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BACKGROUND: Merkel cell carcinoma (MCC) is a highly malignant skin cancer. Despite major treatment improvements during the last decade, up to 50% of patients do not respond to therapy or develop recurrent disease. For these patients, alternative treatment options are urgently needed. Here, we assessed the efficacy of the combination of the BCL-2 inhibitor Navitoclax and the PI3K p110α inhibitor Alpelisib in MCC cell lines. METHODS: The expression of BCL-2 was assessed by immunohistochemistry in MCC and MCC cell lines. Treatment with Navitoclax and Alpelisib alone and in combination was performed on four MCC cell lines. The decrease of cell viability during treatment was assessed by XTT assay and visualized for the combinations by 3D combinatorial index plotting. The increase of apoptotic cells was determined by cleaved PARP Western blotting and Annexin V staining. RESULTS: Some 94% of MCCs and all three MCPyV-positive cell lines showed BCL-2 expression. Navitoclax monotreatment was shown to be highly effective when treating BCL-2-positive cell lines (IC50-values ranging from 96.0 to 323.0 nM). The combination of Alpelisib and Navitoclax resulted in even stronger synergistic and prolonged inhibitions of MCC cell viability through apoptosis up to 4 days. DISCUSSION: Our results show that the anti-apoptotic BCL-2 is frequently expressed in MCC and MCC cell lines. Inhibition of BCL-2 by Navitoclax in combination with Alpelisib revealed a strong synergy and prolonged inhibition of MCC cell viability and induction of apoptosis. The combination of Navitoclax and Alpelisib is a novel potential treatment option for MCC patients.
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OBJECTIVE: We aimed to study the occurrence and development of axonal pathology and the influence of astrocytes in vanishing white matter. METHODS: Axons and myelin were analyzed using electron microscopy and immunohistochemistry on Eif2b4 and Eif2b5 single- and double-mutant mice and patient brain tissue. In addition, astrocyte-forebrain co-culture studies were performed. RESULTS: In the corpus callosum of Eif2b5-mutant mice, myelin sheath thickness, axonal diameter, and G-ratio developed normally up to 4 months. At 7 months, however, axons had become thinner, while in control mice axonal diameters had increased further. Myelin sheath thickness remained close to normal, resulting in an abnormally low G-ratio in Eif2b5-mutant mice. In more severely affected Eif2b4-Eif2b5 double-mutants, similar abnormalities were already present at 4 months, while in milder affected Eif2b4 mutants, few abnormalities were observed at 7 months. Additionally, from 2 months onward an increased percentage of thin, unmyelinated axons and increased axonal density were present in Eif2b5-mutant mice. Co-cultures showed that Eif2b5 mutant astrocytes induced increased axonal density, also in control forebrain tissue, and that control astrocytes induced normal axonal density, also in mutant forebrain tissue. In vanishing white matter patient brains, axons and myelin sheaths were thinner than normal in moderately and severely affected white matter. In mutant mice and patients, signs of axonal transport defects and cytoskeletal abnormalities were minimal. INTERPRETATION: In vanishing white matter, axons are initially normal and atrophy later. Astrocytes are central in this process. If therapy becomes available, axonal pathology may be prevented with early intervention.
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The glial stress protein alpha B-crystallin (HSPB5) is an endogenous agonist for Toll-like receptor 2 in CD14+ cells. Following systemic administration, HSPB5 acts as a potent inhibitor of neuroinflammation in animal models and reduces lesion development in multiple sclerosis patients. Here, we show that systemically administered HSPB5 rapidly crosses the blood-brain barrier, implicating microglia as additional targets for HSPB5 along with peripheral monocytes and macrophages. To compare key players in the HSPB5-induced protective response of human macrophages and microglia, we applied weighted gene co-expression network analysis on transcript expression data obtained 1 and 4 h after activation. This approach identified networks of genes that are co-expressed in all datasets, thus reducing the complexity of the nonsynchronous waves of transcripts that appear after activation by HSPB5. In both cell types, HSPB5 activates a network of highly connected genes that appear to be functionally equivalent and consistent with the therapeutic effects of HSPB5 in vivo, since both networks include factors that suppress apoptosis, the production of proinflammatory factors, and the development of adaptive immunity. Yet, hub genes at the core of the network in either cell type were strikingly different. They prominently feature the well-known tolerance-promoting programmed-death ligand 1 as a key player in the macrophage response to HSPB5, and the immune-regulatory enzyme cyclooxygenase-2 (COX-2) in that of microglia. This latter finding indicates that despite its reputation as a potential target for nonsteroidal anti-inflammatory drugs, microglial COX-2 plays a central role in the therapeutic effects of HSPB5 during neuroinflammation. GLIA 2017;65:460-473.
