RESUMO
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: 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
A significant proportion of multiple sclerosis (MS) patients have functionally relevant cerebellar deficits, which significantly contribute to disability. Although clinical and experimental studies have been conducted to understand the pathophysiology of cerebellar dysfunction in MS, no electrophysiological and morphological studies have investigated potential alterations of synaptic connections of cerebellar Purkinje cells (PC). For this reason we analyzed cerebellar PC GABAergic connectivity in mice with MOG((35-55))-induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. We observed a strong reduction in the frequency of the spontaneous inhibitory post-synaptic currents (IPSCs) recorded from PCs during the symptomatic phase of the disease, and in presence of prominent microglia activation not only in the white matter (WM) but also in the molecular layer (ML). The massive GABAergic innervation on PCs from basket and stellate cells was reduced and associated to a decrease of the number of these inhibitory interneurons. On the contrary no significant loss of the PCs could be detected. Incubation of interleukin-1beta (IL-1ß) was sufficient to mimic the electrophysiological alterations observed in EAE mice. We thus suggest that microglia and pro-inflammatory cytokines, together with a degeneration of basket and stellate cells and their synaptic terminals, contribute to impair GABAergic transmission on PCs during EAE. Our results support a growing body of evidence that GABAergic signaling is compromised in EAE and in MS, and show a selective susceptibility to neuronal and synaptic degeneration of cerebellar inhibitory interneurons.
Assuntos
Encefalomielite Autoimune Experimental/fisiopatologia , Neurônios GABAérgicos/patologia , Células de Purkinje/fisiologia , Transdução de Sinais/fisiologia , Animais , Encefalomielite Autoimune Experimental/patologia , Feminino , Neurônios GABAérgicos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Terminações Pré-Sinápticas/patologia , Terminações Pré-Sinápticas/fisiologia , Células de Purkinje/patologiaRESUMO
Transient receptor potential vanilloid 1 (TRPV1) channels are involved in several inflammatory diseases. However, their action is still controversial, and both pro-inflammatory and anti-inflammatory roles have been described. We used a strain of TRPV1-KO mice to characterize the role of these channels in experimental autoimmune encephalomyelitis (EAE), which models multiple sclerosis (MS) in mice. EAE mice showed higher lethality in the peak phase of the disease and a better recovery of the surviving animals in the chronic stages, compared to their wild-type (WT) counterparts. By means of whole-cell patch clamp experiments in corticostriatal brain slices, we found that the absence of TRPV1 channels exacerbated the defect of glutamate transmission occurring in the peak phase of EAE, and attenuated the alterations of GABA synapses in the chronic phase of EAE, thus paralleling the dual effects of TRPV1-KO on the motor deficits of EAE mice. Furthermore, in slices from non-EAE mice, we found that genetic or pharmacological blockade of TRPV1 channels enhanced the synaptic effects of tumor necrosis factor α (TNF-α) on glutamate-mediated excitatory postsynaptic currents, and prevented the action of interleukin 1ß (IL-1ß) on GABAergic inhibitory postsynaptic currents. Together, our results suggest that TRPV1 channels contrast TNF-α-mediated synaptic deficits in the peak phase of EAE and, in the chronic stages, enhance IL-1ß-induced GABAergic defects. The opposing interplay with the synaptic actions of the two major pro-inflammatory cytokines might explain the bimodal effects of TRPV1 ablation on the motor deficits of EAE, and suggests that the inflammatory milieu determines whether TRPV1 channels exert preferentially aversive or protective effects on neurons during neuroinflammatory diseases.
