RESUMEN
Multiple sclerosis (MS), an autoimmune-driven, inflammatory demyelinating disease of the central nervous system (CNS), causes irreversible accumulation of neurological deficits to a variable extent. Although there are potent disease-modifying agents for its initial relapsing-remitting phase, immunosuppressive therapies show limited efficacy in secondary progressive MS (SPMS). Although modulation of sphingosine-1 phosphate receptors has proven beneficial during SPMS, the underlying mechanisms are poorly understood. In this project, we followed the hypothesis that siponimod, a sphingosine-1 phosphate receptor modulator, exerts protective effects by direct modulation of glia cell function (i.e., either astrocytes, microglia, or oligodendrocytes). To this end, we used the toxin-mediated, nonautoimmune MS animal model of cuprizone (Cup) intoxication. On the histological level, siponimod ameliorated cuprizone-induced oligodendrocyte degeneration, demyelination, and axonal injury. Protective effects were evident as well using GE180 translocator protein 18-kDa (TSPO) imaging with positron emission tomography (PET)/computed tomography (CT) imaging or next generation sequencing (NGS). Siponimod also ameliorated the cuprizone-induced pathologies in Rag1-deficient mice, demonstrating that the protection is independent of T and B cell modulation. Proinflammatory responses in primary mixed astrocytes/microglia cell cultures were not modulated by siponimod, suggesting that other cell types than microglia and astrocytes are targeted. Of note, siponimod completely lost its protective effects in S1pr5-deficient mice, suggesting direct protection of degenerating oligodendrocytes. Our study demonstrates that siponimod exerts protective effects in the brain in a S1PR5-dependent manner. This finding is not just relevant in the context of MS but in other neuropathologies as well, characterized by a degeneration of the axon-myelin unit.
Asunto(s)
Azetidinas , Compuestos de Bencilo , Esclerosis Múltiple Crónica Progresiva , Oligodendroglía , Receptores de Esfingosina-1-Fosfato , Esfingosina , Animales , Azetidinas/farmacología , Compuestos de Bencilo/farmacología , Cuprizona , Modelos Animales de Enfermedad , Proteínas de Homeodominio/genética , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple Crónica Progresiva/tratamiento farmacológico , Oligodendroglía/efectos de los fármacos , Esfingosina/farmacología , Esfingosina/uso terapéutico , Receptores de Esfingosina-1-Fosfato/metabolismoRESUMEN
Accumulating evidence suggests that a degenerative processes within the brain can trigger the formation of new, focal inflammatory lesions in Multiple Sclerosis (MS). Here, we used a novel pre-clinical MS animal model to test whether the amelioration of degenerative brain events reduces the secondary recruitment of peripheral immune cells and, in consequence, inflammatory lesion development. Neural degeneration was induced by a 3â¯weeks cuprizone intoxication period. To mitigate the cuprizone-induced pathology, animals were treated with Laquinimod (25â¯mg/kg) during the cuprizone-intoxication period. At the beginning of week 6, encephalitogenic T cell development in peripheral lymphoid organs was induced by the immunization with myelin oligodendrocyte glycoprotein 35-55 peptide (i.e., Cup/EAE). Demyelination, axonal injury and reactive gliosis were determined by immunohistochemistry. Positron emission tomography (PET) imaging was performed to analyze glia activation in vivo. Vehicle-treated cuprizone mice displayed extensive callosal demyelination, glia activation and enhanced TSPO-ligand binding. This cuprizone-induced pathology was profoundly ameliorated in mice treated with Laquinimod. In vehicle-treated Cup/EAE mice, the cuprizone-induced pathology triggered massive peripheral immune cell recruitment into the forebrain, evidenced by multifocal perivascular inflammation, glia activation and neuro-axonal injury. While anti myelin oligodendrocyte glycoprotein 35-55 peptide immune responses were comparable in vehicle- and Laquinimod-treated Cup/EAE mice, the cuprizone-triggered immune cell recruitment was ameliorated by the Laquinimod treatment. This study clearly illustrates that amelioration of a primary brain-intrinsic degenerative process secondary halts peripheral immune cell recruitment and, in consequence, inflammatory lesion development. These findings have important consequences for the interpretation of the results of clinical studies.
Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/patología , Encefalitis/inmunología , Encefalitis/patología , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Quinolonas/administración & dosificación , Animales , Cuprizona/administración & dosificación , Modelos Animales de Enfermedad , Encefalitis/inducido químicamente , Femenino , Gliosis/inducido químicamente , Gliosis/patología , Ratones Endogámicos C57BLRESUMEN
Oligodendrocyte degeneration is a hallmark of multiple sclerosis pathology, and protecting oligodendrocytes and myelin is likely to be of clinical relevance. Traditionally, oligodendrocyte and myelin degeneration are viewed as a direct consequence of an inflammatory attack, but metabolic defects might be equally important. Appropriate animal models to study the interplay of inflammation and metabolic injury are, therefore, needed. Here, we describe that in spite of its immunosuppressive effects, a continuous intoxication with cuprizone allows the induction of active experimental autoimmune encephalomyelitis (EAE) by myelin oligodendrocyte glycoprotein (MOG35-55) immunization. Although the clinical severity of EAE is ameliorated in cuprizone-intoxicated mice, the recruitment of granulocytes, and especially, CD3+ lymphocytes into the forebrain is triggered by the cuprizone insult. Such combined lesions are further characterized by oligodendrocyte apoptosis and microglia activation, closely mimicking type III multiple sclerosis lesions. In summary, we provide a protocol that allows to study the direct interplay of immune-mediated and metabolic oligodendrocyte injury and its consequences for the cerebral white and grey matters.
Asunto(s)
Cuprizona/toxicidad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Administración Oral , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Cuprizona/administración & dosificación , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Ratones , Ratones Endogámicos C57BL , Glicoproteína Mielina-Oligodendrócito/inmunología , Oligodendroglía/efectos de los fármacos , Oligodendroglía/inmunología , Oligodendroglía/patología , Fragmentos de Péptidos/inmunologíaRESUMEN
Obesity is characterized by immoderate fat accumulation leading to an elevated risk of neurodegenerative disorders, along with a host of metabolic disturbances. Chronic neuroinflammation is a main factor linking obesity and the propensity for neurodegenerative disorders. To determine the cerebrometabolic effects of diet-induced obesity (DIO) in female mice fed a long-term (24 weeks) high-fat diet (HFD, 60% fat) compared to a group on a control diet (CD, 20% fat), we used in vivo PET imaging with the radiotracer [18F]FDG as a marker for brain glucose metabolism. In addition, we determined the effects of DIO on cerebral neuroinflammation using translocator protein 18 kDa (TSPO)-sensitive PET imaging with [18F]GE-180. Finally, we performed complementary post mortem histological and biochemical analyses of TSPO and further microglial (Iba1, TMEM119) and astroglial (GFAP) markers as well as cerebral expression analyses of cytokines (e.g., Interleukin (IL)-1ß). We showed the development of a peripheral DIO phenotype, characterized by increased body weight, visceral fat, free triglycerides and leptin in plasma, as well as increased fasted blood glucose levels. Furthermore, we found obesity-associated hypermetabolic changes in brain glucose metabolism in the HFD group. Our main findings with respect to neuroinflammation were that neither [18F]GE-180 PET nor histological analyses of brain samples seem fit to detect the predicted cerebral inflammation response, despite clear evidence of perturbed brain metabolism along with elevated IL-1ß expression. These results could be interpreted as a metabolically activated state in brain-resident immune cells due to a long-term HFD.
Asunto(s)
Dieta Alta en Grasa , Enfermedades Neurodegenerativas , Ratones , Femenino , Animales , Dieta Alta en Grasa/efectos adversos , Enfermedades Neuroinflamatorias , Obesidad/diagnóstico por imagen , Obesidad/metabolismo , Proteínas Portadoras , Glucosa , Tomografía de Emisión de Positrones/métodos , Ratones Endogámicos C57BLRESUMEN
Oligodendrocytes, the myelinating cells of the central nervous system, orchestrate several key cellular functions in the brain and spinal cord, including axon insulation, energy transfer to neurons, and, eventually, modulation of immune responses. There is growing interest for obtaining reliable markers that can specifically label oligodendroglia and their progeny. In many studies, anti-CC1 antibodies, presumably recognizing the protein adenomatous polyposis coli (APC), are used to label mature, myelinating oligodendrocytes. However, it has been discussed whether anti-CC1 antibodies could recognize as well, under pathological conditions, other cell populations, particularly astrocytes. In this study, we used transgenic mice in which astrocytes are labeled by the enhanced green fluorescent protein (eGFP) under the control of the human glial fibrillary acidic protein (GFAP) promoter. By detailed co-localization studies we were able to demonstrate that a significant proportion of eGFP-expressing cells co-express markers of the oligodendrocyte lineage, such as the transcription factor Oligodendrocyte Transcription Factor 2 (OLIG2); the NG2 proteoglycan, also known as chrondroitin sulfate proteoglycan 4 (CSPG4); or APC. The current finding that the GFAP promoter drives transgene expression in cells of the oligodendrocyte lineage should be considered when interpreting results from co-localization studies.
