental autoimmune encephalomyelitis (EAE), animal model of MS. Mice were housed in standard or wheel-equipped cages starting from the day of immunization and throughout the disease course. Although running activity was reduced during the symptomatic phase, exercise significantly ameliorated motor disability. Exercise improved cognition that was assessed through the novel object recognition test and the nest building in presymptomatic and acute stages of the disease, respectively. In the acute phase exercise was shown to prevent EAE-induced synaptic plasticity abnormalities in the CA1 area, by promoting the survival of parvalbumin-positive (PV+) interneurons and by attenuating inflammation. Indeed, exercise significantly reduced microgliosis in the CA1 area, the expression of tumour necrosis factor (TNF) in microglia and, to a lesser extent, the hippocampal level of interleukin 1 beta (IL-1ß), previously shown to contribute to aberrant synaptic plasticity in the EAE hippocampus. Notably, exercise exerted a precocious and long-lasting mitigating effect on microgliosis that preceded its neuroprotective action, likely underlying the improved cognitive function observed in both presymptomatic and acute phase EAE mice. Overall, these data provide evidence that regular exercise improves cognitive function and synaptic and neuronal pathology that typically affect EAE/MS brains.
Assuntos
Pessoas com Deficiência , Encefalomielite Autoimune Experimental , Transtornos Motores , Animais , Hipocampo , Humanos , Inflamação , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Extracellular vesicles (EVs) represent a new reality for many physiological and pathological functions as an alternative mode of intercellular communication. This is due to their capacity to interact with distant recipient cells, usually involving delivery of the EVs contents into the target cells. Intensive investigation has targeted the role of EVs in different pathological conditions, including multiple sclerosis (MS). MS is a chronic inflammatory and neurodegenerative disease of the nervous system, one of the main causes of neurological disability in young adults. The fine interplay between the immune and nervous systems is profoundly altered in this disease, and EVs seems to have a relevant impact on MS pathogenesis. Here, we provide an overview of both clinical and preclinical studies showing that EVs released from blood-brain barrier (BBB) endothelial cells, platelets, leukocytes, myeloid cells, astrocytes, and oligodendrocytes are involved in the pathogenesis of MS and of its rodent model experimental autoimmune encephalomyelitis (EAE). Most of the information points to an impact of EVs on BBB damage, on spreading pro-inflammatory signals, and altering neuronal functions, but EVs reparative function of brain damage deserves attention. Finally, we will describe recent advances about EVs as potential therapeutic targets and tools for therapeutic intervention in MS.
Assuntos
Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Vesículas Extracelulares/genética , Esclerose Múltipla/genética , Astrócitos/metabolismo , Plaquetas/metabolismo , Barreira Hematoencefálica/patologia , Células Endoteliais/patologia , Vesículas Extracelulares/metabolismo , Humanos , Leucócitos/metabolismo , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Células Mieloides/metabolismo , Oligodendroglia/metabolismoRESUMO
Growing data from human and animal studies indicate the beneficial effects of exercise on several clinical outcomes in patients with multiple sclerosis (MS), an autoimmune, demyelinating disease, suggesting that it may slow down the disease progression, by reducing brain damage. However, the mechanisms involved are still elusive. Aim of this study was to address the effects of voluntary running wheel in a toxic-demyelinating model of MS, in which demyelination and brain inflammation occur in response to cuprizone (CPZ) treatment. Mice were housed in standard or wheel-equipped cages starting from the day of CPZ or normal chow feeding for three or six weeks and evaluated for weight changes, locomotor skills and neuromuscular functions over the course of the experimental design. Biochemical, molecular biology and immunohistochemical analyses were performed. Exercise prevented early weight loss caused by CPZ, indicating improved wellness in these mice. Both neuromuscular function and motor coordination were significantly enhanced by exercise in CPZ-treated mice. Moreover, exercise induced an early protection against axonal damage and the loss of the myelin associated proteins, myelin basic protein (MBP) and 2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNPase), in the striatum and the corpus callosum, in coincidence of a strongly attenuated microglia activation in both brain areas. Further, during the late phase of the treatment, exercise in CPZ mice reduced the recruitment of new OLs compared to sedentary CPZ mice, likely due to the precocious protection against myelin damage. Overall, these results suggest that life-style interventions can be effective against the demyelinating-inflammatory processes occurring in the brains of MS patients.
