RESUMO
Myelinating oligodendrocytes die in human disease and early in aging. Despite this, the mechanisms that underly oligodendrocyte death are not resolved and it is also not clear whether these mechanisms change as oligodendrocyte lineage cells are undergoing differentiation and maturation. Here, we used a combination of intravital imaging, single-cell ablation, and cuprizone-mediated demyelination, in both female and male mice, to discover that oligodendrocyte maturation dictates the dynamics and mechanisms of cell death. After single-cell phototoxic damage, oligodendrocyte precursor cells underwent programmed cell death within hours, differentiating oligodendrocytes died over several days, while mature oligodendrocytes took weeks to die. Importantly cells at each maturation stage all eventually died but did so with drastically different temporal dynamics and morphological features. Consistent with this, cuprizone treatment initiated a caspase-3-dependent form of rapid cell death in differentiating oligodendrocytes, while mature oligodendrocytes never activated this executioner caspase. Instead, mature oligodendrocytes exhibited delayed cell death which was marked by DNA damage and disruption in poly-ADP-ribose subcellular localization. Thus, oligodendrocyte maturation plays a key role in determining the mechanism of death a cell undergoes in response to the same insult. This means that oligodendrocyte maturation is important to consider when designing strategies for preventing cell death and preserving myelin while also enhancing the survival of new oligodendrocytes in demyelinating conditions.
Assuntos
Cuprizona , Doenças Desmielinizantes , Humanos , Camundongos , Masculino , Feminino , Animais , Cuprizona/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/metabolismo , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Apoptose/fisiologia , Diferenciação Celular , Camundongos Endogâmicos C57BLRESUMO
Dysregulation of oligodendrocyte progenitor cell (OPC) recruitment and oligodendrocyte differentiation contribute to failure of remyelination in human demyelinating diseases such as multiple sclerosis (MS). Deletion of muscarinic receptor enhances OPC differentiation and remyelination. However, the role of ligand-dependent signaling versus constitutive receptor activation is unknown. We hypothesized that dysregulated acetylcholine (ACh) release upon demyelination contributes to ligand-mediated activation hindering myelin repair. Following chronic cuprizone (CPZ)-induced demyelination (male and female mice), we observed a 2.5-fold increase in ACh concentration. This increase in ACh concentration could be attributed to increased ACh synthesis or decreased acetylcholinesterase-/butyrylcholinesterase (BChE)-mediated degradation. Using choline acetyltransferase (ChAT) reporter mice, we identified increased ChAT-GFP expression following both lysolecithin and CPZ demyelination. ChAT-GFP expression was upregulated in a subset of injured and uninjured axons following intraspinal lysolecithin-induced demyelination. In CPZ-demyelinated corpus callosum, ChAT-GFP was observed in Gfap+ astrocytes and axons indicating the potential for neuronal and astrocytic ACh release. BChE expression was significantly decreased in the corpus callosum following CPZ demyelination. This decrease was due to the loss of myelinating oligodendrocytes which were the primary source of BChE. To determine the role of ligand-mediated muscarinic signaling following lysolecithin injection, we administered neostigmine, a cholinesterase inhibitor, to artificially raise ACh. We identified a dose-dependent decrease in mature oligodendrocyte density with no effect on OPC recruitment. Together, these results support a functional role of ligand-mediated activation of muscarinic receptors following demyelination and suggest that dysregulation of ACh homeostasis directly contributes to failure of remyelination in MS.
Assuntos
Doenças Desmielinizantes , Oligodendroglia , Transdução de Sinais , Animais , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia , Camundongos , Oligodendroglia/metabolismo , Oligodendroglia/efeitos dos fármacos , Feminino , Masculino , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Camundongos Endogâmicos C57BL , Acetilcolina/metabolismo , Cuprizona/toxicidade , Lisofosfatidilcolinas/toxicidade , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Colina O-Acetiltransferase/metabolismo , Remielinização/fisiologia , Remielinização/efeitos dos fármacos , Bainha de Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos , Camundongos TransgênicosRESUMO
Sphingosine-1-phosphate receptor (S1PR) modulators are clinically used to treat relapse-remitting multiple sclerosis (MS) and the early phase of progressive MS when inflammation still prevails. In the periphery, S1PR modulators prevent lymphocyte egress from lymph nodes, hence hampering neuroinflammation. Recent findings suggest a role for S1PR modulation in remyelination. As the Giα-coupled S1P1 subtype is the most prominently expressed S1PR in oligodendrocyte precursor cells (OPCs), selective modulation (functional antagonism) of S1P1 may have direct effects on OPC functionality. We hypothesized that functional antagonism of S1P1 by ponesimod induces remyelination by boosting OPC differentiation. In the cuprizone mouse model of demyelination, we found ponesimod to decrease the latency time of visual evoked potentials compared to vehicle conditions, which is indicative of functional remyelination. In addition, the Y maze spontaneous alternations test revealed that ponesimod reversed cuprizone-induced working memory deficits. Myelin basic protein (MBP) immunohistochemistry and transmission electron microscopy of the corpus callosum revealed an increase in myelination upon ponesimod treatment. Moreover, treatment with ponesimod alone or in combination with A971432, an S1P5 monoselective modulator, significantly increased primary mouse OPC differentiation based on O4 immunocytochemistry. In conclusion, S1P1 functional antagonism by ponesimod increases remyelination in the cuprizone model of demyelination and significantly increases OPC differentiation in vitro.