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Ciclo-Oxigenase 2/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Cadeia B de alfa-Cristalina/farmacologia , Animais , Encéfalo/citologia , Células Cultivadas , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Redes Reguladoras de Genes/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tecido Parenquimatoso/citologia , Tecido Parenquimatoso/efeitos dos fármacos , RNA Mensageiro/metabolismo , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismo , Cadeia B de alfa-Cristalina/metabolismoRESUMO
BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disease. In addition to the occurrence of amyloid deposits and widespread tau pathology, AD is associated with a neuroinflammatory response characterized by the activation of microglia and astrocytes. Protein kinase 2 (CK2, former casein kinase II) is involved in a wide variety of cellular processes. Previous studies on CK2 in AD showed controversial results, and the involvement of CK2 in neuroinflammation in AD remains elusive. METHODS: In this study, we used immunohistochemical and immunofluorescent staining methods to investigate the localization of CK2 in the hippocampus and temporal cortex of patients with AD and non-demented controls. We compared protein levels with Western blotting analysis, and we investigated CK2 activity in human U373 astrocytoma cells and human primary adult astrocytes stimulated with IL-1ß or TNF-α. RESULTS: We report increased levels of CK2 in the hippocampus and temporal cortex of AD patients compared to non-demented controls. Immunohistochemical analysis shows CK2 immunoreactivity in astrocytes in AD and control cases. In AD, the presence of CK2 immunoreactive astrocytes is increased. CK2 immunopositive astrocytes are associated with amyloid deposits, suggesting an involvement of CK2 in the neuroinflammatory response. In U373 cells and human primary astrocytes, the selective CK2 inhibitor CX-4945 shows a dose-dependent reduction of the IL-1ß or TNF-α induced MCP-1 and IL-6 secretion. CONCLUSIONS: This data suggests that CK2 in astrocytes is involved in the neuroinflammatory response in AD. The reduction in pro-inflammatory cytokine secretion by human astrocytes using the selective CK2 inhibitor CX-4945 indicates that CK2 could be a potential target to modulate neuroinflammation in AD.
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Doença de Alzheimer/patologia , Astrócitos/enzimologia , Encéfalo/patologia , Idoso , Idoso de 80 Anos ou mais , Amiloide/metabolismo , Astrócitos/efeitos dos fármacos , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Caseína Quinase II/metabolismo , Células Cultivadas , Citocinas/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Naftiridinas/farmacologia , FenazinasRESUMO
INTRODUCTION: The important protective role of small heat-shock proteins (HSPs) in regulating cellular survival and migration, counteracting protein aggregation, preventing apoptosis, and regulating inflammation in the central nervous system is now well-recognized. Yet, their role in the neuroinflammatory disorder multiple sclerosis (MS) is largely undocumented. With the exception of alpha B-crystallin (HSPB5), little is known about the roles of small HSPs in disease. RESULTS: Here, we examined the expression of four small HSPs during lesion development in MS, focussing on their cellular distribution, and regional differences between white matter (WM) and grey matter (GM). It is well known that MS lesions in these areas differ markedly in their pathology, with substantially more intense blood-brain barrier damage, leukocyte infiltration and microglial activation typifying WM but not GM lesions. We analysed transcript levels and protein distribution profiles for HSPB1, HSPB6, HSPB8 and HSPB11 in MS lesions at different stages, comparing them with normal-appearing brain tissue from MS patients and non-neurological controls. During active stages of demyelination in WM, and especially the centre of chronic active MS lesions, we found significantly increased expression of HSPB1, HSPB6 and HSPB8, but not HSPB11. When induced, small HSPs were exclusively found in astrocytes but not in oligodendrocytes, microglia or neurons. Surprisingly, while the numbers of astrocytes displaying high expression of small HSPs were markedly increased in actively demyelinating lesions in WM, no such induction was observed in GM lesions. This difference was particularly obvious in leukocortical lesions covering both WM and GM areas. CONCLUSIONS: Since induction of small HSPs in astrocytes is apparently a secondary response to damage, their differential expression between WM and GM likely reflects differences in mediators that accompany demyelination in either WM or GM during MS. Our findings also suggest that during MS, cortical structures fail to benefit from the protective actions of small HSPs.