Assuntos
Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/prevenção & controle , Interleucina-1beta/fisiologia , Sinapses/fisiologia , Canais de Cátion TRPV/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Modelos Animais de Doenças , Regulação para Baixo/genética , Encefalomielite Autoimune Experimental/patologia , Potenciais Pós-Sinápticos Excitadores/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Inflamação/genética , Inflamação/metabolismo , Inflamação/fisiopatologia , Potenciais Pós-Sinápticos Inibidores/genética , Potenciais Pós-Sinápticos Inibidores/fisiologia , Interleucina-1beta/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fármacos Neuroprotetores/antagonistas & inibidores , Fármacos Neuroprotetores/farmacologia , Técnicas de Cultura de Órgãos , Sinapses/genética , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Regulação para Cima/genéticaRESUMO
Multiple sclerosis (MS) has long been regarded as a chronic inflammatory disease of the white matter that leads to demyelination and eventually to neurodegeneration. In the past decade, several aspects of MS pathogenesis have been challenged, and degenerative changes of the grey matter, which are independent of demyelination, have become a topic of interest. CNS inflammation in MS and experimental autoimmune encephalomyelitis (EAE; a disease model used to study MS in rodents) causes a marked imbalance between GABAergic and glutamatergic transmission, and a loss of synapses, all of which leads to a diffuse 'synaptopathy'. Altered synaptic transmission can occur early in MS and EAE, independently of demyelination and axonal loss, and subsequently causes excitotoxic damage. Inflammation-driven synaptic abnormalities are emerging as a prominent pathogenic mechanism in MS-importantly, they are potentially reversible and, therefore, represent attractive therapeutic targets. In this Review, we focus on the connection between inflammation and synaptopathy in MS and EAE, which sheds light not only on the pathophysiology of MS but also on that of primary neurodegenerative disorders in which inflammatory processes contribute to disease progression.
Assuntos
Encefalite/etiologia , Encefalite/patologia , Esclerose Múltipla/complicações , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/patologia , Sinapses/patologia , Animais , HumanosRESUMO
Type-1 cannabinoid receptors (CB1R) are important regulators of the neurodegenerative damage in multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis (EAE). In GABAergic striatal neurons, CB1R stimulation exerts protective effects by limiting inflammation-induced potentiation of glutamate-mediated spontaneous excitatory postsynaptic currents (sEPSCs). Here we show that CB1R located on GABAergic or on glutamatergic neurons are differentially involved in the pre- and postsynaptic alterations of sEPSCs caused by EAE in the striatum. After induction of EAE, mice selectively lacking CB1R on GABAergic neurons (GABA-CB1R-KO) showed exacerbated alterations of sEPSC duration in GABAergic medium spiny neurons (MSN). On the other hand, EAE-induced alterations of corticostriatal sEPSC frequency were exacerbated only in mice lacking CB1R on glutamatergic neurons (Glu-CB1R-KO), indicating that this subset of receptors controls the effects of inflammation on glutamate release. While EAE severity was enhanced in whole CB1R-KO mice, GABA-CB1R-KO and Glu-CB1R-KO mice had similar motor deficits as the respective wild-type (WT) counterparts. Our results provide further evidence that CB1R are involved in EAE pathophysiology, and suggest that both pre- and postsynaptic alterations of glutamate transmission are important to drive excitotoxic neurodegeneration typical of this disorder.
Assuntos
Corpo Estriado/fisiopatologia , Encefalomielite Autoimune Experimental/fisiopatologia , Ácido Glutâmico/metabolismo , Neurônios/fisiologia , Receptor CB1 de Canabinoide/metabolismo , Transmissão Sináptica/fisiologia , Animais , Progressão da Doença , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Terminações Pré-Sinápticas/fisiologia , Fatores de Tempo , Ácido gama-Aminobutírico/metabolismoRESUMO
Glutamate-mediated excitotoxicity is supposed to induce neurodegeneration in multiple sclerosis (MS). Glatiramer acetate (GA) is an immunomodulatory agent used in MS treatment with potential neuroprotective action. Aim of the present study was to investigate whether GA has effects on glutamate transmission alterations occurring in experimental autoimmune encephalomyelitis (EAE), to disclose a possible mechanism of GA-induced neuroprotection in this mouse model of MS. Single neuron electrophysiological recordings and immunofluorescence analysis of microglia activation were performed in the striatum of EAE mice, treated or not with GA, at different stages of the disease. GA treatment was able to reverse the tumor necrosis factor-α (TNF-α)-induced alterations of striatal glutamate-mediated excitatory postsynaptic currents (EPSCs) of EAE mice. Incubation of striatal slices of control animals with lymphocytes taken from EAE mice treated with GA failed to replicate such an anti-glutamatergic effect, while activated microglial cells stimulated with GA in vitro mimicked the effect of GA treatment of EAE mice. Consistently, EAE mice treated with GA had less microglial activation and less TNF-α expression than untreated EAE animals. Furthermore, direct application of GA to EAE slices replicated the in vivo protective activity of GA. Our results show that GA is neuroprotective against glutamate toxicity independently of its peripheral immunodulatory action, and through direct modulation of microglial activation and TNF-α release in the grey matter of EAE and possibly of MS brains.
