RESUMO
Krabbe's disease is an infantile neurodegenerative disease, which is affected by mutations in the lysosomal enzyme galactocerebrosidase, leading to the accumulation of its metabolite psychosine. We have shown previously that the S1P receptor agonist fingolimod (FTY720) attenuates psychosine-induced glial cell death and demyelination both in vitro and ex vivo models. These data, together with a lack of therapies for Krabbe's disease, prompted the current preclinical study examining the effects of fingolimod in twitcher mice, a murine model of Krabbe's disease. Twitcher mice, both male and female, carrying a natural mutation in the galc gene were given fingolimod via drinking water (1 mg/kg/d). The direct impact of fingolimod administration was assessed via histochemical and biochemical analysis using markers of myelin, astrocytes, microglia, neurons, globoid cells, and immune cells. The effects of fingolimod on twitching behavior and life span were also demonstrated. Our results show that treatment of twitcher mice with fingolimod significantly rescued myelin levels compared with vehicle-treated animals and also regulated astrocyte and microglial reactivity. Furthermore, nonphosphorylated neurofilament levels were decreased, indicating neuroprotective and neurorestorative processes. These protective effects of fingolimod on twitcher mice brain pathology was reflected by an increased life span of fingolimod-treated twitcher mice. These in vivo findings corroborate initial in vitro studies and highlight the potential use of S1P receptors as drug targets for treatment of Krabbe's disease.SIGNIFICANCE STATEMENT This study demonstrates that the administration of the therapy known as fingolimod in a mouse model of Krabbe's disease (namely, the twitcher mouse model) significantly rescues myelin levels. Further, the drug fingolimod also regulates the reactivity of glial cells, astrocytes and microglia, in this mouse model. These protective effects of fingolimod result in an increased life span of twitcher mice.
Assuntos
Doenças Desmielinizantes/tratamento farmacológico , Cloridrato de Fingolimode/uso terapêutico , Leucodistrofia de Células Globoides/tratamento farmacológico , Animais , Astrócitos/metabolismo , Comportamento Animal , Feminino , Leucodistrofia de Células Globoides/psicologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Microglia/metabolismo , Bainha de Mielina/metabolismo , Proteínas de Neurofilamentos/metabolismo , Neurônios/metabolismo , Fosforilação , Pró-Proteína Convertases/efeitos dos fármacos , Células de Purkinje/metabolismo , Serina Endopeptidases/efeitos dos fármacosRESUMO
Sphingosine 1-phosphate receptors (S1PR) are G protein-coupled and compose a family with five subtypes, S1P1R-S1P5R. The drug Gilenya® (Novartis, Basel, Switzerland) (Fingolimod; FTY720) targets S1PRs and was the first oral therapy for patients with relapsing-remitting multiple sclerosis (MS). The phosphorylated form of FTY720 (pFTY720) binds S1PRs causing initial agonism, then subsequent receptor internalization and functional antagonism. Internalization of S1P1R attenuates sphingosine 1-phosphate (S1P)-mediated egress of lymphocytes from lymph nodes, limiting aberrant immune function in MS. pFTY720 also exerts direct actions on neurons and glial cells which express S1PRs. In this study, we investigated the regulation of pro-inflammatory chemokine release by S1PRs in enriched astrocytes and microglial cultures. Astrocytes and microglia were stimulated with lipopolysaccharide (LPS) and increases in C-X-C motif chemokine 5 (CXCL5), also known as LIX (lipopolysaccharide-induced CXC chemokine) expression were quantified. Results showed that pFTY720 attenuated LPS-induced CXCL5 (LIX) protein release from astrocytes, as did the S1P1R selective agonist, SEW2871. In addition, pFTY720 blocked messenger ribonucleic acid (mRNA) transcription of the chemokines, (i) CXCL5/LIX, (ii) C-X-C motif chemokine 10 (CXCL10) also known as interferon gamma-induced protein 10 (IP10) and (iii) chemokine (C-C motif) ligand 2 (CCL2) also known as monocyte chemoattractant protein 1 (MCP1). Interestingly, inhibition of sphingosine kinase attenuated LPS-induced increases in mRNA levels of all three chemokines, suggesting that LPS-TLR4 (Toll-like receptor 4) signalling may enhance chemokine expression via S1P-S1PR transactivation. Lastly, these observations were not limited to astrocytes since we also found that pFTY720 attenuated LPS-induced release of CXCL5 from microglia. These data highlight a role for S1PR signalling in regulating the levels of chemokines in glial cells and support the notion that pFTY720 efficacy in multiple sclerosis may involve the direct modulation of astrocytes and microglia.