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Regulação da Expressão Gênica/fisiologia , Substância Cinzenta/metabolismo , Proteínas de Choque Térmico Pequenas/metabolismo , Esclerose Múltipla/patologia , Substância Branca/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Sistema Nervoso Central/patologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Antígenos HLA-DR/metabolismo , Proteínas de Choque Térmico Pequenas/genética , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Proteína Proteolipídica de Mielina/metabolismo , RNA Mensageiro/metabolismo , Estatísticas não ParamétricasRESUMO
Similar to macrophages, microglia adopt diverse activation states and contribute to repair and tissue damage in multiple sclerosis. Using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, we show that in vitro M1-polarized (proinflammatory) human adult microglia express the distinctive markers CD74, CD40, CD86, and CCR7, whereas M2 (anti-inflammatory) microglia express mannose receptor and the anti-inflammatory cytokine CCL22. The expression of these markers was assessed in clusters of activated microglia in normal-appearing white matter (preactive lesions) and areas of remyelination, representing reparative multiple sclerosis lesions. We show that activated microglia in preactive and remyelinating lesions express CD74, CD40, CD86, and the M2 markers CCL22 and CD209, but not mannose receptor. To examine whether this intermediate microglia profile is static or dynamic and thus susceptible to changes in the microenvironment, we polarized microglia into M1 or M2 phenotype in vitro and then subsequently treated them with the opposing polarization regimen. These studies revealed that expression of CD40, CXCL10, and mannose receptor is dynamic and that microglia, like macrophages, can switch between M1 and M2 phenotypic profiles. Taken together, our data define the differential activation states of microglia during lesion development in multiple sclerosis-affected CNS tissues and underscore the plasticity of human adult microglia in vitro.
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Encéfalo/patologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Microglia/patologia , Esclerose Múltipla/patologia , Proteína Proteolipídica de Mielina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/genética , Antígenos CD/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Humanos , Macrófagos/patologia , Masculino , Microglia/metabolismo , Pessoa de Meia-Idade , Proteína Proteolipídica de Mielina/genética , RNA Mensageiro/metabolismo , Estatísticas não Paramétricas , TranscriptomaRESUMO
Activated microglia and macrophages play a key role in driving demyelination during multiple sclerosis (MS), but the factors responsible for their activation remain poorly understood. Here, we present evidence for a dual-trigger role of IFN-γ and alpha B-crystallin (HSPB5) in this context. In MS-affected brain tissue, accumulation of the molecular chaperone HSPB5 by stressed oligodendrocytes is a frequent event. We have shown before that this triggers a TLR2-mediated protective response in surrounding microglia, the molecular signature of which is widespread in normal-appearing brain tissue during MS. Here, we show that IFN-γ, which can be released by infiltrated T cells, changes the protective response of microglia and macrophages to HSPB5 into a robust pro-inflammatory classical response. Exposure of cultured microglia and macrophages to IFN-γ abrogated subsequent IL-10 induction by HSPB5, and strongly promoted HSPB5-triggered release of TNF-α, IL-6, IL-12, IL-1ß and reactive oxygen and nitrogen species. In addition, high levels of CXCL9, CXCL10, CXL11, several guanylate-binding proteins and the ubiquitin-like protein FAT10 were induced by combined activation with IFN-γ and HSPB5. As immunohistochemical markers for microglia and macrophages exposed to both IFN-γ and HSPB5, these latter factors were found to be selectively expressed in inflammatory infiltrates in areas of demyelination during MS. In contrast, they were absent from activated microglia in normal-appearing brain tissue. Together, our data suggest that inflammatory demyelination during MS is selectively associated with IFN-γ-induced re-programming of an otherwise protective response of microglia and macrophages to the endogenous TLR2 agonist HSPB5.