Assuntos
Cuprizona , Doenças Desmielinizantes , Tiazóis , Camundongos , Animais , Cuprizona/toxicidade , Receptores de Esfingosina-1-Fosfato/metabolismo , Oligodendroglia , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/tratamento farmacológico , Potenciais Evocados Visuais , Diferenciação Celular/fisiologia , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismo , Modelos Animais de DoençasRESUMO
Multiple sclerosis is a chronic inflammatory disease in which disability results from the disruption of myelin and axons. During the initial stages of the disease, injured myelin is replaced by mature myelinating oligodendrocytes that differentiate from oligodendrocyte precursor cells. However, myelin repair fails in secondary and chronic progressive stages of the disease and with ageing, as the environment becomes progressively more hostile. This may be attributable to inhibitory molecules in the multiple sclerosis environment including activation of the p38MAPK family of kinases. We explored oligodendrocyte precursor cell differentiation and myelin repair using animals with conditional ablation of p38MAPKγ from oligodendrocyte precursors. We found that p38γMAPK ablation accelerated oligodendrocyte precursor cell differentiation and myelination. This resulted in an increase in both the total number of oligodendrocytes and the migration of progenitors ex vivo and faster remyelination in the cuprizone model of demyelination/remyelination. Consistent with its role as an inhibitor of myelination, p38γMAPK was significantly downregulated as oligodendrocyte precursor cells matured into oligodendrocytes. Notably, p38γMAPK was enriched in multiple sclerosis lesions from patients. Oligodendrocyte progenitors expressed high levels of p38γMAPK in areas of failed remyelination but did not express detectable levels of p38γMAPK in areas where remyelination was apparent. Our data suggest that p38γ could be targeted to improve myelin repair in multiple sclerosis.
Assuntos
Esclerose Múltipla , Bainha de Mielina , Oligodendroglia , Remielinização , Animais , Remielinização/fisiologia , Esclerose Múltipla/patologia , Esclerose Múltipla/metabolismo , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Camundongos , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Humanos , Proteína Quinase 12 Ativada por Mitógeno/metabolismo , Proteína Quinase 12 Ativada por Mitógeno/genética , Diferenciação Celular/fisiologia , Cuprizona/toxicidade , Camundongos Endogâmicos C57BL , Masculino , Feminino , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/metabolismo , Células Precursoras de Oligodendrócitos/metabolismo , Células Precursoras de Oligodendrócitos/patologia , Camundongos TransgênicosRESUMO
In the adult brain, activity-dependent myelin plasticity is required for proper learning and memory consolidation. Myelin loss, alteration, or even subtle structural modifications can therefore compromise the network activity, leading to functional impairment. In multiple sclerosis, spontaneous myelin repair process is possible, but it is heterogeneous among patients, sometimes leading to functional recovery, often more visible at the motor level than at the cognitive level. In cuprizone-treated mouse model, massive brain demyelination is followed by spontaneous and robust remyelination. However, reformed myelin, although functional, may not exhibit the same morphological characteristics as developmental myelin, which can have an impact on the activity of neural networks. In this context, we used the cuprizone-treated mouse model to analyze the structural, functional, and cognitive long-term effects of transient demyelination. Our results show that an episode of demyelination induces despite remyelination long-term cognitive impairment, such as deficits in spatial working memory, social memory, cognitive flexibility, and hyperactivity. These deficits were associated with a reduction in myelin content in the medial prefrontal cortex (mPFC) and hippocampus (HPC), as well as structural myelin modifications, suggesting that the remyelination process may be imperfect in these structures. In vivo electrophysiological recordings showed that the demyelination episode altered the synchronization of HPC-mPFC activity, which is crucial for memory processes. Altogether, our data indicate that the myelin repair process following transient demyelination does not allow the complete recovery of the initial myelin properties in cortical structures. These subtle modifications alter network features, leading to prolonged cognitive deficits in mice.