Assuntos
Encéfalo/patologia , Doenças Autoimunes Desmielinizantes do Sistema Nervoso Central/patologia , Condicionamento Físico Animal/fisiologia , Animais , Encéfalo/metabolismo , Cuprizona/toxicidade , Doenças Autoimunes Desmielinizantes do Sistema Nervoso Central/induzido quimicamente , Doenças Autoimunes Desmielinizantes do Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Inibidores da Monoaminoxidase/toxicidade , Esclerose MúltiplaRESUMO
MicroRNAs (miRNA) play an important role in post-transcriptional gene regulation of several physiological and pathological processes. In multiple sclerosis (MS), a chronic inflammatory and degenerative disease of the CNS, and in its mouse model, the experimental autoimmune encephalomyelitis (EAE), miRNA dysregulation has been mainly related to immune system dysfunction and white matter (WM) pathology. However, little is known about their role in gray matter pathology. Here, we explored miRNA involvement in the inflammation-driven alterations of synaptic structure and function, collectively known as synaptopathy, a neuropathological process contributing to excitotoxic neurodegeneration in MS/EAE. Particularly, we observed that miR-142-3p is increased in the CSF of patients with active MS and in EAE brains. We propose miR-142-3p as a molecular mediator of the IL-1ß-dependent downregulation of the glial glutamate-aspartate transporter (GLAST), which causes an enhancement of the glutamatergic transmission in the EAE cerebellum. The synaptic abnormalities mediated by IL-1ß and the clinical and neuropathological manifestations of EAE disappeared in miR-142 knock-out mice. Furthermore, we observed that in vivo miR-142-3p inhibition, either by a preventive and local treatment or by a therapeutic and systemic strategy, abolished IL-1ß- and GLAST-dependent synaptopathy in EAE wild-type mice. Consistently, miR-142-3p was responsible for the glutamatergic synaptic alterations caused by CSF of patients with MS, and CSF levels of miR-142-3p correlated with prospective MS disease progression. Our findings highlight miR-142-3p as key molecular player in IL-1ß-mediated synaptic dysfunction, possibly leading to excitotoxic damage in both EAE and MS diseases. Inhibition of miR-142-3p could be neuroprotective in MS. SIGNIFICANCE STATEMENT: Current studies suggest the role of glutamate excitotoxicity in the development and progression of multiple sclerosis (MS) and of its mouse model experimental autoimmune encephalomyelitis (EAE). The molecular mechanisms linking inflammation and synaptic alterations in MS/EAE are still unknown. Here, we identified miR-142-3p as a determinant molecular actor in inflammation-dependent synaptopathy typical of both MS and EAE. miR-142-3p was upregulated in the CSF of MS patients and in EAE cerebellum. Inhibition of miR-142-3p, locally in EAE brain and in a MS chimeric ex vivo model, recovered glutamatergic synaptic enhancement typical of EAE/MS. We proved that miR-142-3p promoted the IL-1ß-dependent glutamate dysfunction by targeting glutamate-aspartate transporter (GLAST), a crucial glial transporter involved in glutamate homeostasis. Finally, we suggest miR-142-3p as a negative prognostic factor in patients with relapsing-remitting multiple sclerosis.
Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Interleucina-1beta/biossíntese , MicroRNAs/biossíntese , Esclerose Múltipla Recidivante-Remitente/metabolismo , Sinapses/metabolismo , Adulto , Animais , Células Cultivadas , Encefalomielite Autoimune Experimental/patologia , Feminino , Técnicas de Introdução de Genes , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/líquido cefalorraquidiano , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/líquido cefalorraquidiano , Esclerose Múltipla Recidivante-Remitente/diagnóstico , Sinapses/patologiaRESUMO
BACKGROUND: Laquinimod is an immunomodulatory drug under clinical investigation for the treatment of the progressive form of multiple sclerosis (MS) with both anti-inflammatory and neuroprotective effects. Excitotoxicity, a prominent pathophysiological feature of MS and of its animal model, experimental autoimmune encephalomyelitis (EAE), involves glutamate transporter (GluT) dysfunction in glial cells. The aim of this study was to assess whether laquinimod might exert direct neuroprotective effects by interfering with the mechanisms of excitotoxicity linked to GluT function impairments in EAE. METHODS: Osmotic minipumps allowing continuous intracerebroventricular (icv) infusion of laquinimod for 4 weeks were implanted into C57BL/6 mice before EAE induction. EAE cerebella were taken to perform western blot and qPCR experiments. For ex vivo experiments, EAE cerebellar slices were incubated with laquinimod before performing electrophysiology, western blot, and qPCR. RESULTS: In vivo treatment with laquinimod attenuated EAE clinical score at the peak of the disease, without remarkable effects on inflammatory markers. In vitro application of laquinimod to EAE cerebellar slices prevented EAE-linked glutamatergic alterations without mitigating astrogliosis and inflammation. Moreover, such treatment induced an increase of Slcla3 mRNA coding for the glial glutamate-aspartate transporter (GLAST) without affecting the protein content. Concomitantly, laquinimod significantly increased the levels of the glial glutamate transporter 1 (GLT-1) protein and pharmacological blockade of GLT-1 function fully abolished laquinimod anti-excitotoxic effect. CONCLUSIONS: Overall, our results suggest that laquinimod protects against glutamate excitotoxicity of the cerebellum of EAE mice by bursting the expression of glial glutamate transporters, independently of its anti-inflammatory effects.
Assuntos
Anti-Inflamatórios/administração & dosagem , Encefalomielite Autoimune Experimental/tratamento farmacológico , Transportador 2 de Aminoácido Excitatório/biossíntese , Ácido Glutâmico/metabolismo , Quinolonas/administração & dosagem , Animais , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Cerebelo/patologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Transportador 1 de Aminoácido Excitatório/biossíntese , Feminino , Infusões Intraventriculares , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Distribuição AleatóriaRESUMO
BACKGROUND: In the early phases of relapsing-remitting multiple sclerosis (RR-MS), a clear correlation between brain lesion load and clinical disability is often lacking, originating the so-called clinico-radiological paradox. Different factors may contribute to such discrepancy. In particular, synaptic plasticity may reduce the clinical expression of brain damage producing enduring enhancement of synaptic strength largely dependent on neurotrophin-induced protein synthesis. Cytokines released by the immune cells during acute inflammation can alter synaptic transmission and plasticity possibly influencing the clinical course of MS. In addition, immune cells may promote brain repair during the post-acute phases, by secreting different growth factors involved in neuronal and oligodendroglial cell survival. Platelet-derived growth factor (PDGF) is a neurotrophic factor that could be particularly involved in clinical recovery. Indeed, PDGF promotes long-term potentiation of synaptic activity in vitro and in MS and could therefore represent a key factor improving the clinical compensation of new brain lesions. The aim of the present study is to explore whether cerebrospinal fluid (CSF) PDGF concentrations at the time of diagnosis may influence the clinical course of RR-MS. METHODS: At the time of diagnosis, we measured in 100 consecutive early MS patients the CSF concentrations of PDGF, of the main pro- and anti-inflammatory cytokines, and of reliable markers of neuronal damage. Clinical and radiological parameters of disease activity were prospectively collected during follow-up. RESULTS: CSF PDGF levels were positively correlated with prolonged relapse-free survival. Radiological markers of disease activity, biochemical markers of neuronal damage, and clinical parameters of disease progression were instead not influenced by PDGF concentrations. Higher CSF PDGF levels were associated with an anti-inflammatory milieu within the central nervous system. CONCLUSIONS: Our results suggest that PDGF could promote a more prolonged relapse-free period during the course of RR-MS, without influencing inflammation reactivation and inflammation-driven neuronal damage and likely enhancing adaptive plasticity.