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Interferon gama/metabolismo , Macrófagos/fisiologia , Microglia/fisiologia , Esclerose Múltipla/imunologia , Cadeia B de alfa-Cristalina/metabolismo , Encéfalo/imunologia , Encéfalo/patologia , Células Cultivadas , Quimiocina CXCL10/metabolismo , Quimiocina CXCL11/metabolismo , Quimiocina CXCL9/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Macrófagos/patologia , Microglia/patologia , Esclerose Múltipla/patologia , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitinas/metabolismoRESUMO
Progressive multiple sclerosis is associated with metabolic failure of the axon and excitotoxicity that leads to chronic neurodegeneration. Global sodium-channel blockade causes side effects that can limit its use for neuroprotection in multiple sclerosis. Through selective targeting of drugs to lesions we aimed to improve the potential therapeutic window for treatment. This was assessed in the relapsing-progressive experimental autoimmune encephalomyelitis ABH mouse model of multiple sclerosis using conventional sodium channel blockers and a novel central nervous system-excluded sodium channel blocker (CFM6104) that was synthesized with properties that selectively target the inflammatory penumbra in experimental autoimmune encephalomyelitis lesions. Carbamazepine and oxcarbazepine were not immunosuppressive in lymphocyte-driven autoimmunity, but slowed the accumulation of disability in experimental autoimmune encephalomyelitis when administered during periods of the inflammatory penumbra after active lesion formation, and was shown to limit the development of neurodegeneration during optic neuritis in myelin-specific T cell receptor transgenic mice. CFM6104 was shown to be a state-selective, sodium channel blocker and a fluorescent p-glycoprotein substrate that was traceable. This compound was >90% excluded from the central nervous system in normal mice, but entered the central nervous system during the inflammatory phase in experimental autoimmune encephalomyelitis mice. This occurs after the focal and selective downregulation of endothelial p-glycoprotein at the blood-brain barrier that occurs in both experimental autoimmune encephalomyelitis and multiple sclerosis lesions. CFM6104 significantly slowed down the accumulation of disability and nerve loss in experimental autoimmune encephalomyelitis. Therapeutic-targeting of drugs to lesions may reduce the potential side effect profile of neuroprotective agents that can influence neurotransmission. This class of agents inhibit microglial activity and neural sodium loading, which are both thought to contribute to progressive neurodegeneration in multiple sclerosis and possibly other neurodegenerative diseases.
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Benzamidas/uso terapêutico , Indazóis/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Oxidiazóis/uso terapêutico , Bloqueadores dos Canais de Sódio/uso terapêutico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Bancos de Espécimes Biológicos , Encéfalo/patologia , Carbamazepina/farmacologia , Proteínas de Transporte/metabolismo , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Sistemas de Liberação de Medicamentos , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Imuno-Histoquímica , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/fisiopatologia , Neurite Óptica/fisiopatologia , Linfócitos T/efeitos dos fármacos , Uveíte/fisiopatologia , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
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.
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Autoantígenos/imunologia , Encefalomielite Autoimune Experimental/imunologia , Proteínas de Neurofilamentos/imunologia , Medula Espinal/imunologia , Medula Espinal/patologia , Animais , Encefalomielite Autoimune Experimental/patologia , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito T/imunologia , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Microscopia Confocal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos TRESUMO
Microglial nodules are frequently observed in the normal-appearing white matter of multiple sclerosis (MS) patients. Previously, we have shown that these clusters, which we call "preactive MS lesions," are closely associated with stressed oligodendrocytes and myelin sheaths that contain markedly elevated levels of the small stress protein alpha-B-crystallin (HspB5). Here, we show that microglia in these lesions express the recently identified receptors for HspB5, that is, CD14, Toll-like receptor family 1 and 2 (TLR1 and TLR2), and several molecular markers of the microglial response to HspB5. These markers were identified by genome-wide transcript profiling of 12 primary human microglial cultures at 2 time points after exposure to HspB5. These data indicate that HspB5 activates production by microglia of an array of chemokines, immune-regulatory mediators, and a striking number of antiviral genes that are generally inducible by type I interferons. Together, our data suggest that preactive MS lesions are at least in part driven by HspB5 derived from stressed oligodendrocytes and may reflect a local attempt to restore tissue homeostasis.