Assuntos
Disfunção Cognitiva , Doenças Desmielinizantes , Humanos , Animais , Camundongos , Bainha de Mielina , Doenças Desmielinizantes/induzido quimicamente , Cuprizona/toxicidade , Encéfalo , Modelos Animais de Doenças , Disfunção Cognitiva/induzido quimicamente , Camundongos Endogâmicos C57BL , Oligodendroglia/fisiologiaRESUMO
Extracellular vesicles (EVs) are involved in diverse cellular functions, playing a significant role in cell-to-cell communication in both physiological conditions and pathological scenarios. Therefore, EVs represent a promising therapeutic strategy. Oligodendrocytes (OLs) are myelinating glial cells developed from oligodendrocyte progenitor cells (OPCs) and damaged in chronic demyelinating diseases such as multiple sclerosis (MS). Glycoprotein transferrin (Tf) plays a critical role in iron homeostasis and has pro-differentiating effects on OLs in vivo and in vitro. In the current work, we evaluated the use of EVs as transporters of Tf to the central nervous system (CNS) through the intranasal (IN) route. For the in vitro mechanistic studies, we used rat plasma EVs. Our results show that EVTf enter OPCs through clathrin-caveolae and cholesterol-rich lipid raft endocytic pathways, releasing the cargo and exerting a pro-maturation effect on OPCs. These effects were also observed in vivo using the animal model of demyelination induced by cuprizone (CPZ). In this model, IN administered Tf-loaded EVs isolated from mouse plasma reached the brain parenchyma, internalizing into OPCs, promoting their differentiation, and accelerating remyelination. Furthermore, in vivo experiments demonstrated that EVs protected the Tf cargo and significantly reduced the amount of Tf required to induce remyelination as compared to soluble Tf. Collectively, these findings unveil EVs as functional nanocarriers of Tf to induce remyelination.
Assuntos
Doenças Desmielinizantes , Vesículas Extracelulares , Camundongos , Ratos , Animais , Transferrina/metabolismo , Doenças Desmielinizantes/patologia , Oligodendroglia/metabolismo , Encéfalo/metabolismo , Diferenciação Celular/fisiologia , Cuprizona/toxicidade , Vesículas Extracelulares/metabolismo , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismoRESUMO
Multiple sclerosis is an autoimmune disease of the central nervous system (CNS) characterized by demyelination, axonal damage and, for the majority of people, a decline in neurological function in the long-term. Remyelination could assist in the protection of axons and their functional recovery, but such therapies are not, as yet, available. The TAM (Tyro3, Axl, and MERTK) receptor ligand GAS6 potentiates myelination in vitro and promotes recovery in pre-clinical models of MS. However, it has remained unclear which TAM receptor is responsible for transducing this effect and whether post-translational modification of GAS6 is required. In this study, we show that the promotion of myelination requires post-translational modification of the GLA domain of GAS6 via vitamin K-dependent γ-carboxylation. We also confirmed that the intracerebroventricular provision of GAS6 for 2 weeks to demyelinated wild-type (WT) mice challenged with cuprizone increased the density of myelinated axons in the corpus callosum by over 2-fold compared with vehicle control. Conversely, the provision of GAS6 to Tyro3 KO mice did not significantly improve the density of myelinated axons. The improvement in remyelination following the provision of GAS6 to WT mice was also accompanied by an increased density of CC1+ve mature oligodendrocytes compared with vehicle control, whereas this improvement was not observed in the absence of Tyro3. This effect occurs independent of any influence on microglial activation. This work therefore establishes that the remyelinative activity of GAS6 is dependent on Tyro3 and includes potentiation of oligodendrocyte numbers.
Assuntos
Cuprizona , Doenças Desmielinizantes , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Proteína Tirosina Quinases , Remielinização , Animais , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Remielinização/fisiologia , Remielinização/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/metabolismo , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Cuprizona/toxicidade , Camundongos , Modelos Animais de Doenças , Bainha de Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Masculino , FemininoRESUMO
The kappa opioid receptor has been identified as a promising therapeutic target for promoting remyelination. In the current study, we evaluated the ability of nalfurafine to promote oligodendrocyte progenitor cell (OPC) differentiation and myelination in vitro, and its efficacy in an extended, cuprizone-induced demyelination model. Primary mouse (C57BL/6J) OPC-containing cultures were treated with nalfurafine (0.6-200 nM), clemastine (0.01-100 µM), T3 (30 ng/mL), or vehicle for 5 days. Using immunocytochemistry and confocal microscopy, we found that nalfurafine treatment increased OPC differentiation, oligodendrocyte (OL) morphological complexity, and myelination of nanofibers in vitro. Adult male mice (C57BL/6J) were given a diet containing 0.2% cuprizone and administered rapamycin (10 mg/kg) once daily for 12 weeks followed by 6 weeks of treatment with nalfurafine (0.01 or 0.1 mg/kg), clemastine (10 mg/kg), or vehicle. We quantified the number of OLs using immunofluorescence, gross myelination using black gold staining, and myelin thickness using electron microscopy. Cuprizone + rapamycin treatment produced extensive demyelination and was accompanied by a loss of mature OLs, which was partially reversed by therapeutic administration of nalfurafine. We also assessed these mice for functional behavioral changes in open-field, horizontal bar, and mouse motor skill sequence tests (complex wheel running). Cuprizone + rapamycin treatment resulted in hyperlocomotion, poorer horizontal bar scores, and less distance traveled on the running wheels. Partial recovery was observed on both the horizontal bar and complex running wheel tests over time, which was facilitated by nalfurafine treatment. Taken together, these data highlight the potential of nalfurafine as a remyelination-promoting therapeutic.
