RESUMEN
We demonstrate the impact of a disrupted molecular clock in Bmal1-deficient (Bmal1-/-) mice on migration of neural progenitor cells (NPCs). Proliferation of NPCs in rostral migratory stream (RMS) was reduced in Bmal1-/- mice, consistent with our earlier studies on adult neurogenesis in hippocampus. However, a significantly higher number of NPCs from Bmal1-/- mice reached the olfactory bulb as compared to wild-type littermates (Bmal1+/+ mice), indicating a higher migration velocity in Bmal1-/- mice. In isolated NPCs from Bmal1-/- mice, not only migration velocity and expression pattern of genes involved in detoxification of reactive oxygen species were affected, but also RNA oxidation of catalase was increased and catalase protein levels were decreased. Bmal1+/+ migration phenotype could be restored by treatment with catalase, while treatment of NPCs from Bmal1+/+ mice with hydrogen peroxide mimicked Bmal1-/- migration phenotype. Thus, we conclude that Bmal1 deficiency affects NPC migration as a consequence of dysregulated detoxification of reactive oxygen species.
Asunto(s)
Factores de Transcripción ARNTL/deficiencia , Movimiento Celular , Células-Madre Neurales/metabolismo , Neurogénesis , Bulbo Olfatorio/metabolismo , Factores de Transcripción ARNTL/genética , Animales , Catalasa/metabolismo , Células Cultivadas , Cofilina 1/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genotipo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Bulbo Olfatorio/citología , Estrés Oxidativo , Fenotipo , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
Oligodendrocytes are integral to efficient neuronal signaling. Loss of myelinating oligodendrocytes is a central feature of many neurological diseases, including multiple sclerosis (MS). The results of neuropathological studies suggest that oligodendrocytes react with differing sensitivity to toxic insults, with some cells dying early during lesion development and some cells being resistant for weeks. This proposed graded vulnerability has never been demonstrated but provides an attractive window for therapeutic interventions. Furthermore, the biochemical pathways associated with graded oligodendrocyte vulnerability have not been well explored. We used immunohistochemistry and serial block-face scanning electron microscopy (3D-SEM) to show that cuprizone-induced metabolic stress results in an "out of phase" degeneration of oligodendrocytes. Although expression induction of stress response transcription factors in oligodendrocytes occurs within days, subsequent oligodendrocyte apoptosis continues for weeks. In line with the idea of an out of phase degeneration of oligodendrocytes, detailed ultrastructural reconstructions of the axon-myelin unit demonstrate demyelination of single internodes. In parallel, genome wide array analyses revealed an active unfolded protein response early after initiation of the cuprizone intoxication. In addition to the cytoprotective pathways, the pro-apoptotic transcription factor DNA damage-inducible transcript 3 (DDIT3) was induced early in oligodendrocytes. In advanced lesions, DDIT3 was as well expressed by activated astrocytes. Toxin-induced oligodendrocyte apoptosis, demyelination, microgliosis, astrocytosis, and acute axonal damage were less intense in the Ddit3-null mutants. This study identifies DDIT3 as an important regulator of graded oligodendrocyte vulnerability in a MS animal model. Interference with this stress cascade might offer a promising therapeutic approach for demyelinating disorders.