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Encéfalo/efeitos dos fármacos , Microglia/efeitos dos fármacos , Esclerose Múltipla/metabolismo , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Cadeia B de alfa-Cristalina/farmacologia , Idoso , Idoso de 80 Anos ou mais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Axônios/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Humanos , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Microglia/imunologia , Microglia/metabolismo , Microglia/patologia , Pessoa de Meia-Idade , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Fibras Nervosas Mielinizadas/metabolismo , Fibras Nervosas Mielinizadas/patologia , Receptor 1 Toll-Like/genética , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Cadeia B de alfa-Cristalina/metabolismoRESUMO
As an extracellular protein, the small heat-shock protein alpha B-crystallin (HSPB5) has anti-inflammatory effects in several mouse models of inflammation. Here, we show that these effects are associated with the ability of HSPB5 to activate an immune-regulatory response in macrophages via endosomal/phagosomal CD14 and Toll-like receptors 1 and 2. Humans, however, possess natural antibodies against HSPB5 that block receptor binding. To protect it from these antibodies, we encapsulated HSPB5 in porous PLGA microparticles. We document here size, morphology, protein loading and release characteristics of such microparticles. Apart from effectively protecting HSPB5 from neutralization, PLGA microparticles also strongly promoted macrophage targeting of HSPB via phagocytosis. As a result, HSPB5 in porous PLGA microparticles was more than 100-fold more effective in activating macrophages than free soluble protein. Yet, the immune-regulatory nature of the macrophage response, as documented here by microarray transcript profiling, remained the same. In mice developing cigarette smoke-induced COPD, HSPB5-loaded PLGA microparticles were selectively taken up by alveolar macrophages upon intratracheal administration, and significantly suppressed lung infiltration by lymphocytes and neutrophils. In contrast, 30-fold higher doses of free soluble HSPB5 remained ineffective. Our data indicate that porous HSPB5-PLGA microparticles hold considerable promise as an anti-inflammatory biomaterial for humans.
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Anti-Inflamatórios/administração & dosagem , Pulmão/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Pneumonia/complicações , Pneumonia/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/complicações , Cadeia B de alfa-Cristalina/administração & dosagem , Animais , Anti-Inflamatórios/imunologia , Anti-Inflamatórios/uso terapêutico , Linhagem Celular , Portadores de Fármacos/química , Proteínas de Choque Térmico Pequenas/administração & dosagem , Proteínas de Choque Térmico Pequenas/imunologia , Proteínas de Choque Térmico Pequenas/uso terapêutico , Humanos , Ácido Láctico/química , Receptores de Lipopolissacarídeos/imunologia , Pulmão/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia/imunologia , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Receptor 1 Toll-Like/imunologia , Receptor 2 Toll-Like/imunologia , Cadeia B de alfa-Cristalina/imunologia , Cadeia B de alfa-Cristalina/uso terapêuticoRESUMO
There is growing evidence that mitochondrial dysfunction and associated reactive oxygen species (ROS) formation contribute to neurodegenerative processes in multiple sclerosis (MS). Here, we investigated whether alterations in transcriptional regulators of key mitochondrial proteins underlie mitochondrial dysfunction in MS cortex and contribute to neuronal loss. Hereto, we analyzed the expression of mitochondrial transcriptional (co-)factors and proteins involved in mitochondrial redox balance regulation in normal-appearing grey matter (NAGM) samples of cingulate gyrus and/or frontal cortex from 15 MS patients and nine controls matched for age, gender and post-mortem interval. PGC-1α, a transcriptional co-activator and master regulator of mitochondrial function, was consistently and significantly decreased in pyramidal neurons in the deeper layers of MS cortex. Reduced PGC-1α levels coincided with reduced expression of oxidative phosphorylation subunits and a decrease in gene and protein expression of various mitochondrial antioxidants and uncoupling proteins (UCPs) 4 and 5. Short-hairpin RNA-mediated silencing of PGC-1α in a neuronal cell line confirmed that reduced levels of PGC-1α resulted in a decrease in transcription of OxPhos subunits, mitochondrial antioxidants and UCPs. Moreover, PGC-1α silencing resulted in a decreased mitochondrial membrane potential, increased ROS formation and enhanced susceptibility to ROS-induced cell death. Importantly, we found extensive neuronal loss in NAGM from cingulate gyrus and frontal cortex of MS patients, which significantly correlated with the extent of PGC-1α decrease. Taken together, our data indicate that reduced neuronal PGC-1α expression in MS cortex partly underlies mitochondrial dysfunction in MS grey matter and thereby contributes to neurodegeneration in MS cortex.