Assuntos
Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/prevenção & controle , Imunossupressores/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Peptídeos/uso terapêutico , Sinapses/patologia , Animais , Células Cultivadas , Feminino , Acetato de Glatiramer , Imunossupressores/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/farmacologia , Peptídeos/farmacologia , Sinapses/efeitos dos fármacosRESUMO
Alterations of glutamate-mediated synaptic transmission occur in both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), the animal model of MS. Here we investigated whether intracerebroventricular (Icv) administration of cladribine has effects on EAE. Icv infusion of cladribine reduced the clinical deficits of EAE mice and reversed EAE-induced enhancement of excitatory postsynaptic current (sEPSC) frequency, a neurophysiological measure of glutamatergic synaptopathy associated with central inflammation. Cladribine failed to interfere with EAE-induced microglial and astroglial activation, but blocked EAE synaptic alterations by interfering with interleukin-1ß effects. Cladribine possesses neuroprotective properties in experimental MS that are independent of its peripheral immunosuppressant action.
Assuntos
Cladribina/uso terapêutico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Imunossupressores/uso terapêutico , Interleucina-1beta/metabolismo , Sinapses/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Córtex Cerebral/citologia , Quelantes/farmacologia , Corpo Estriado/citologia , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Ácido Glutâmico/farmacologia , Imunossupressores/farmacologia , Técnicas In Vitro , Interleucina-1beta/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/efeitos dos fármacos , Técnicas de Patch-ClampRESUMO
Multiple sclerosis (MS) causes a variety of motor and sensory deficits and it is also associated with mood disturbances. It is unclear if anxiety and depression in MS entirely reflect a subjective reaction to a chronic disease causing motor disability or rather depend on specific effects of neuroinflammation in neuronal circuits. To answer this question, behavioral, electrophysiological, and immunofluorescence experiments were performed in mice with experimental autoimmune encephalomyelitis (EAE), which models MS in mice. First, we observed high anxiety indexes in EAE mice, preceding the appearance of motor defects. Then, we demonstrated that tumor necrosis factor α (TNF-α) has a crucial role in anxiety associated with neuroinflammation. In fact, intracerebroventricular (icv) administration of etanercept, an inhibitor of TNF-α signaling, resulted in anxiolytic-like effects in EAE-mice. Accordingly, icv injection of TNF-α induced per se overt anxious behavior in control mice. Moreover, we propose the striatum as one of the brain regions potentially involved in EAE anxious behavior. We observed that before disease onset EAE striatum presents elevated TNF-α levels and strong activated microglia, early signs of inflammation associated with alterations of striatal excitatory postsynaptic currents (EPSCs). Interestingly, etanercept corrected the synaptic defects of pre-symptomatic EAE mice while icv injection of TNF-α in non-EAE mice altered EPSCs, thus mimicking the synaptic effects of EAE. In conclusion, anxiety characterizes EAE course since the very early phases of the disease. TNF-α released from activated microglia mediates this effect likely through the modulation of striatal excitatory synaptic transmission.