Assuntos
Esclerose Múltipla/líquido cefalorraquidiano , Fator de Crescimento Derivado de Plaquetas/líquido cefalorraquidiano , Adulto , Citocinas/líquido cefalorraquidiano , Avaliação da Deficiência , Feminino , Seguimentos , Humanos , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/diagnóstico por imagem , Exame Neurológico , Estatísticas não Paramétricas , Adulto JovemRESUMO
Cytokines are constitutively released in the healthy brain by resident myeloid cells to keep proper synaptic plasticity, either in the form of Hebbian synaptic plasticity or of homeostatic plasticity. However, when cytokines dramatically increase, establishing a status of neuroinflammation, the synaptic action of such molecules remarkably interferes with brain circuits of learning and cognition and contributes to excitotoxicity and neurodegeneration. Among others, interleukin-1ß (IL-1ß) and tumor necrosis factor (TNF) are the best studied proinflammatory cytokines in both physiological and pathological conditions and have been invariably associated with long-term potentiation (LTP) (Hebbian synaptic plasticity) and synaptic scaling (homeostatic plasticity), respectively. Multiple sclerosis (MS) is the prototypical neuroinflammatory disease, in which inflammation triggers excitotoxic mechanisms contributing to neurodegeneration. IL-ß and TNF are increased in the brain of MS patients and contribute to induce the changes in synaptic plasticity occurring in MS patients and its animal model, the experimental autoimmune encephalomyelitis (EAE). This review will introduce and discuss current evidence of the role of IL-1ß and TNF in the regulation of synaptic strength at both physiological and pathological levels, in particular speculating on their involvement in the synaptic plasticity changes observed in the EAE brain.
Assuntos
Encefalite/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Interleucina-1beta/metabolismo , Esclerose Múltipla/metabolismo , Plasticidade Neuronal/fisiologia , Sinapses/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Encefalite/patologia , Encefalomielite Autoimune Experimental/patologia , Humanos , Esclerose Múltipla/patologiaRESUMO
Interferon-γ (IFN-γ) has been implicated in the pathogenesis of multiple sclerosis (MS) and in its animal model, experimental autoimmune encephalomyelitis (EAE). The type-1 cannabinoid receptors (CB1Rs) are heavily involved in MS pathophysiology, and a growing body of evidence suggests that mood disturbances reflect specific effects of proinflammatory cytokines on neuronal activity. Here, we investigated whether IFN-γ could exert a role in the anxiety- and depressive-like behavior observed in mice with EAE, and in the modulation of CB1Rs. Anxiety and depression in fact are often diagnosed in MS, and have already been shown to depend on cannabinoid system. We performed biochemical, behavioral and electrophysiological experiments to assess the role of IFN-γ on mood control and on synaptic transmission in mice. Intracerebroventricular delivery of IFN-γ caused a depressive- and anxiety-like behavior in mice, associated with the selective dysfunction of CB1Rs controlling GABA transmission in the striatum. EAE induction was associated with increased striatal expression of IFN-γ, and with CB1R transmission deficits, which were rescued by pharmacological blockade of IFN-γ. IFN-γ was unable to replicate the effects of EAE on excitatory and inhibitory transmission in the striatum, but mimicked the effects of EAE on CB1R function in this brain area. Overall these results indicate that IFN-γ exerts a relevant control on mood, through the modulation of CB1R function. A better understanding of the biological pathways underling the psychological disorders during neuroinflammatory conditions is crucial for developing effective therapeutic strategies.