Asunto(s)
Enfermedades Desmielinizantes/patología , Regulación de la Expresión Génica/genética , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Factor de Transcripción CHOP/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Astrocitos/patología , Proteínas de Unión al Calcio , Células Cultivadas , Cuerpo Calloso/metabolismo , Cuerpo Calloso/patología , Cuerpo Calloso/ultraestructura , Cuprizona/toxicidad , Enfermedades Desmielinizantes/inducido químicamente , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos , Microscopía Electrónica de Rastreo , Inhibidores de la Monoaminooxidasa/toxicidad , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/ultraestructura , Factor de Transcripción CHOP/genéticaRESUMEN
The extent of remyelination in multiple sclerosis lesions is often incomplete. Injury to oligodendrocyte progenitor cells can be a contributing factor for such incomplete remyelination. The precise mechanisms underlying insufficient repair remain to be defined, but oxidative stress appears to be involved. Here, we used immortalized oligodendrocyte cell lines as model systems to investigate a causal relation of oxidative stress and endoplasmic reticulum stress signaling cascades. OLN93 and OliNeu cells were subjected to chemical hypoxia by blocking the respiratory chain at various levels. Mitochondrial membrane potential and oxidative stress levels were quantified by flow cytometry. Endoplasmic reticulum stress was monitored by the expression induction of activating transcription factor 3 and 4 (Atf3, Atf4), DNA damage-inducible transcript 3 protein (Ddit3), and glucose-regulated protein 94. Lentiviral silencing of nuclear factor (erythroid-derived 2)-like 2 or kelch-like ECH-associated protein 1 was applied to study the relevance of NRF2 for endoplasmic reticulum stress responses. We demonstrate that inhibition of the respiratory chain induces oxidative stress in cultured oligodendrocytes which is paralleled by the expression induction of distinct mediators of the endoplasmic reticulum stress response, namely Atf3, Atf4, and Ddit3. Atf3 and Ddit3 expression induction is potentiated in kelch-like ECH-associated protein 1-deficient cells and absent in cells lacking the oxidative stress-related transcription factor NRF2. This study provides strong evidence that oxidative stress in oligodendrocytes activates endoplasmic reticulum stress response in a NRF2-dependent manner and, in consequence, might regulate oligodendrocyte degeneration in multiple sclerosis and other neurological disorders.
Asunto(s)
Estrés del Retículo Endoplásmico , Factor 2 Relacionado con NF-E2/metabolismo , Oligodendroglía/metabolismo , Estrés Oxidativo , Factor de Transcripción Activador 3/metabolismo , Animales , Hipoxia de la Célula , Línea Celular , Transporte de Electrón , Potencial de la Membrana Mitocondrial , Ratas , Transducción de Señal , Factor de Transcripción CHOP/metabolismoRESUMEN
17ß-Estradiol (E2) and progesterone (P) are neuroprotective in acute brain injury by attenuating neuropathophysiological processes and regulating local glial function. Besides controlling brain-intrinsic immune responses, astrocytes are cellular targets for sex steroids in health and disease and typically resist to hypoxic damage. In this in vitro study, we aimed at uncovering astroglia-specific reactions to sublethal hypoxic conditions and astroglia-specific effects of both sex steroid hormones on these parameters. Short-term hypoxia for 3 h increased reactive oxygen species production, but had no influence on cell viability of cerebral cortical rat astroglia. Astrocytes expressed classical estrogen receptors (ER), progesterone receptor (PR), and a set of nonclassical steroid hormone receptors. Hypoxia specifically induced ERα and PR isoform A gene expression. Oxygen deprivation increased gene expression of aquaporin-4 (AQP4), hypoxia-inducible factor 1α (Hif1α), and cyclooxygenase-2 (COX2). The application of E2 and P selectively prevented this induction. Effects on protein levels of these genes appeared to be delayed. These data show that astrocytes change their receptivity for sex steroid hormones by switching steroid hormone receptor expression and that E2 and P modify or antagonize proinflammatory COX2 synthesis, edema-promoting AQP4 expression, and the Hif1α increase. In vivo studies have to address whether these cell responses contribute to steroid-mediated neuroprotection in stroke.