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Córtex Cerebral/patologia , Proteínas de Choque Térmico/fisiologia , Mitocôndrias/patologia , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Neurônios/patologia , Fatores de Transcrição/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Contagem de Células , Regulação para Baixo , Feminino , Vetores Genéticos , Giro do Cíngulo/patologia , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Humanos , Imuno-Histoquímica , Lentivirus/genética , Masculino , Pessoa de Meia-Idade , Oxirredução , Fosforilação Oxidativa , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Células Piramidais/patologia , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Bancos de Tecidos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: In brain tissues from multiple sclerosis (MS) patients, clusters of activated HLA-DR-expressing microglia, also referred to as preactive lesions, are located throughout the normal-appearing white matter. The aim of this study was to gain more insight into the frequency, distribution and cellular architecture of preactive lesions using a large cohort of well-characterized MS brain samples. METHODS: Here, we document the frequency of preactive lesions and their association with distinct white matter lesions in a cohort of 21 MS patients. Immunohistochemistry was used to gain further insight into the cellular and molecular composition of preactive lesions. RESULTS: Preactive lesions were observed in a majority of MS patients (67%) irrespective of disease duration, gender or subtype of disease. Microglial clusters were predominantly observed in the vicinity of active demyelinating lesions and are not associated with T cell infiltrates, axonal alterations, activated astrocytes or blood-brain barrier disruption. Microglia in preactive lesions consistently express interleukin-10 and TNF-α, but not interleukin-4, whereas matrix metalloproteases-2 and -9 are virtually absent in microglial nodules. Interestingly, key subunits of the free-radical-generating enzyme NADPH oxidase-2 were abundantly expressed in microglial clusters. CONCLUSIONS: The high frequency of preactive lesions suggests that it is unlikely that most of them will progress into full-blown demyelinating lesions. Preactive lesions are not associated with blood-brain barrier disruption, suggesting that an intrinsic trigger of innate immune activation, rather than extrinsic factors crossing a damaged blood-brain barrier, induces the formation of clusters of activated microglia.
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Encéfalo/imunologia , Encéfalo/metabolismo , Microglia/imunologia , Microglia/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Encéfalo/patologia , Estudos de Coortes , Humanos , Imunidade Inata , Microglia/citologia , Pessoa de Meia-Idade , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Fibras Nervosas Mielinizadas/imunologia , Fibras Nervosas Mielinizadas/metabolismoRESUMO
In multiple sclerosis (MS), a chronic inflammatory relapsing demyelinating disease, failure to control or repair damage leads to progressive neurological dysfunction and neurodegeneration. Implantation of neural stem cells (NSCs) has been shown to promote repair and functional recovery in the acute experimental autoimmune encephalomyelitis (EAE) animal model for MS; the major therapeutic mechanism of these NSCs appeared to be immune regulation. In the present study, we examined the efficacy of intraventricularly injected NSCs in chronic relapsing experimental autoimmune encephalomyelitis (CREAE), the animal disease model that is widely accepted to mimic most closely recurrent inflammatory demyelination lesions as observed in relapsing-remitting MS. In addition, we assessed whether priming these NSCs to become oligodendrocyte precursor cells (OPCs) by transient overexpression of Olig2 would further promote functional recovery, for example, by contributing to actual remyelination. Upon injection at the onset of the acute phase or the relapse phase of CREAE, NSCs as well as Olig2-NSCs directly migrated toward active lesions in the spinal cord as visualized by in vivo bioluminescence and biofluorescence imaging, and once in the spinal cord, the majority of Olig2-NSCs, in contrast to NSCs, differentiated into OPCs. The survival of Olig2-NSCs was significantly higher than that of injected control NSCs, which remained undifferentiated. Nevertheless, both Olig2-NSCs and NSC significantly reduced the clinical signs of acute and relapsing disease and, in case of Olig2-NSCs, even completely abrogated relapsing disease when administered early after onset of acute disease. We provide the first evidence that NSCs and in particular NSC-derived OPCs (Olig2-NSCs) ameliorate established chronic relapsing EAE in mice. Our experimental data in established neurological disease in mice indicate that such therapy may be effective in relapsing-remitting MS preventing chronic progressive disease.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Encefalomielite Autoimune Experimental/terapia , Esclerose Múltipla/terapia , Proteínas do Tecido Nervoso/biossíntese , Células-Tronco Neurais/transplante , Oligodendroglia/transplante , Transplante de Células-Tronco/métodos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/administração & dosagem , Diferenciação Celular/fisiologia , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Feminino , Injeções Intraventriculares , Camundongos , Esclerose Múltipla/patologia , Bainha de Mielina/patologia , Proteínas do Tecido Nervoso/administração & dosagem , Células-Tronco Neurais/metabolismo , Fator de Transcrição 2 de Oligodendrócitos , Oligodendroglia/metabolismo , TransfecçãoRESUMO
There is now compelling evidence that members of the family of small heat shock proteins (HSP) can be secreted by a variety of different types of cells. Secretion of small HSP may at times represent altruistic delivery of supporting and stabilizing factors from one cell to another. A probably more general effect of extracellular small HSP, however, is exerted by their ability to activate macrophages and macrophage-like cells. When doing so, small HSP induce an immune-regulatory state of activation, stimulating macrophages to suppress inflammation. For this reason, small HSP deserve consideration as broadly applicable therapeutic agents for inflammatory disorders. In one particular case, however, adaptive immune responses to the small HSP itself may subvert the protective quality of the innate immune response it triggers. This situation only applies to alpha B-crystallin, and is unique for humans as well. In this special case, local concentrations of alpha B-crystallin determine the balance between protective innate responses and destructive adaptive responses, the latter of which are held responsible for the development of multiple sclerosis lesions. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.