Assuntos
Cuprizona , Doenças Desmielinizantes , Camundongos Endogâmicos C57BL , Morfinanos , Bainha de Mielina , Sirolimo , Compostos de Espiro , Animais , Morfinanos/farmacologia , Masculino , Compostos de Espiro/farmacologia , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/tratamento farmacológico , Camundongos , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Bainha de Mielina/metabolismo , Sirolimo/farmacologia , Cuprizona/toxicidade , Células Cultivadas , Modelos Animais de Doenças , Células Precursoras de Oligodendrócitos/efeitos dos fármacos , Células Precursoras de Oligodendrócitos/metabolismo , Diferenciação Celular/efeitos dos fármacosRESUMO
Neuroinflammation coupled with demyelination and neuro-axonal damage in the central nervous system (CNS) contribute to disease advancement in progressive multiple sclerosis (P-MS). Inflammasome activation accompanied by proteolytic cleavage of gasdermin D (GSDMD) results in cellular hyperactivation and lytic death. Using multiple experimental platforms, we investigated the actions of GSDMD within the CNS and its contributions to P-MS. Brain tissues from persons with P-MS showed significantly increased expression of GSDMD, NINJ1, IL-1ß, and -18 within chronic active demyelinating lesions compared to MS normal appearing white matter and nonMS (control) white matter. Conditioned media (CM) from stimulated GSDMD+/+ human macrophages caused significantly greater cytotoxicity of oligodendroglial and neuronal cells, compared to CM from GSDMD-/- macrophages. Oligodendrocytes and CNS macrophages displayed increased Gsdmd immunoreactivity in the central corpus callosum (CCC) of cuprizone (CPZ)-exposed Gsdmd+/+ mice, associated with greater demyelination and reduced oligodendrocyte precursor cell proliferation, compared to CPZ-exposed Gsdmd-/- animals. CPZ-exposed Gsdmd+/+ mice exhibited significantly increased G-ratios and reduced axonal densities in the CCC compared to CPZ-exposed Gsdmd-/- mice. Proteomic analyses revealed increased brain complement C1q proteins and hexokinases in CPZ-exposed Gsdmd-/- animals. [18F]FDG PET imaging showed increased glucose metabolism in the hippocampus and whole brain with intact neurobehavioral performance in Gsdmd-/- animals after CPZ exposure. GSDMD activation in CNS macrophages and oligodendrocytes contributes to inflammatory demyelination and neuroaxonal injury, offering mechanistic and potential therapeutic insights into P-MS pathogenesis.
Assuntos
Gasderminas , Esclerose Múltipla Crônica Progressiva , Esclerose Múltipla , Animais , Humanos , Camundongos , Moléculas de Adesão Celular Neuronais , Cuprizona/uso terapêutico , Cuprizona/toxicidade , Modelos Animais de Doenças , Gasderminas/metabolismo , Camundongos Endogâmicos C57BL , Microglia/patologia , Esclerose Múltipla/patologia , Esclerose Múltipla Crônica Progressiva/patologia , Fatores de Crescimento Neural , Oligodendroglia , ProteômicaRESUMO
The molecular composition of myelin membranes determines their structure and function. Even minute changes to the biochemical balance can have profound consequences for axonal conduction and the synchronicity of neural networks. Hypothesizing that the earliest indication of myelin injury involves changes in the composition and/or polarity of its constituent lipids, we developed a sensitive spectroscopic technique for defining the chemical polarity of myelin lipids in fixed frozen tissue sections from rodent and human. The method uses a simple staining procedure involving the lipophilic dye Nile Red, whose fluorescence spectrum varies according to the chemical polarity of the microenvironment into which the dye embeds. Nile Red spectroscopy identified histologically intact yet biochemically altered myelin in prelesioned tissues, including mouse white matter following subdemyelinating cuprizone intoxication, as well as normal-appearing white matter in multiple sclerosis brain. Nile Red spectroscopy offers a relatively simple yet highly sensitive technique for detecting subtle myelin changes.