Asunto(s)
Acuaporina 4/metabolismo , Astrocitos/efectos de los fármacos , Corteza Cerebral/citología , Ciclooxigenasa 2/metabolismo , Estradiol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Progesterona/farmacología , Análisis de Varianza , Animales , Animales Recién Nacidos , Acuaporina 4/genética , Astrocitos/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ciclooxigenasa 2/genética , Ensayo de Inmunoadsorción Enzimática , Proteína Ácida Fibrilar de la Glía/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , ARN Mensajero/metabolismo , RatasRESUMEN
BACKGROUND: Autoimmunity to neuronal proteins occurs in several neurological syndromes, where cellular and humoral responses are directed to surface as well as intracellular antigens. Similar to myelin autoimmunity, pathogenic immune response to neuroaxonal components such as neurofilaments may contribute to neurodegeneration in multiple sclerosis. METHODS: We studied the immune response to the axonal protein neurofilament light (NF-L) in the experimental autoimmune encephalomyelitis animal model of multiple sclerosis. To examine the association between T cells and axonal damage, pathology studies were performed on NF-L immunized mice. The interaction of T cells and axons was analyzed by confocal microscopy of central nervous system tissues and T-cell and antibody responses to immunodominant epitopes identified in ABH (H2-Ag7) and SJL/J (H2-As) mice. These epitopes, algorithm-predicted peptides and encephalitogenic motifs within NF-L were screened for encephalitogenicity. RESULTS: Confocal microscopy revealed both CD4+ and CD8+ T cells alongside damaged axons in the lesions of NF-L immunized mice. CD4+ T cells dominated the areas of axonal injury in the dorsal column of spastic mice in which the expression of granzyme B and perforin was detected. Identified NF-L epitopes induced mild neurological signs similar to the observed with the NF-L protein, yet distinct from those characteristic of neurological disease induced with myelin oligodendrocyte glycoprotein. CONCLUSIONS: Our data suggest that CD4+ T cells are associated with spasticity, axonal damage and neurodegeneration in NF-L immunized mice. In addition, defined T-cell epitopes in the NF-L protein might be involved in the pathogenesis of the disease.
Asunto(s)
Autoantígenos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Proteínas de Neurofilamentos/inmunología , Médula Espinal/inmunología , Médula Espinal/patología , Animales , Encefalomielitis Autoinmune Experimental/patología , Ensayo de Inmunoadsorción Enzimática , Epítopos de Linfocito T/inmunología , Femenino , Inmunohistoquímica , Masculino , Ratones , Microscopía Confocal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos TRESUMEN
Cuprizone [bis(cyclohexylidenehydrazide)]-induced toxic demyelination is an experimental approach frequently used to study de- and re-myelination in the central nervous system. In this model, mice are fed with the copper chelator cuprizone which leads to oligodendrocyte apoptosis and subsequent microgliosis, astrocytosis, and demyelination. The underlying mechanisms of cuprizone-induced oligodendrocyte death are still unknown. We analysed differences in amino acid levels after short-term cuprizone exposure (i.e., 4 days). Furthermore, an amino acid response (AAR) pathway activated in oligodendrocytes after cuprizone intoxication was evaluated. Short-term cuprizone exposure resulted in a selective decrease of alanine, glycine, and proline plasma levels, which was paralleled by an increase of apoptotic cells in the liver and a decrease of alanine aminotransferase in the serum. These parameters were paralleled by oligodendrocyte apoptosis and the induction of an AAR with increased expression of the transcription factors ATF-3 and ATF-4 (activating transcription factor-3 and -4). Immunohistochemistry revealed that ATF-3 is exclusively expressed by oligodendrocytes and localized to the nuclear compartment. Our results suggest that cuprizone-induced liver dysfunction results in amino acid starvation and in consequence to the activation of an AAR. We propose that this stress response modulates oligodendrocyte viability in the cuprizone animal model.
