RESUMEN
The complement system is a key driver of neuroinflammation. Activation of complement by all pathways, results in the formation of the anaphylatoxin C5a and the membrane attack complex (MAC). Both initiate pro-inflammatory responses which can contribute to neurological disease. In this study, we delineate the specific roles of C5a receptor signaling and MAC formation during the progression of experimental autoimmune encephalomyelitis (EAE)-mediated neuroinflammation. MAC inhibition was achieved by subcutaneous administration of an antisense oligonucleotide specifically targeting murine C6 mRNA (5 mg/kg). The C5a receptor 1 (C5aR1) was inhibited with the C5a receptor antagonist PMX205 (1.5 mg/kg). Both treatments were administered systemically and started after disease onset, at the symptomatic phase when lymphocytes are activated. We found that antisense-mediated knockdown of C6 expression outside the central nervous system prevented relapse of disease by impeding the activation of parenchymal neuroinflammatory responses, including the Nod-like receptor protein 3 (NLRP3) inflammasome. Furthermore, C6 antisense-mediated MAC inhibition protected from relapse-induced axonal and synaptic damage. In contrast, inhibition of C5aR1-mediated inflammation diminished expression of major pro-inflammatory mediators, but unlike C6 inhibition, it did not stop progression of neurological disability completely. Our study suggests that MAC is a key driver of neuroinflammation in this model, thereby MAC inhibition might be a relevant treatment for chronic neuroinflammatory diseases.
Asunto(s)
Antiinflamatorios/uso terapéutico , Complejo de Ataque a Membrana del Sistema Complemento/antagonistas & inhibidores , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Encefalitis/tratamiento farmacológico , Encefalitis/etiología , Encefalomielitis Autoinmune Experimental/complicaciones , Animales , Antiinflamatorios/química , Axones/efectos de los fármacos , Axones/patología , Axones/ultraestructura , Activación de Complemento , Complejo de Ataque a Membrana del Sistema Complemento/química , Modelos Animales de Enfermedad , Exorribonucleasas/uso terapéutico , Masculino , Ratones , Microscopía Electrónica , Modelos Biológicos , Péptidos Cíclicos/uso terapéutico , ARN Mensajero/metabolismo , Receptor de Anafilatoxina C5a/antagonistas & inhibidores , Receptor de Anafilatoxina C5a/química , Receptor de Anafilatoxina C5a/metabolismo , Sinaptofisina/metabolismo , Sinaptofisina/ultraestructuraRESUMEN
Numerous mouse myelin mutants are available to analyze the biology of the peripheral nervous system related to health and disease in vivo. However, robust in vitro biochemical characterizations of players in peripheral nerve processes are still not possible due to the limited growth capacities of Schwann cells. In order to generate cell lines from peripheral nerves that are amenable to experimental manipulation, we have isolated Schwann cells from transgenic mice (H-2Kb-tsA58) carrying the temperature sensitive SV40 large T oncogene under the control of the interferon gamma (IFNgamma) H-2Kb promoter. These cells are immortalized at 33 degrees C when the SV40 large T antigen has a stable conformation. At the non-permissive temperature of 37 degrees C and in the absence of IFNgamma, the growth rate of the cultures reduces and typical Schwann cell markers such as p75(NGFR) become upregulated. The conditionally immortalized Schwann cells allow genetic manipulation as demonstrated here by the generation of a stable eGFP expressing cell line. They regain their characteristic non-immortalized properties at non-permissive temperature and differentiate to myelin-forming cells when seeded on dorsal root ganglia neurons. The Schwann cell lines derived are valuable tools for in vitro studies involving demyelinating diseases.
Asunto(s)
Vaina de Mielina/metabolismo , Fibras Nerviosas Mielínicas/metabolismo , Nervios Periféricos/metabolismo , Células de Schwann/metabolismo , Animales , Animales Recién Nacidos , Antígenos Transformadores de Poliomavirus/genética , Técnicas de Cultivo de Célula , Diferenciación Celular/genética , Línea Celular Transformada , Separación Celular , Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/metabolismo , Técnica del Anticuerpo Fluorescente , Ganglios Espinales/citología , Ganglios Espinales/crecimiento & desarrollo , Ganglios Espinales/metabolismo , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Interferón gamma/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Vaina de Mielina/ultraestructura , Fibras Nerviosas Mielínicas/ultraestructura , Nervios Periféricos/citología , Nervios Periféricos/crecimiento & desarrollo , Regiones Promotoras Genéticas/genética , Receptor de Factor de Crecimiento Nervioso/genética , Células de Schwann/citología , Temperatura , Transfección/métodos , Regulación hacia Arriba/genéticaRESUMEN
Patients with schwannomatosis develop multiple schwannomas but no vestibular schwannomas diagnostic of neurofibromatosis type 2. We report an inactivating germline mutation in exon 1 of the tumor-suppressor gene INI1 in a father and daughter who both had schwannomatosis. Inactivation of the wild-type INI1 allele, by a second mutation in exon 5 or by clear loss, was found in two of four investigated schwannomas from these patients. All four schwannomas displayed complete loss of nuclear INI1 protein expression in part of the cells. Although the exact oncogenetic mechanism in these schwannomas remains to be elucidated, our findings suggest that INI1 is the predisposing gene in familial schwannomatosis.