Asunto(s)
Linaje de la Célula , Genes Reporteros , Proteína Ácida Fibrilar de la Glía/genética , Proteínas Fluorescentes Verdes/genética , Oligodendroglía/metabolismo , Animales , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Endogámicos C57BL , Oligodendroglía/citología , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransgenesRESUMEN
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system. Various pre-clinical models with different specific features of the disease are available to study MS pathogenesis and to develop new therapeutic options. During the last decade, the model of toxic demyelination induced by cuprizone has become more and more popular, and it has contributed substantially to our understanding of distinct yet important aspects of the MS pathology. Here, we aim to provide a practical guide on how to use the cuprizone model and which pitfalls should be avoided.
Asunto(s)
Cuprizona/toxicidad , Modelos Animales de Enfermedad , Esclerosis Múltiple/inducido químicamente , Esclerosis Múltiple/patología , Animales , Peso Corporal/efectos de los fármacos , Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/patología , Regulación de la Expresión Génica , Esclerosis Múltiple/genéticaRESUMEN
Multiple sclerosis (MS) is a neuroinflammatory disorder of the central nervous system (CNS), and represents one of the main causes of disability in young adults. On the histopathological level, the disease is characterized by inflammatory demyelination and diffuse neurodegeneration. Although on the surface the development of new inflammatory CNS lesions in MS may appear consistent with a primary recruitment of peripheral immune cells, questions have been raised as to whether lymphocyte and/or monocyte invasion into the brain are really at the root of inflammatory lesion development. In this review article, we discuss a less appreciated inflammation-neurodegeneration interplay, that is: Neurodegeneration can trigger the formation of new, focal inflammatory lesions. We summarize old and recent findings suggesting that new inflammatory lesions develop at sites of focal or diffuse degenerative processes within the CNS. Such a concept is discussed in the context of the EXPAND trial, showing that siponimod exerts anti-inflammatory and neuroprotective activities in secondary progressive MS patients. The verification or rejection of such a concept is vital for the development of new therapeutic strategies for progressive MS.
Asunto(s)
Antiinflamatorios/farmacología , Azetidinas/farmacología , Compuestos de Bencilo/farmacología , Sistema Nervioso Central/efectos de los fármacos , Esclerosis Múltiple Crónica Progresiva/tratamiento farmacológico , Neuroprotección/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Adolescente , Adulto , Antiinflamatorios/uso terapéutico , Azetidinas/uso terapéutico , Compuestos de Bencilo/uso terapéutico , Sistema Nervioso Central/patología , Progresión de la Enfermedad , Humanos , Persona de Mediana Edad , Fármacos Neuroprotectores/uso terapéutico , Adulto JovenRESUMEN
Matrix metalloproteinase-9 (GELB) as a member of gelatinases plays key role in the destruction of blood-brain barrier (BBB), T cells migration into the CNS, and demyelination induction. Considering remyelination induction in response to tart cherry extract and pure p-coumaric acid ingestion via tracking MMP9 gene expression in the cuprizone mouse model. Firstly, predicting the chemical interaction between p-coumaric acid and MMP9 protein was conducted through PASS and Swiss dock web services. Next, the content of p-coumaric acid in the tart cherry extract was analyzed by HPLC. Later, mice (male, female) were categorized into two groups: standard, cuprizone. After the demyelination period, mice classified into four groups: standard, natural chow, tart cherry extract, p-coumaric acid. Finally, brains were extracted from the skull, and MMP9 gene expression was evaluated by real time RT-PCR. Bioinformatics analysis displayed p-coumaric acid has potent inhibitory effect on MMP9 gene expression (Pa=0.818) with estimated ΔG (kcal/mol) -8.10. In addition, during the demyelination period, MMP9 expression was increased significantly in the male group that is related to myelin destruction. However, MMP9 was declined throughout remyelination in both male and female. It's remarkable that pure p-coumaric acid and tart cherry extract ingestion could decrease the gene expression ratio more than natural chow. According to the results, it's deduced the male mouse is more appropriate gender for demyelination induction via cuprizone. In addition, tart cherry extract and pure p-coumaric acid ingestion could decrease MMP9 gene expression level considerably during remyelination.