Asunto(s)
Cuprizona/toxicidad , Conducta Alimentaria , Oligodendroglía/metabolismo , Oligodendroglía/patología , Estrés Fisiológico/efectos de los fármacos , Factor de Transcripción Activador 3/genética , Factor de Transcripción Activador 3/metabolismo , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Aminoácidos/sangre , Aminoácidos/deficiencia , Animales , Cuerpo Calloso/efectos de los fármacos , Cuerpo Calloso/metabolismo , Cuerpo Calloso/patología , Cuprizona/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito/metabolismo , Glicoproteína Asociada a Mielina/metabolismo , Oligodendroglía/efectos de los fármacos , Factores de TiempoRESUMEN
In multiple sclerosis, conduction block in demyelinated axons underlies early neurological symptoms, whereas axonal transection is believed to be responsible for more permanent later deficits. Approved treatments for the disease are immunoregulatory and reduce the rate of lesion formation and clinical exacerbation, but are only partially effective in preventing the onset of disability. Remyelination is a term for the re-generation of the nerve's myelin sheath and is a subject of active medical research. Remyelination capacity varies from patient to patient or even from lesion to lesion in one and the same patient. Efforts to understand the causes for remyelination failure have prompted research into the biology of remyelination and the complex molecular factors that regulate remyelination. In the current review article we address challenges of remyelination research with a special focus on histo-pathological studies using brain biopsy and autopsy material. We summarize our current knowledge about extent of remyelination in multiple sclerosis patients and its relation to disease duration, lesion type, inflammation, affected brain region and gender. Furthermore we will address which step(s) of the oligodendrocyte maturation program is impaired and, thus, could be a feasible target for therapeutic interventions. Specifically mentioned will be the distribution of oligodendrocyte progenitor cells in demyelinated multiple sclerosis plaques and therapeutic approaches which aim to boost intrinsic properties of progenitor cells or to supply progenitors by cell transplantation approaches. This comprehensive overview is complemented by recent findings suggesting that U.S. Food and Drug Administration-approved treatment options, such as FTY720 (Gilenya®) or glatiramer acetate (Copaxone®) might boost myelin repair.
Asunto(s)
Axones/metabolismo , Encéfalo/metabolismo , Esclerosis Múltiple/metabolismo , Vaina de Mielina/metabolismo , Degeneración Nerviosa/prevención & control , Animales , Axones/efectos de los fármacos , Axones/inmunología , Axones/patología , Encéfalo/efectos de los fármacos , Encéfalo/inmunología , Encéfalo/patología , Femenino , Humanos , Inmunosupresores/farmacología , Masculino , Terapia Molecular Dirigida , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/patología , Esclerosis Múltiple/fisiopatología , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/inmunología , Vaina de Mielina/patología , Degeneración Nerviosa/etiología , Oligodendroglía/efectos de los fármacos , Oligodendroglía/inmunología , Oligodendroglía/metabolismo , Oligodendroglía/patología , Especificidad de Órganos , Remisión Espontánea , Factores SexualesRESUMEN
The mitochondrion is the unquestionable cellular compartment that actively preserves most of the cell functions, such as lipid metabolism, ion homeostasis, energy and ROS production, steroid biosynthesis, and control of apoptotic signaling. Thus, this cell organelle depicts a major drop-in centre for regulatory processes within a cell irrespective of the organ or tissue. However, brain tissue is unique in spite of everything due to its extremely high energy demand and sensitivity to oxidative stress. This makes brain cells, in particular neurons, considerably vulnerable against toxins and challenges that attack the mitochondrial structural organization and energetic performance. Estrogens are known to regulate a multitude of cellular functions in neural cells under physiological conditions but also play a protective role under neuropathological circumstances. In recent years, it became evident that estrogens affect distinct cellular processes by interfering with the bioenergetic mitochondrial compartment. According to the general view, estrogens indirectly regulate the mitochondrion through the control of genomic transcription of mitochondrial-located proteins and modulation of cytoplasmic signaling cascades that act upon mitochondrial physiology. More recent but still arguable data suggest that estrogens might directly signal to the mitochondrion either through classical steroid receptors or novel types of receptors/proteins associated with the mitochondrial compartment. This would allow estrogens to more rapidly modulate the function of a mitochondrion than hitherto discussed. Assuming that this novel perception of steroid action is correct, estrogen might influence the energetic control centre through long-lasting nuclear-associated processes and rapid mitochondria-intrinsic temporary mechanisms. In this article, we would like to particularly accentuate the novel conceptual approach of this duality comprising that estrogens govern the mitochondrial structural integrity and functional capacity by different cellular signaling routes. This article is part of a Special Issue entitled 'Neurosteroids'.
