RESUMO
Mucolipidosis IV (MLIV) is an orphan neurodevelopmental disease that causes severe neurologic dysfunction and loss of vision. Currently there is no therapy for MLIV. It is caused by loss of function of the lysosomal channel mucolipin-1, also known as TRPML1. Knockout of the Mcoln1 gene in a mouse model mirrors clinical and neuropathologic signs in humans. Using this model, we previously observed robust activation of microglia and astrocytes in early symptomatic stages of disease. Here we investigate the consequence of mucolipin-1 loss on astrocyte inflammatory activation in vivo and in vitro and apply a pharmacologic approach to restore Mcoln1-/- astrocyte homeostasis using a clinically approved immunomodulator, fingolimod. We found that Mcoln1-/- mice over-express numerous pro-inflammatory cytokines, some of which were also over-expressed in astrocyte cultures. Changes in the cytokine profile in Mcoln1-/- astrocytes are concomitant with changes in phospho-protein signaling, including activation of PI3K/Akt and MAPK pathways. Fingolimod promotes cytokine homeostasis, down-regulates signaling within the PI3K/Akt and MAPK pathways and restores the lysosomal compartment in Mcoln1-/- astrocytes. These data suggest that fingolimod is a promising candidate for preclinical evaluation in our MLIV mouse model, which, in case of success, can be rapidly translated into clinical trial.
Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Encéfalo/efeitos dos fármacos , Cloridrato de Fingolimode/farmacologia , Mucolipidoses/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalite/tratamento farmacológico , Encefalite/genética , Encefalite/metabolismo , Encefalite/patologia , Feminino , Regulação da Expressão Gênica , Proteínas de Membrana Lisossomal/metabolismo , Masculino , Camundongos Knockout , Mucolipidoses/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
The objective of this study was to develop an in vivo model for locally induced epilepsy. Epilepsy is a prominent neurological disorder that affects millions of people worldwide. Patients may experience either global seizures, affecting the entire brain, or focal seizures, affecting only one brain region. The majority of epileptic patients experience focal seizures but they go undiagnosed because such seizures can be difficult to detect. To better understand the effects of focal epilepsy on the neurochemistry of a brain region with high seizure diathesis, an animal model for locally induced seizures in the hippocampus was developed. In this model, two seizure events were chemically induced by administering the epileptogenic agent, 3-mercaptopropionic acid (3-MPA), to the hippocampus to disturb the balance between excitatory and inhibitory neurotransmitters in the brain. Microdialysis was used for local delivery of 3-MPA as well as for collection of dialysate for neurochemical analyses. Two periods of seizures separated by varying inter-seizure recovery times were employed, and changes in the release of the excitatory transmitter, glutamate, were measured. Significant differences in glutamate release were observed between the first and second seizure episodes. Diminished glutamate biosynthesis, enhanced glutamate re-uptake, and/or neuronal death were considered possible causes of the attenuated glutamate release during the second seizure episode. Biochemical measurements were indicative that a combination of these factors led to the attenuation in glutamate release.