Assuntos
Ansiedade/induzido quimicamente , Corpo Estriado/efeitos dos fármacos , Depressão/induzido quimicamente , Interferon gama/farmacologia , Nootrópicos/farmacologia , Receptor CB1 de Canabinoide/metabolismo , Afeto/efeitos dos fármacos , Afeto/fisiologia , Animais , Ansiedade/metabolismo , Corpo Estriado/metabolismo , Depressão/metabolismo , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/psicologia , Feminino , Neurônios GABAérgicos/efeitos dos fármacos , Neurônios GABAérgicos/metabolismo , Fatores Imunológicos/farmacologia , Infusões Intraventriculares , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos , Distribuição Aleatória , Técnicas de Cultura de TecidosRESUMO
BACKGROUND: Data from multiple sclerosis (MS) and the MS rodent model, experimental autoimmune encephalomyelitis (EAE), highlighted an inflammation-dependent synaptopathy at the basis of the neurodegenerative damage causing irreversible disability in these disorders. This synaptopathy is characterized by an imbalance between glutamatergic and GABAergic transmission and has been proposed to be a potential therapeutic target. Siponimod (BAF312), a selective sphingosine 1-phosphate1,5 receptor modulator, is currently under investigation in a clinical trial in secondary progressive MS patients. We investigated whether siponimod, in addition to its peripheral immune modulation, may exert direct neuroprotective effects in the central nervous system (CNS) of mice with chronic progressive EAE. METHODS: Minipumps allowing continuous intracerebroventricular (icv) infusion of siponimod for 4 weeks were implanted into C57BL/6 mice subjected to MOG35-55-induced EAE. Electrophysiology, immunohistochemistry, western blot, qPCR experiments, and peripheral lymphocyte counts were performed. In addition, the effect of siponimod on activated microglia was assessed in vitro to confirm the direct effect of the drug on CNS-resident immune cells. RESULTS: Siponimod administration (0.45 µg/day) induced a significant beneficial effect on EAE clinical scores with minimal effect on peripheral lymphocyte counts. Siponimod rescued defective GABAergic transmission in the striatum of EAE, without correcting the EAE-induced alterations of glutamatergic transmission. We observed a significant attenuation of astrogliosis and microgliosis together with reduced lymphocyte infiltration in the striatum of EAE mice treated with siponimod. Interestingly, siponimod reduced the release of IL-6 and RANTES from activated microglial cells in vitro, which might explain the reduced lymphocyte infiltration. Furthermore, the loss of parvalbumin-positive (PV+) GABAergic interneurons typical of EAE brains was rescued by siponimod treatment, providing a plausible explanation of the selective effects of this drug on inhibitory synaptic transmission. CONCLUSIONS: Altogether, our results show that siponimod has neuroprotective effects in the CNS of EAE mice, which are likely independent of its peripheral immune effect, suggesting that this drug could be effective in limiting neurodegenerative pathological processes in MS.
Assuntos
Azetidinas/uso terapêutico , Compostos de Benzil/uso terapêutico , Encefalomielite Autoimune Experimental/complicações , Degeneração Neural , Fármacos Neuroprotetores/uso terapêutico , Sinapses/fisiologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Azetidinas/farmacologia , Compostos de Benzil/farmacologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Transformada , Córtex Cerebral/citologia , Citocinas/farmacologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Adjuvante de Freund/imunologia , Adjuvante de Freund/toxicidade , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Glicoproteína Mielina-Oligodendrócito/imunologia , Glicoproteína Mielina-Oligodendrócito/toxicidade , Degeneração Neural/etiologia , Degeneração Neural/patologia , Degeneração Neural/prevenção & controle , Condução Nervosa/efeitos dos fármacos , Condução Nervosa/fisiologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/toxicidade , Sinapses/efeitos dos fármacos , Potenciais Sinápticos/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T/patologia , Substância Branca/efeitos dos fármacos , Substância Branca/patologiaRESUMO
BACKGROUND: Mood disorders, including anxiety and depression, are frequently diagnosed in multiple sclerosis (MS) patients, even independently of the disabling symptoms associated with the disease. Anatomical, biochemical, and pharmacological evidence indicates that type-1 cannabinoid receptor (CB1R) is implicated in the control of emotional behavior and is modulated during inflammatory neurodegenerative diseases such as MS and experimental autoimmune encephalomyelitis (EAE). METHODS: We investigated whether CB1R could exert a role in anxiety-like behavior in mice with EAE. We performed behavioral, pharmacological, and electrophysiological experiments to explore the link between central inflammation, mood, and CB1R function in EAE. RESULTS: We observed that EAE-induced anxiety was associated with the downregulation of CB1R-mediated control of striatal GABA synaptic transmission and was exacerbated in mice lacking CB1R (CB1R-KO mice). Central blockade of interleukin-1ß (IL-1ß) reversed the anxiety-like phenotype of EAE mice, an effect associated with the concomitant rescue of dopamine (DA)-regulated spontaneous behavior, and DA-CB1R neurotransmission, leading to the rescue of striatal CB1R sensitivity. CONCLUSIONS: Overall, results of the present investigation indicate that synaptic dysfunction linked to CB1R is involved in EAE-related anxiety and motivation-based behavior and contribute to clarify the complex neurobiological mechanisms underlying mood disorders associated to MS.