Assuntos
Esclerose Múltipla/patologia , Bainha de Mielina/química , Oligodendroglia/patologia , Oxazinas/química , Espectrometria de Fluorescência/métodos , Idoso , Animais , Estudos de Casos e Controles , Linhagem Celular , Cuprizona/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia , Corantes Fluorescentes , Substância Cinzenta/química , Substância Cinzenta/citologia , Humanos , Lipídeos/química , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Oligodendroglia/química , Substância Branca/química , Substância Branca/citologiaRESUMO
NOX4 is a major reactive oxygen species-producing enzyme that modulates cell stress responses. We here examined the effect of Nox4 deletion on demyelination-remyelination, the most common pathological change in the brain. We used a model of cuprizone (CPZ)-associated demyelination-remyelination in wild-type and Nox4-deficient (Nox4-/- ) mice. While the CPZ-induced demyelination in the corpus callosum after 4 weeks of CPZ intoxication was slightly less pronounced in Nox4-/- mice than that in wild-type mice, remyelination following CPZ withdrawal was significantly enhanced in Nox4-/- mice with an increased accumulation of IBA1-positive microglia/macrophages in the demyelinating corpus callosum. Consistently, locomotor function, as assessed by the beam walking test, was significantly better during the remyelination phase in Nox4-/- mice. Nox4 deletion did not affect autonomous growth of primary-culture oligodendrocyte precursor cells. Although Nox4 expression was higher in cultured macrophages than in microglia, Nox4-/- microglia and macrophages both showed enhanced phagocytic capacity of myelin debris and produced increased amounts of trophic factors upon phagocytosis. The expression of trophic factors was higher, in parallel with the accumulation of IBA1-positive cells, in the corpus callosum in Nox4-/- mice than that in wild-type mice. Nox4 deletion suppressed phagocytosis-induced increase in mitochondrial membrane potential, enhancing phagocytic capacity of macrophages. Treatment with culture medium of Nox4-/- macrophages engulfing myelin debris, but not that of Nox4-/- astrocytes, enhanced cell growth and expression of myelin-associated proteins in cultured oligodendrocyte precursor cells. Collectively, Nox4 deletion promoted remyelination after CPZ-induced demyelination by enhancing microglia/macrophage-mediated clearance of myelin debris and the production of trophic factors leading to oligodendrogenesis.
Assuntos
Doenças Desmielinizantes , Remielinização , Animais , Camundongos , Microglia/metabolismo , Cuprizona/toxicidade , Doenças Desmielinizantes/patologia , Bainha de Mielina/metabolismo , Macrófagos/metabolismo , Corpo Caloso/patologia , Proteínas da Mielina/metabolismo , Camundongos Endogâmicos C57BL , Oligodendroglia/metabolismo , Modelos Animais de Doenças , NADPH Oxidase 4/metabolismoRESUMO
There is an urgent need for therapies that target the multicellular pathology of central nervous system (CNS) disease. Modified, nonanticoagulant heparins mimic the heparan sulfate glycan family and are known regulators of multiple cellular processes. In vitro studies have demonstrated that low sulfated modified heparin mimetics (LS-mHeps) drive repair after CNS demyelination. Herein, we test LS-mHep7 (an in vitro lead compound) in experimental autoimmune encephalomyelitis (EAE) and cuprizone-induced demyelination. In EAE, LS-mHep7 treatment resulted in faster recovery and rapidly reduced inflammation which was accompanied by restoration of animal weight. LS-mHep7 treatment had no effect on remyelination or on OLIG2 positive oligodendrocyte numbers within the corpus callosum in the cuprizone model. Further in vitro investigation confirmed that LS-mHep7 likely mediates its pro-repair effect in the EAE model by sequestering inflammatory cytokines, such as CCL5 which are upregulated during immune-mediated inflammatory attacks. These data support the future clinical translation of this next generation modified heparin as a treatment for CNS diseases with active immune system involvement.
Assuntos
Doenças do Sistema Nervoso Central , Encefalomielite Autoimune Experimental , Animais , Camundongos , Cuprizona/toxicidade , Sulfatos/efeitos adversos , Oligodendroglia/patologia , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Corpo Caloso/patologia , Doenças do Sistema Nervoso Central/patologia , Heparitina Sulfato/uso terapêutico , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Bainha de Mielina/patologiaRESUMO
Demyelination in the central nervous system (CNS) is a hallmark of many neurodegenerative diseases such as multiple sclerosis (MS) and others. Here, we studied astrocytes during de- and remyelination in the cuprizone mouse model. To this end, we exploited the ribosomal tagging (RiboTag) technology that is based on Cre-mediated cell type-selective HA-tagging of ribosomes. Analyses were performed in the corpus callosum of GFAP-Cre+/- Rpl22HA/wt mice 5 weeks after cuprizone feeding, at the peak of demyelination, and 0.5 and 2 weeks after cuprizone withdrawal, when remyelination and tissue repair is initiated. After 5 weeks of cuprizone feeding, reactive astrocytes showed inflammatory signatures with enhanced expression of genes that modulate leukocyte migration (Tlr2, Cd86, Parp14) and they produced the chemokine CXCL10, as verified by histology. Furthermore, demyelination-induced reactive astrocytes expressed numerous ligands including Cx3cl1, Csf1, Il34, and Gas6 that act on homeostatic as well as activated microglia and thus potentially mediate activation and recruitment of microglia and enhancement of their phagocytotic activity. During early remyelination, HA-tagged cells displayed reduced inflammatory response signatures, as indicated by shutdown of CXCL10 production, and enhanced expression of osteopontin (SPP1) as well as of factors that are relevant for tissue remodeling (Timp1), regeneration and axonal repair. During late remyelination, the signatures shifted towards resolving inflammation by active suppression of lymphocyte activation and differentiation and support of glia cell differentiation. In conclusion, we detected highly dynamic astroglial transcriptomic signatures in the cuprizone model, which reflects excessive communication among glia cells and highlights different astrocyte functions during neurodegeneration and regeneration.