Assuntos
Astrócitos/metabolismo , Quimiocina CXCL5/metabolismo , Inflamação/metabolismo , Microglia/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Feminino , Cloridrato de Fingolimode/administração & dosagem , Humanos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/administração & dosagem , Masculino , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos Wistar , Receptores de Lisoesfingolipídeo/agonistas , Transdução de SinaisRESUMO
Oxidative stress is a pathological condition defined as an imbalance between production and removal of reactive oxygen species. This process causes structural cell damage, disrupts DNA repair and induces mitochondrial dysfunction. Many in vitro studies have used direct bolus application of H2 O2 to investigate the role of oxidative stress in cell culture. In this study, using mouse organotypic cerebellar slice cultures, the effects of H2 O2 -induced oxidative stress on myelination state were examined, using bolus concentrations of H2 O2 (0.1-1 mM) and low-continuous H2 O2 (â¼20 µM) generated from glucose oxidase and catalase (GOX-CAT). Using these models, the potential therapeutic effects of pFTY720, an oral therapy used in multiple sclerosis, was also examined. We found bolus treatment of H2 O2 (0.5 mM) and, for the first time, low-continuous H2 O2 (GOX-CAT) to induce demyelination in organotypic slices. Both bolus H2 O2 and GOX-CAT treatments significantly decreased vimentin expression in these slice cultures as well as increased cell death in isolated astrocyte cultures. Importantly, pre-treatment with pFTY720 significantly attenuated both bolus H2 O2 and GOX-CAT-induced demyelination and the GOX-CAT-induced decrease in vimentin in cerebellar slices, without altering levels of the proinflammatory cytokines such as IL-6 and CX3CL1. We also observed increased SMI-32 immunoreactivity in the white matter tract induced by GOX-CAT indicating axonal damage, which was remarkably attenuated by pFTY720. Taken together, this data establishes a novel GOX-CAT model of demyelination and demonstrates that pFTY720 can act independently of inflammatory cytokines to attenuate decreases in vimentin, as well as axonal damage and demyelination induced by oxidative stress.
Assuntos
Doenças Desmielinizantes/etiologia , Doenças Desmielinizantes/metabolismo , Estresse Oxidativo/fisiologia , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/fisiologia , Encéfalo/citologia , Proteínas de Ligação ao Cálcio , Células Cultivadas , Cerebelo/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Doenças Desmielinizantes/tratamento farmacológico , Feminino , Feto , Cloridrato de Fingolimode/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/toxicidade , Imunossupressores/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos , Glicoproteína Mielina-Oligodendrócito/metabolismo , Proteínas de Neurofilamentos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Receptores de Lisoesfingolipídeo/genética , Proteínas Repressoras/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
BACKGROUND: Fractalkine/CX3CR1 signalling has been implicated in many neurodegenerative and neurological diseases of the central nervous system (CNS). This signalling pathway plays an important role in regulating reactive oxygen species (ROS), as well as itself being altered in conditions of oxidative stress. Here, we investigated the effects of recombinant fractalkine (rCX3CL1) in models of hydrogen peroxide (H2O2)-induced demyelination and astrocyte toxicity, within organotypic cerebellar slice cultures. METHODS: Organotypic cerebellar slice cultures were generated from postnatal day 10 C57BL/6J mice to assess myelination. Immunohistochemistry was used to measure the degree of myelination. Fluorescent images were obtained using a leica SP8 confocal microscope and data analysed using ImageJ software. RESULTS: We show here, for the first time, that rCX3CL1 significantly attenuated bolus H2O2-induced demyelination as measured by expression of myelin basic protein (MBP) and attenuated reduced vimentin expression. Using the GOX-CAT system to continuously generate low levels of H2O2 and induce demyelination, we observed similar protective effects of rCX3CL1 on MBP and MOG fluorescence, although in this model, the decrease in vimentin expression was not altered. CONCLUSIONS: This data indicates possible protective effects of fractalkine signalling in oxidative stress-induced demyelination in the central nervous system. This opens up the possibility of fractalkine receptor (CX3CR1) modulation as a potential new target for protecting against oxidative stress-induced demyelination in both inflammatory and non-inflammatory nervous system disorders.
Assuntos
Cerebelo/efeitos dos fármacos , Cerebelo/patologia , Quimiocina CX3CL1/uso terapêutico , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/prevenção & controle , Peróxido de Hidrogênio/toxicidade , Animais , Animais Recém-Nascidos , Receptor 1 de Quimiocina CX3C/metabolismo , Catalase/toxicidade , Morte Celular/efeitos dos fármacos , Feminino , Gliose/induzido quimicamente , Gliose/prevenção & controle , Glucose Oxidase/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Básica da Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito/metabolismo , Proteínas de Neurofilamentos/metabolismo , Técnicas de Cultura de Órgãos , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: The fractalkine (CX3CR1) ligand is expressed in astrocytes and reported to be neuroprotective. When cleaved from the membrane, soluble fractalkine (sCX3CL1) activates the receptor CX3CR1. Although somewhat controversial, CX3CR1 is reported to be expressed in neurons and microglia. The membrane-bound form of CX3CL1 additionally acts as an adhesion molecule for microglia and infiltrating white blood cells. Much research has been done on the role of fractalkine in neuronal cells; however, little is known about the regulation of the CX3CL1 ligand in astrocytes. METHODS: The mechanisms involved in the up-regulation and cleavage of CX3CL1 from human astrocytes were investigated using immunocytochemistry, Q-PCR and ELISA. All statistical analysis was performed using GraphPad Prism 5. RESULTS: A combination of ADAM17 (TACE) and ADAM10 protease inhibitors was found to attenuate IL-1ß-, TNF-α- and IFN-γ-induced sCX3CL1 levels in astrocytes. A specific ADAM10 (but not ADAM17) inhibitor also attenuated these effects, suggesting ADAM10 proteases induce release of sCX3CL1 from stimulated human astrocytes. A p38 MAPK inhibitor also attenuated the levels of sCX3CL1 upon treatment with IL-1ß, TNF-α or IFN-γ. In addition, an IKKß inhibitor significantly reduced the levels of sCX3CL1 induced by IL-1ß or TNF-α in a concentration-dependent manner, suggesting a role for the NF-kB pathway. CONCLUSIONS: In conclusion, this study shows that the release of soluble astrocytic fractalkine is regulated by ADAM10 proteases with p38 MAPK also playing a role in the fractalkine shedding event. These findings are important for understanding the role of CX3CL1 in healthy and stimulated astrocytes and may benefit our understanding of this pathway in neuro-inflammatory and neurodegenerative diseases.
Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Quimiocina CX3CL1/metabolismo , Citocinas/farmacologia , Proteínas de Membrana/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Células Cultivadas , Quimiocina CX3CL1/genética , Meios de Cultura Livres de Soro/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Humanos , Metaloproteinases da Matriz/metabolismo , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
Interneuronal transfer and brain spreading of pathogenic proteins are features of neurodegenerative diseases. Pathophysiological conditions and mechanisms affecting this spreading remain poorly understood. This study investigated the relationship between neuronal activity and interneuronal transfer of α-synuclein, a Parkinson-associated protein, and elucidated mechanisms underlying this relationship. In a mouse model of α-synuclein brain spreading, hyperactivity augmented and hypoactivity attenuated protein transfer. Important features of neuronal hyperactivity reported here were an exacerbation of oxidative and nitrative reactions, pronounced accumulation of nitrated α-synuclein, and increased protein aggregation. Data also pointed to mitochondria as key targets and likely sources of reactive oxygen and nitrogen species within hyperactive neurons. Rescue experiments designed to counteract the increased burden of reactive oxygen species reversed hyperactivity-induced α-synuclein nitration, aggregation, and interneuronal transfer, providing first evidence of a causal link between these pathological effects of neuronal stimulation and indicating a mechanistic role of oxidant stress in hyperactivity-induced α-synuclein spreading.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Animais , Encéfalo/metabolismo , Camundongos , Neurônios/metabolismo , Oxidantes , Doença de Parkinson/metabolismoRESUMO
Krabbe disease is a fatal rare inherited lipid storage disorder affecting 1:100,000 births. This illness is caused by mutations in the galc gene encoding for the enzyme galactosylceramidase (GALC). Dysfunction of GALC has been linked to the toxic build-up of the galactolipid, galactosylsphingosine (psychosine), which induces cell death of oligodendrocytes. Previous studies show that phospholipase A2 (PLA2) may play a role in psychosine induce cell death. Here, we demonstrate that non-selective inhibition of cPLA2/sPLA2 and selective inhibition of cPLA2, but not sPLA2, also attenuates psychosine-induced cell death of human astrocytes. This study shows that extracellular calcium is required for psychosine induced cell death, but intracellular calcium release, reactive oxygen species or release of soluble factors are not involved. These findings suggest a cell autonomous effect, at least in human astrocytes. Supporting a role for PLA2 in psychosine-induced cell death of oligodendrocytes and astrocytes, the results show inhibition of PLA2 attenuates psychosine-induced decrease in the expression of astrocyte marker vimentin as well as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) and the neuronal marker SMI-32 in organotypic slice cultures. These findings provide further mechanistic details of psychosine-induced death of glia and suggest a role for PLA2 in the process. This work also supports the proposal that novel drugs for Krabbe disease may require testing on astrocytes as well as oligodendrocytes for more holistic prediction of pre-clinical and clinical efficacy.
Assuntos
Astrócitos/fisiologia , Doenças Desmielinizantes , Neurônios/patologia , Fosfolipases A2/fisiologia , Psicosina/fisiologia , Animais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Fosfolipases A2/efeitos dos fármacosRESUMO
EBI2 is a G protein-coupled receptor activated by oxysterol 7α, 25-dihydroxycholesterol (7α25HC) and regulates T cell-dependant antibody response and B cell migration. We recently found EBI2 is expressed in human astrocytes, regulates intracellular signalling and modulates astrocyte migration. Here, we report that LPS treatment of mouse astrocytes alters mRNA levels of EBI2 and oxysterols suggesting that the EBI2 signalling pathway is sensitive to LPS-mediated immune challenge. We also find that conditioned media obtained from LPS-stimulated mouse astrocytes induces macrophage migration, which is inhibited by the EBI2 antagonist NIBR189. These results demonstrate a role for the EBI2 signalling pathway in astrocytes as a sensor for immune challenge and for communication with innate immune cells such as macrophages.