Assuntos
Proteínas de Choque Térmico Pequenas/fisiologia , Imunidade Inata , Mediadores da Inflamação/fisiologia , Esclerose Múltipla/metabolismo , Imunidade Adaptativa , Animais , Exossomos/metabolismo , Proteínas de Choque Térmico Pequenas/metabolismo , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/fisiologia , Esclerose Múltipla/imunologiaRESUMO
Neuroaxonal degeneration is a pathological hallmark of multiple sclerosis (MS) contributing to irreversible neurological disability. Pathological mechanisms leading to axonal damage include autoimmunity to neuronal antigens. In actively demyelinating lesions, myelin is phagocytosed by microglia and blood-borne macrophages, whereas the fate of degenerating or damaged axons is unclear. Phagocytosis is essential for clearing neuronal debris to allow repair and regeneration. However, phagocytosis may lead to antigen presentation and autoimmunity, as has been described for neuroaxonal antigens. Despite this notion, it is unknown whether phagocytosis of neuronal antigens occurs in MS. Here, we show using novel, well-characterized antibodies to axonal antigens, that axonal damage is associated with HLA-DR expressing microglia/macrophages engulfing axonal bulbs, indicative of axonal damage. Neuronal proteins were frequently observed inside HLA-DR(+) cells in areas of axonal damage. In vitro, phagocytosis of neurofilament light (NF-L), present in white and gray matter, was observed in human microglia. The number of NF-L or myelin basic protein (MBP) positive cells was quantified using the mouse macrophage cell line J774.2. Intracellular colocalization of NF-L with the lysosomal membrane protein LAMP1 was observed using confocal microscopy confirming that NF-L is taken up and degraded by the cell. In vivo, NF-L and MBP was observed in cerebrospinal fluid cells from patients with MS, suggesting neuronal debris is drained by this route after axonal damage. In summary, neuroaxonal debris is engulfed, phagocytosed, and degraded by HLA-DR(+) cells. Although uptake is essential for clearing neuronal debris, phagocytic cells could also play a role in augmenting autoimmunity to neuronal antigens.
Assuntos
Microglia/fisiologia , Esclerose Múltipla/patologia , Neurônios/patologia , Fagocitose/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Catepsina D/farmacologia , Catepsinas/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Antígenos HLA-DR/metabolismo , Humanos , Masculino , Camundongos , Microglia/efeitos dos fármacos , Microscopia Confocal , Pessoa de Meia-Idade , Esclerose Múltipla/líquido cefalorraquidiano , Proteína Básica da Mielina/líquido cefalorraquidiano , Proteína Básica da Mielina/metabolismo , Fibras Nervosas Mielinizadas/metabolismo , Fibras Nervosas Mielinizadas/patologia , Proteínas de Neurofilamentos/líquido cefalorraquidiano , Proteínas de Neurofilamentos/efeitos dos fármacos , Proteínas de Neurofilamentos/metabolismo , Neurônios/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Fatores de TempoRESUMO
Hippocampal pathology was shown to be extensive in multiple sclerosis (MS) and is associated with memory impairment. In this post-mortem study, we investigated hippocampal tissue from MS and Alzheimer's disease (AD) patients and compared these to non-neurological controls. By means of biochemical assessment, (immuno)histochemistry and western blot analyses, we detected substantial alterations in the cholinergic neurotransmitter system in the MS hippocampus, which were different from those in AD hippocampus. In MS hippocampus, activity and protein expression of choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, was decreased, while the activity and protein expression of acetylcholinesterase (AChE), the acetylcholine degrading enzyme, was found to be unaltered. In contrast, in AD hippocampus both ChAT and AChE enzyme activity and protein expression was decreased. Our findings reveal an MS-specific cholinergic imbalance in the hippocampus, which may be instrumental in terms of future treatment options for memory problems in this disease.