Assuntos
Cuprizona , Doenças Desmielinizantes , Camundongos , Animais , Cuprizona/toxicidade , Astrócitos/metabolismo , Doenças Desmielinizantes/patologia , Neuroglia/metabolismo , Corpo Caloso/patologia , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismoRESUMO
Multiple sclerosis (MS) is a focal inflammatory and demyelinating disease. The inflammatory infiltrates consist of macrophages/microglia, T and B cells. Remyelination (RM) is an endogenous repair process which frequently fails in MS patients. In earlier studies, T cells either promoted or impaired RM. Here, we used the combined cuprizone/MOG-EAE model to further dissect the functional role of T cells for RM. The combination of MOG immunization with cuprizone feeding targeted T cells to the corpus callosum and increased the extent of axonal injury. Global gene expression analyses demonstrated significant changes in the inflammatory environment; however, additional MOG immunization did not alter the course of RM. Our results suggest that the inflammatory environment in the combined model affects axons and oligodendrocytes differently and that oligodendroglial lineage cells might be less susceptible to T cell mediated injury.
Assuntos
Doenças Desmielinizantes , Esclerose Múltipla , Remielinização , Animais , Camundongos , Axônios , Corpo Caloso/metabolismo , Cuprizona/toxicidade , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Esclerose Múltipla/metabolismo , Bainha de Mielina/fisiologia , Oligodendroglia/metabolismo , Remielinização/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
BACKGROUND: To investigate the association of ihMT (inhom signals with the demyelination and remyelination phases of the acute cuprizone mouse model in comparison with histology, and to assess the extent of tissue damage and repair from MRI data. METHODS: Acute demyelination by feeding 0.2% cuprizone for five weeks, followed by a four-week remyelination period was applied on genetically modified plp-GFP mice. Animals were scanned at different time points of the demyelination and remyelination phases of the cuprizone model using a multimodal MRI protocol, including ihMT T1D-filters, MPF (Macromolecular Proton Fraction) and R1 (longitudinal relaxation rate). For histology, plp-GFP (proteolipid protein - Green Fluorescent Protein) microscopy and LFB (Luxol Fast Blue) staining were employed as references for the myelin content. Comparison of MRI with histology was performed in the medial corpus callosum (mCC) and cerebral cortex (CTX) at two brain levels whereas ROI-wise and voxel-based analyses of the MRI metrics allowed investigating in vivo the spatial extent of myelin alterations. RESULTS: IhMT high-pass T1D-filters, targeted toward long T1D components, showed significant temporal variations in the mCC consistent with the effects induced by the cuprizone toxin. In addition, the corresponding signals correlated strongly and significantly with the myelin content assessed by GFP fluorescence and LFB staining over the demyelination and the remyelination phases. The signal of the band-pass T1D-filter, which isolates short T1D components, showed changes over time that were poorly correlated with histology, hence suggesting a sensitivity to pathological processes possibly not related to myelin. Although MPF was also highly correlated to histology, ihMT high-pass T1D-filters showed better capability to characterize the spatial-temporal patterns during the demyelination and remyelination phases of the acute cuprizone model (e.g., rostro-caudal gradient of demyelination in the mCC previously described in the literature). CONCLUSIONS: IhMT sequences selective for long T1D components are specific and sensitive in vivo markers of demyelination and remyelination and have successfully captured the spatially heterogeneous pattern of the demyelination and remyelination mechanisms in the cuprizone model. Interestingly, differences in signal variations between the ihMT high-pass and band-pass T1D-filter, suggest a sensitivity of the ihMT sequences targeted to short T1Ds to alterations other than those of myelin. Future studies will need to further address these differences by examining more closely the origin of the short T1D components and the variation of each T1D component in pathology.