Assuntos
Ansiedade/etiologia , Ansiedade/metabolismo , Encefalomielite Autoimune Experimental/complicações , Interleucina-1beta/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Anfetamina/farmacologia , Animais , Antirreumáticos/administração & dosagem , Antirreumáticos/farmacologia , Ansiedade/patologia , Estimulantes do Sistema Nervoso Central/farmacologia , Córtex Cerebral/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Adaptação à Escuridão/efeitos dos fármacos , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/tratamento farmacológico , Antagonistas de Aminoácidos Excitatórios/farmacologia , Comportamento Exploratório/efeitos dos fármacos , Feminino , Proteína Antagonista do Receptor de Interleucina 1/administração & dosagem , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito/imunologia , Glicoproteína Mielina-Oligodendrócito/toxicidade , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/toxicidade , Receptor CB1 de Canabinoide/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Mood disturbances are frequent in patients with multiple sclerosis (MS), even in non-disabled patients and in the remitting stages of the disease. It is still largely unknown how the pathophysiological process on MS causes anxiety and depression, but the dopaminergic system is likely involved. Aim of the present study was to investigate depressive-like behavior in mice with experimental autoimmune encephalomyelitis (EAE), a model of MS, and its possible link to dopaminergic neurotransmission. Behavioral, amperometric and biochemical experiments were performed to determine the role of inflammation in mood control in EAE. First, we assessed the independence of mood alterations from motor disability during the acute phase of the disease, by showing a depressive-like behavior in EAE mice with mild clinical score and preserved motor skills (mild-EAE). Second, we linked such behavioral changes to the selective increased striatal expression of interleukin-1beta (IL-1ß) in a context of mild inflammation and to dopaminergic system alterations. Indeed, in the striatum of EAE mice, we observed an impairment of dopamine (DA) neurotransmission, since DA release was reduced and signaling through DA D1- and D2-like receptors was unbalanced. In conclusion, the present study provides first evidence of the link between the depressive-like behavior and the alteration of dopaminergic system in EAE mice, raising the possibility that IL-1ß driven dysfunction of dopaminergic signaling might play a role in mood disturbances also in MS patients.
Assuntos
Corpo Estriado/imunologia , Depressão/metabolismo , Dopamina/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/psicologia , Interleucina-1beta/metabolismo , Doença Aguda , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/imunologia , Tonsila do Cerebelo/patologia , Animais , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Depressão/tratamento farmacológico , Depressão/patologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/imunologia , Hipocampo/patologia , Interleucina-1beta/antagonistas & inibidores , Camundongos Endogâmicos C57BL , Destreza Motora , RNA Mensageiro/metabolismo , Distribuição Aleatória , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Índice de Gravidade de Doença , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
Cerebellar deficit contributes significantly to disability in multiple sclerosis (MS). Several clinical and experimental studies have investigated the pathophysiology of cerebellar dysfunction in this neuroinflammatory disorder, but the cellular and molecular mechanisms are still unclear. In experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, proinflammatory cytokines, together with a degeneration of inhibitory neurons, contribute to impair GABAergic transmission at Purkinje cells (PCs). Here, we investigated glutamatergic transmission to gain insight into the pathophysiology of cerebellar dysfunction in EAE. Electrophysiological recordings from PCs showed increased duration of spontaneous excitatory postsynaptic currents (EPSCs) during the symptomatic phase of EAE, suggesting an alteration of glutamate uptake played by Bergmann glia. We indeed observed an impaired functioning of the glutamate-aspartate transporter/excitatory amino acid transporter 1 (GLAST/EAAT1) in EAE cerebellum caused by protein downregulation and in correlation with prominent astroglia activation. We have also demonstrated that the proinflammatory cytokine interleukin-1ß (IL-1ß), released by a subset of activated microglia/macrophages and infiltrating lymphocytes, was involved directly in such synaptic alteration. In fact, brief incubation of IL-1ß in normal cerebellar slices replicated EAE modifications through a rapid GLAST/EAAT1 downregulation, whereas incubation of an IL-1 receptor antagonist (IL-1ra) in EAE slices reduced spontaneous EPSC alterations. Finally, EAE mice treated with intracerebroventricular IL-1ra showed normal glutamatergic and GABAergic transmissions, along with GLAST/EAAT1 normalization, milder inflammation, and reduced motor deficits. These results highlight the crucial role played by the proinflammatory IL-1ß in triggering molecular and synaptic events involved in neurodegenerative processes that characterize neuroinflammatory diseases such as MS.
Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Ácido Glutâmico/metabolismo , Interleucina-1beta/farmacologia , Células de Purkinje/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Animais , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Encefalomielite Autoimune Experimental/fisiopatologia , Transportador 1 de Aminoácido Excitatório/metabolismo , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Camundongos , Células de Purkinje/metabolismo , Sinapses/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
The endocannabinoid system (ECS) is critically involved in the pathophysiology of Multiple Sclerosis (MS), a neuroinflammatory and neurodegenerative disease of the central nervous system (CNS). Over the past decade, researchers have extensively studied the neuroprotective and anti-inflammatory effects of the ECS. Inhibiting the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) has emerged as a promising strategy to mitigate brain damage in MS. In this study, we investigated the effects of a novel reversible MAGL inhibitor (MAGLi 432) on C57/BL6 female mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. We assessed its implications on motor disability, neuroinflammation, and synaptic dysfunction. Systemic in vivo treatment with MAGLi 432 resulted in a less severe EAE disease, accompanied by increased 2-AG levels and decreased levels of arachidonic acid (AA) and prostaglandins (PGs) in the brain. Additionally, MAGLi 432 reduced both astrogliosis and microgliosis, as evidenced by decreased microglia/macrophage density and a less reactive morphology. Flow cytometry analysis further revealed fewer infiltrating CD45+ and CD3+ cells in the brains of MAGLi 432-treated EAE mice. Finally, MAGLi treatment counteracted the striatal synaptic hyperexcitability promoted by EAE neuroinflammation. In conclusion, MAGL inhibition significantly ameliorated EAE clinical disability and striatal inflammatory synaptopathy through potent anti-inflammatory effects. These findings provide new mechanistic insights into the neuroprotective role of the ECS during neuroinflammation and highlight the therapeutic potential of MAGLi-based drugs in mitigating MS-related inflammatory and neurodegenerative brain damage.
Assuntos
Ácidos Araquidônicos , Encefalomielite Autoimune Experimental , Endocanabinoides , Glicerídeos , Camundongos Endogâmicos C57BL , Animais , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Glicerídeos/metabolismo , Camundongos , Endocanabinoides/metabolismo , Ácidos Araquidônicos/farmacologia , Ácidos Araquidônicos/metabolismo , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/patologia , Sinapses/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/metabolismoRESUMO
Proinflammatory cytokines are implicated in promoting neurodegeneration in multiple sclerosis (MS) by affecting excitatory and inhibitory transmission at central synapses. Conversely, the synaptic effects of anti-inflammatory molecules remain underexplored, despite their potential neuroprotective properties and their presence in the cerebrospinal fluid (CSF) of patients. In a study involving 184 newly diagnosed relapsing-remitting (RR)-MS patients, we investigated whether CSF levels of the anti-inflammatory interleukin (IL)-10 were linked to disease severity and neurodegeneration measures. Additionally, we examined IL-10 impact on synaptic transmission in striatal medium spiny neurons and its role in counteracting inflammatory synaptopathy induced by IL-1ß in female C57BL/6 mice with experimental autoimmune encephalomyelitis (EAE). Our findings revealed a significant positive correlation between IL-10 CSF levels and changes in EDSS (Expanded Disability Status Scale) scores one year after MS diagnosis. Moreover, IL-10 levels in the CSF were positively correlated with volumes of specific subcortical brain structures, such as the nucleus caudate. In both MS patients' CSF and EAE mice striatum, IL-10 and IL-1ß expressions were upregulated, suggesting possible antagonistic effects of these cytokines. Notably, IL-10 exhibited the ability to decrease glutamate transmission, increase GABA transmission in the striatum, and reverse IL-1ß-induced abnormal synaptic transmission in EAE. In conclusion, our data suggest that IL-10 exerts direct neuroprotective effects in MS patients by modulating both excitatory and inhibitory transmission and attenuating IL-1ß-induced inflammatory synaptopathy. These findings underscore the potential therapeutic significance of IL-10 in mitigating neurodegeneration in MS.