Asunto(s)
Estrógenos/fisiología , Mitocondrias/fisiología , Animales , Encéfalo/metabolismo , Metabolismo Energético/fisiología , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Proteínas Mitocondriales/fisiología , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Receptores de Estrógenos/fisiología , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Tyrosine kinase inhibitors (TKIs) are effective in treating malignant disorders and were lately suggested to have an impact on non-malignant diseases. However, in some inflammatory conditions like rheumatoid arthritis (RA) the in vivo effect seemed to be moderate. As most TKIs are taken up actively into cells by cell membrane transporters, this study aimed to evaluate the role of such transporters for the accumulation of the TKI Imatinib mesylates in RA synovial fibroblasts as well as their regulation under inflammatory conditions. METHODOLOGY/PRINCIPAL FINDINGS: The transport and accumulation of Imatinib was investigated in transporter-transfected HEK293 cells and human RA synovial fibroblasts (hRASF). Transporter expression was quantified by qRT-PCR. In transfection experiments, hMATE1 showed the highest apparent affinity for Imatinib among all known Imatinib transporters. Experiments quantifying the Imatinib uptake in the presence of specific transporter inhibitors and after siRNA knockdown of hMATE1 indeed identified hMATE1 to mediate Imatinib transport in hRASF. The anti-proliferative effect of Imatinib on PDGF stimulated hRASF was quantified by cell counting and directly correlated with the uptake activity of hMATE1. Expression of hMATE1 was investigated by Western blot and immuno-fluorescence. Imatinib transport under disease-relevant conditions, such as an altered pH and following stimulation with different cytokines, was also investigated by HPLC. The uptake was significantly reduced by an acidic extracellular pH as well as by the cytokines TNFα, IL-1ß and IL-6, which all decreased the expression of hMATE1-mRNA and protein. CONCLUSION/SIGNIFICANCE: The regulation of Imatinib uptake via hMATE1 in hRASF and resulting effects on their proliferation may explain moderate in vivo effects on RA. Moreover, our results suggest that investigating transporter mediated drug processing under normal and pathological conditions is important for developing intracellular acting drugs used in inflammatory diseases.
Asunto(s)
Artritis Reumatoide/enzimología , Artritis Reumatoide/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Benzamidas/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Citocinas/farmacología , Humanos , Concentración de Iones de Hidrógeno , Mesilato de Imatinib , Interleucina-1beta/farmacología , Interleucina-6/farmacología , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas , Pirimidinas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Parkinson's disease is a neurodegenerative disorder characterized by a sex and brain region specificity, showing a higher incidence in men than in women, which is caused by cell death of mainly dopaminergic neurons in the mesencephalon. Mitochondrial toxins are often used to trigger and mimic neurodegenerative processes. Thus, systemic application of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces Parkinsonian symptoms, indicating a causative or consequent involvement of mitochondria. Therefore, mitochondria of neural cells may demonstrate a sex and brain region specificity with respect to structural and functional characteristics of these organelles during toxic and degenerative processes. The application of MPTP in vivo and its toxic derivative 1-methyl-4-phenylpyridinium (MPP(+) ) in vitro represent a well-accepted experimental model of Parkinson's disease. Aside from the known effects of MPP(+) on mitochondria and neural cell survivability and with respect to the supportive role of astrocytes for neuronal function, we aimed to demonstrate the involvement of cytochrome c oxidase subunit IV isoform expression in energy and reactive oxygen species production taking part in an impairment of astrocyte survival. MPP(+) caused a specific increase of COX IV-2 transcript and protein levels in male mesencephalic astrocytes, accompanied by decreased ATP and increased reactive oxygen species levels and elevated apoptotic cell death, which were more pronounced in mesencephalic than in cortical astrocytes from male than from female mice. Our data suggest that MPP(+) acts on astrocytes in a sex- and brain region-specific manner involving cytochrome c oxidase isoform expression in an impairment of energy production and elevated oxidative stress levels, which represent hallmarks of neurodegenerative diseases.