Assuntos
Doenças Desmielinizantes , Remielinização , Animais , Camundongos , Cuprizona/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/diagnóstico por imagem , Doenças Desmielinizantes/metabolismo , Imageamento por Ressonância Magnética/métodos , Bainha de Mielina/metabolismo , Camundongos Endogâmicos C57BL , Modelos Animais de DoençasRESUMO
Astrocytes are the most abundant glial cell type in the brain, where they participate in various homeostatic functions. Transcriptomically, diverse astrocyte subpopulations play distinct roles during development and disease progression. However, the biochemical identification of astrocyte subtypes, especially by membrane surface protein glycosylation, remains poorly investigated. Protein tyrosine phosphatase receptor type zeta (PTPRZ) is a highly expressed membrane protein in CNS glia cells that can be modified with diverse glycosylation, including the unique HNK-1 capped O-mannosyl (O-Man) core M2 glycan mediated by brain-specific branching enzyme GnT-IX. Although PTPRZ modified with HNK-1 capped O-Man glycans (HNK-1-O-Man+ PTPRZ) is increased in reactive astrocytes of demyelination model mice, whether such astrocytes emerge in a broad range of disease-associated conditions or are limited to conditions associated with demyelination remains unclear. Here, we show that HNK-1-O-Man+ PTPRZ localizes in hypertrophic astrocytes of damaged brain areas in patients with multiple sclerosis. Furthermore, we show that astrocytes expressing HNK-1-O-Man+ PTPRZ are present in two demyelination mouse models (cuprizone-fed mice and a vanishing white matter disease model), while traumatic brain injury does not induce glycosylation. Administration of cuprizone to Aldh1l1-eGFP and Olig2KICreER/+ ;Rosa26eGFP mice revealed that cells expressing HNK-1-O-Man+ PTPRZ are derived from cells in the astrocyte lineage. Notably, GnT-IX but not PTPRZ mRNA was up-regulated in astrocytes isolated from the corpus callosum of cuprizone model mice. These results suggest that the unique PTPRZ glycosylation plays a key role in the patterning of demyelination-associated astrocytes.
Assuntos
Astrócitos , Doenças Desmielinizantes , Animais , Camundongos , Astrócitos/metabolismo , Encéfalo/metabolismo , Cuprizona/toxicidade , Cuprizona/metabolismo , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/genética , Modelos Animais de Doenças , Glicosilação , Camundongos Endogâmicos C57BL , Polissacarídeos/metabolismo , Proteínas Tirosina Fosfatases/metabolismoRESUMO
Multiple sclerosis (MS) is the most common demyelinating disease that attacks the central nervous system. Dietary intake of cuprizone (CPZ) produces demyelination resembling that of patients with MS. Given the role of the vagus nerve in gut-microbiota-brain axis in development of MS, we performed this study to investigate whether subdiaphragmatic vagotomy (SDV) affects demyelination in CPZ-treated mice. SDV significantly ameliorated demyelination and microglial activation in the brain compared with sham-operated CPZ-treated mice. Furthermore, 16S ribosomal RNA analysis revealed that SDV significantly improved the abnormal gut microbiota composition of CPZ-treated mice. An untargeted metabolomic analysis demonstrated that SDV significantly improved abnormal blood levels of metabolites in CPZ-treated mice compared with sham-operated CPZ-treated mice. Notably, there were correlations between demyelination or microglial activation in the brain and the relative abundance of several microbiome populations, suggesting a link between gut microbiota and the brain. There were also correlations between demyelination or microglial activation in the brain and blood levels of metabolites. Together, these data suggest that CPZ produces demyelination in the brain through the gut-microbiota-brain axis via the subdiaphragmatic vagus nerve.
Assuntos
Doenças Desmielinizantes , Microbiota , Esclerose Múltipla , Animais , Camundongos , Encéfalo/metabolismo , Cuprizona/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Esclerose Múltipla/metabolismo , Nervo Vago/metabolismoRESUMO
Triggering receptor expressed on myeloid cells 2 (TREM2) is a cell-surface immunoreceptor expressed on microglia, osteoclasts, dendritic cells and macrophages. Heterozygous loss-of-function mutations in TREM2, including mutations enhancing shedding form the cell surface, have been associated with myelin/neuronal loss and neuroinflammation in neurodegenerative diseases, such as Alzheimer`s disease and Frontotemporal Dementia. Using the cuprizone model, we investigated the involvement of soluble and cleavage-reduced TREM2 on central myelination processes in cleavage-reduced (TREM2-IPD), soluble-only (TREM2-sol), knockout (TREM2-KO) and wild-type (WT) mice. The TREM2-sol mouse is a new model with selective elimination of plasma membrane TREM2 and a reduced expression of soluble TREM2. In the acute cuprizone model demyelination and remyelination events were reflected by a T2-weighted signal intensity change in magnetic resonance imaging (MRI), most prominently in the external capsule (EC). In contrast to WT and TREM2-IPD, TREM2-sol and TREM2-KO showed an additional increase in MRI signal during the recovery phase. Histological analyses of TREM2-IPD animals revealed no recovery of neuroinflammation as well as of the lysosomal marker LAMP-1 and displayed enhanced cytokine/chemokine levels in the brain. TREM2-sol and, to a much lesser extent, TREM2-KO, however, despite presenting reduced levels of some cytokines/chemokines, showed persistent microgliosis and astrocytosis during recovery, with both homeostatic (TMEM119) as well as activated (LAMP-1) microglia markers increased. This was accompanied, specifically in the EC, by no myelin recovery, with appearance of myelin debris and axonal pathology, while oligodendrocytes recovered. In the chronic model consisting of 12-week cuprizone administration followed by 3-week recovery TREM2-IPD displayed sustained microgliosis and enhanced remyelination in the recovery phase. Taken together, our data suggest that sustained microglia activation led to increased remyelination, whereas microglia without plasma membrane TREM2 and only soluble TREM2 had reduced phagocytic activity despite efficient lysosomal function, as observed in bone marrow-derived macrophages, leading to a dysfunctional phenotype with improper myelin debris removal, lack of remyelination and axonal pathology following cuprizone intoxication.