Assuntos
Acetilcolinesterase/metabolismo , Doença de Alzheimer/patologia , Colina O-Acetiltransferase/metabolismo , Hipocampo/enzimologia , Esclerose Múltipla/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/complicações , Feminino , Hipocampo/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/complicaçõesRESUMO
Experimental autoimmune encephalomyelitis (EAE) is a spectrum of neurological disorders in laboratory animals that is used to model multiple sclerosis (MS). However, few agents have translated from efficacy in EAE to the treatment of human disease. Although this may reflect species differences in pathological disease mechanisms, importantly it may also relate to the practice of how drugs and models are currently used. This often bears very little resemblance to the clinical scenarios where treatments are investigated, such that lack of appreciation of the biology of disease may doom drugs to failure. The use of EAE is critically appraised with the aim of provoking thought, improving laboratory practise and aiding researchers and reviewers to address quality issues when undertaking, reporting and interpreting animal studies related to MS research. This is important as many researchers using EAE could and should do more to improve the quality of the studies.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Imunossupressores/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Pesquisa Translacional Biomédica , Animais , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/fisiopatologia , Humanos , Esclerose Múltipla/patologia , Esclerose Múltipla/fisiopatologia , Controle de Qualidade , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Especificidade da Espécie , Fatores de Tempo , Pesquisa Translacional Biomédica/normasRESUMO
Multiple Sclerosis (MS) is a neuroinflammatory disease mainly affecting young adults. A major pathological hallmark of MS is the presence of demyelinated lesions in the central nervous system. In the active phase of the disease, astrocytes become activated, migrate and contribute to local tissue remodeling that ultimately can result in an astroglial scar. This process is facilitated by extracellular matrix proteins, including fibronectin. Tissue Transglutaminase (TG2) is a multifunctional enzyme with a ubiquitous tissue distribution and it has been shown that inflammatory cytokines can induce TG2 activity. In addition, TG2 is known to mediate cell adhesion and migration. We therefore hypothesized that TG2 is present in MS lesions and plays a role in cell adhesion and/or migration. Our studies showed that TG2 immunoreactivity appeared in astrocytes in active and chronic active MS lesions. These TG2 positive astrocytes partly co-localized with fibronectin. Additional in vitro studies showed that TG2 mediated astrocytoma adhesion to and migration on the extracellular matrix protein fibronectin. We therefore speculate that TG2 mediates the enhanced interaction of astrocytes with fibronectin in the extracellular matrix of MS lesions, thereby contributing to astrocyte adhesion and migration, and thus in tissue remodeling and possibly glial scarring.
Assuntos
Astrócitos/metabolismo , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Esclerose Múltipla/enzimologia , Bainha de Mielina/metabolismo , Transglutaminases/metabolismo , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Astrócitos/patologia , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Fibronectinas/metabolismo , Proteínas de Ligação ao GTP , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/patologia , Bainha de Mielina/patologia , Proteína 2 Glutamina gama-Glutamiltransferase , RNA Interferente PequenoRESUMO
Oxidative stress plays a major role in multiple sclerosis (MS), a chronic inflammatory central nervous system (CNS) disease. Invading leukocytes contribute to cell damage and demyelination by producing excessive amounts of cytotoxic mediators, including reactive oxygen species (ROS). To counteract the damaging effects of ROS the CNS is endowed with a repertoire of endogenous antioxidant enzymes, which are regulated by the transcription factor NF-E2-related factor 2 (Nrf2). Upon exposure to ROS, Nrf2 translocates to the nucleus allowing transcriptional activation of various antioxidant enzymes. DJ1 is a protein that is involved in the stabilization of Nrf2 and hence acts as a positive regulator of Nrf2-driven antioxidant protection. Here, we investigate the (sub)cellular localization of Nrf2 and DJ1 in various MS lesion stages and show that Nrf2 is strikingly upregulated in active MS lesions, in both the nucleus and the cytoplasm of infiltrating macrophages and to a lesser extent in reactive astrocytes. Simultaneously, DJ1 protein expression is predominantly increased in astrocytes in both active and chronic inactive MS lesions compared to control brain tissue and normal-appearing white matter. Together, our findings suggest that persistent Nrf2-mediated transcription occurs in active MS lesions, but that this endogenous response is insufficient to prevent ROS-induced cellular damage, which is abundant in inflammatory MS lesions.