RESUMO
Background: Secondary progressive multiple sclerosis (SPMS) is defined by the irreversible accumulation of disability following a relapsing-remitting MS (RRMS) course. Despite treatments advances, a reliable tool able to capture the transition from RRMS to SPMS is lacking. A T cell chimeric MS model demonstrated that T cells derived from relapsing patients exacerbate excitatory transmission of central neurons, a synaptotoxic event absent during remitting stages. We hypothesized the re-emergence of T cell synaptotoxicity during SPMS and investigated the synaptoprotective effects of siponimod, a sphingosine 1-phosphate receptor (S1PR) modulator, known to reduce grey matter damage in SPMS patients. Methods: Data from healthy controls (HC), SPMS patients, and siponimod-treated SPMS patients were collected. Chimeric experiments were performed incubating human T cells on murine cortico-striatal slices, and recording spontaneous glutamatergic activity from striatal neurons. Homologous chimeric experiments were executed incubating EAE mice T cells with siponimod and specific S1PR agonists or antagonists to identify the receptor involved in siponimod-mediated synaptic recovery. Results: SPMS patient-derived T cells significantly increased the striatal excitatory synaptic transmission (n=40 synapses) compared to HC T cells (n=55 synapses), mimicking the glutamatergic alterations observed in active RRMS-T cells. Siponimod treatment rescued SPMS T cells synaptotoxicity (n=51 synapses). Homologous chimeric experiments highlighted S1P5R involvement in the siponimod's protective effects. Conclusion: Transition from RRMS to SPMS involves the reappearance of T cell-mediated synaptotoxicity. Siponimod counteracts T cell-induced excitotoxicity, emphasizing the significance of inflammatory synaptopathy in progressive MS and its potential as a promising pharmacological target.
Assuntos
Azetidinas , Compostos de Benzil , Esclerose Múltipla Crônica Progressiva , Sinapses , Linfócitos T , Humanos , Animais , Camundongos , Feminino , Esclerose Múltipla Crônica Progressiva/imunologia , Esclerose Múltipla Crônica Progressiva/tratamento farmacológico , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/efeitos dos fármacos , Azetidinas/farmacologia , Azetidinas/uso terapêutico , Compostos de Benzil/farmacologia , Compostos de Benzil/uso terapêutico , Masculino , Adulto , Sinapses/metabolismo , Pessoa de Meia-Idade , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Moduladores do Receptor de Esfingosina 1 Fosfato/farmacologia , Moduladores do Receptor de Esfingosina 1 Fosfato/uso terapêutico , Camundongos Endogâmicos C57BL , Receptores de Esfingosina-1-Fosfato/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologiaRESUMO
OBJECTIVE: Accumulating evidence suggests that dysfunctional adipose tissue (AT) plays a major role in the risk of developing multiple sclerosis (MS), the most common immune-mediated and demyelinating disease of the central nervous system. However, the contribution of adipose tissue to the etiology and progression of MS is still obscure. This study aimed at deciphering the responses of AT in experimental autoimmune encephalomyelitis (EAE), the best characterized animal model of MS. RESULTS AND METHODS: We observed a significant AT loss in EAE mice at the onset of disease, with a significant infiltration of M1-like macrophages and fibrosis in the AT, resembling a cachectic phenotype. Through an integrative and multilayered approach, we identified lipocalin2 (LCN2) as the key molecule released by dysfunctional adipocytes through redox-dependent mechanism. Adipose-derived LCN2 shapes the pro-inflammatory macrophage phenotype, and the genetic deficiency of LCN2 specifically in AT reduced weight loss as well as inflammatory macrophage infiltration in spinal cord in EAE mice. Mature adipocytes downregulating LCN2 reduced lipolytic response to inflammatory stimuli (e.g. TNFα) through an ATGL-mediated mechanism. CONCLUSIONS: Overall data highlighted a role LCN2 in exacerbating inflammatory phenotype in EAE model, suggesting a pathogenic role of dysfunctional AT in MS.