Assuntos
Doenças Desmielinizantes , Glicoproteínas de Membrana , Receptores Imunológicos , Animais , Camundongos , Cuprizona/toxicidade , Citocinas/metabolismo , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Modelos Genéticos , Bainha de Mielina/metabolismo , Doenças Neuroinflamatórias , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismoRESUMO
Multiple sclerosis (MS) is an inflammatory-mediated demyelinating disease of the central nervous system (CNS). Although studies have demonstrated that microglia facilitate remyelination in demyelinating diseases, the underlying mechanisms are still not fully characterized. We found that aryl hydrocarbon receptor (AhR), an environment sensor, was upregulated within the corpus callosum in the cuprizone model of CNS demyelination, and upregulated AhR was mainly confined to microglia. Deletion of AhR in adult microglia inhibited efficient remyelination. Transcriptome analysis using RNA-seq revealed that AhR-deficient microglia displayed impaired gene expression signatures associated with lysosome and phagocytotic pathways. Furthermore, AhR-deficient microglia showed impaired clearance of myelin debris and defected phagocytic capacity. Further investigation of target genes of AhR revealed that spleen tyrosine kinase (SYK) is the downstream effector of AhR and mediated the phagocytic capacity of microglia. Additionally, AhR deficiency in microglia aggravated CNS inflammation during demyelination. Altogether, our study highlights an essential role for AhR in microglial phagocytic function and suggests the therapeutic potential of AhR in demyelinating diseases.
Assuntos
Doenças Desmielinizantes , Receptores de Hidrocarboneto Arílico , Remielinização , Animais , Camundongos , Corpo Caloso/metabolismo , Cuprizona/toxicidade , Doenças Desmielinizantes/tratamento farmacológico , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Bainha de Mielina/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Remielinização/fisiologiaRESUMO
Cuprizone causes consistent demyelination and oligodendrocyte damage in the mouse brain. Cu,Zn-superoxide dismutase 1 (SOD1) has neuroprotective potential against various neurological disorders, such as transient cerebral ischemia and traumatic brain injury. In this study, we investigated whether SOD1 has neuroprotective effects against cuprizone-induced demyelination and adult hippocampal neurogenesis in C57BL/6 mice, using the PEP-1-SOD1 fusion protein to facilitate the delivery of SOD1 protein into hippocampal neurons. Eight weeks feeding of cuprizone-supplemented (0.2%) diets caused a significant decrease in myelin basic protein (MBP) expression in the stratum lacunosum-moleculare of the CA1 region, the polymorphic layer of the dentate gyrus, and the corpus callosum, while ionized calcium-binding adapter molecule 1 (Iba-1)-immunoreactive microglia showed activated and phagocytic phenotypes. In addition, cuprizone treatment reduced proliferating cells and neuroblasts as shown using Ki67 and doublecortin immunostaining. Treatment with PEP-1-SOD1 to normal mice did not show any significant changes in MBP expression and Iba-1-immunoreactive microglia. However, Ki67-positive proliferating cells and doublecortin-immunoreactive neuroblasts were significantly decreased. Simultaneous treatment with PEP-1-SOD1 and cuprizone-supplemented diets did not ameliorate the MBP reduction in these regions, but mitigated the increase of Iba-1 immunoreactivity in the corpus callosum and alleviated the reduction of MBP in corpus callosum and proliferating cells, not neuroblasts, in the dentate gyrus. In conclusion, PEP-1-SOD1 treatment only has partial effects to reduce cuprizone-induced demyelination and microglial activation in the hippocampus and corpus callosum and has minimal effects on proliferating cells in the dentate gyrus.