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1.
Glia ; 67(7): 1320-1332, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30815939

RESUMO

Oligodendrocytes (OLs) are the myelinating glia of the central nervous system. Injury to OLs causes myelin loss. In demyelinating diseases, such as multiple sclerosis, the remyelination is hindered principally due to a failure of the oligodendrocyte precursor cells (OPCs) to differentiate into mature OLs. To identify inducers of OPC to OL differentiation, a high-throughput screening based on myelin basic protein expression using neural progenitor cells-derived OPCs has been performed and, PD0325901-an MEK (MAPK kinase) inhibitor-is found to significantly enhance OPC to OL differentiation in a dose- and time-dependent manner. Other MEK inhibitors also display similar effect, indicating blockade of MAPK-ERK signaling is sufficient to induce OPC differentiation into OLs. PD0325901 facilitates the formation of myelin sheaths in OPC-neuron co-culture in vitro. And in experimental autoimmune encephalomyelitis model and cuprizone-induced demyelination model, PD0325901 displays significant therapeutic effect by promoting myelin regeneration. Our results suggest that targeting the MAPK-ERK pathway might be an intriguing way to develop new therapies for demyelinating diseases.


Assuntos
Doenças Desmielinizantes/enzimologia , Encefalomielite Autoimune Experimental/enzimologia , Sistema de Sinalização das MAP Quinases/fisiologia , Oligodendroglia/enzimologia , Recuperação de Função Fisiológica/fisiologia , Remielinização/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Técnicas de Cocultura , Cuprizona/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/tratamento farmacológico , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Oligodendroglia/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Remielinização/efeitos dos fármacos
2.
J Clin Invest ; 129(3): 1240-1256, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30620337

RESUMO

Sphingolipid imbalance is the culprit in a variety of neurological diseases, some affecting the myelin sheath. We have used whole-exome sequencing in patients with undetermined leukoencephalopathies to uncover the endoplasmic reticulum lipid desaturase DEGS1 as the causative gene in 19 patients from 13 unrelated families. Shared features among the cases include severe motor arrest, early nystagmus, dystonia, spasticity, and profound failure to thrive. MRI showed hypomyelination, thinning of the corpus callosum, and progressive thalamic and cerebellar atrophy, suggesting a critical role of DEGS1 in myelin development and maintenance. This enzyme converts dihydroceramide (DhCer) into ceramide (Cer) in the final step of the de novo biosynthesis pathway. We detected a marked increase of the substrate DhCer and DhCer/Cer ratios in patients' fibroblasts and muscle. Further, we used a knockdown approach for disease modeling in Danio rerio, followed by a preclinical test with the first-line treatment for multiple sclerosis, fingolimod (FTY720, Gilenya). The enzymatic inhibition of Cer synthase by fingolimod, 1 step prior to DEGS1 in the pathway, reduced the critical DhCer/Cer imbalance and the severe locomotor disability, increasing the number of myelinating oligodendrocytes in a zebrafish model. These proof-of-concept results pave the way to clinical translation.


Assuntos
Animais Geneticamente Modificados , Encéfalo , Cloridrato de Fingolimode/farmacologia , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Encéfalo/enzimologia , Encéfalo/patologia , Modelos Animais de Doenças , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/tratamento farmacológico , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/enzimologia , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Humanos , Locomoção/efeitos dos fármacos , Oligodendroglia/enzimologia , Oligodendroglia/patologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética
3.
Mol Neurobiol ; 56(5): 3380-3392, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30121936

RESUMO

The extracellular protein tissue inhibitor of metalloproteinase (TIMP)-1 is both a matrix metalloproteinase (MMP) inhibitor and a trophic factor. Mice lacking TIMP-1 exhibit delayed central nervous system myelination during postnatal development and impaired remyelination following immune-mediated injury in adulthood. We have previously determined that the trophic action of TIMP-1 on oligodendrocyte progenitor cells (OPCs) to mature into oligodendrocytes is independent of its MMP inhibitory function. However, the mechanism by which TIMP-1 promotes OPC differentiation is not known. To address this gap in our understanding, herein, we report that TIMP-1 signals via a CD63/ß1-integrin receptor complex to activate Akt (protein kinase B) to promote ß-catenin signaling in OPCs. The regulation of ß-catenin by TIMP-1 to promote OPC differentiation was counteracted, but not abrogated, by canonical signaling evoked by Wnt7a. These data provide a previously uncharacterized trophic action of TIMP-1 to regulate oligodendrocyte maturation via a CD63/ß1-integrin/Akt pathway mechanism. These findings contribute to our emerging understanding on the role of TIMP-1 as a growth factor expressed to promote CNS myelination during development and induced in the adult to promote myelin repair.


Assuntos
Diferenciação Celular , Oligodendroglia/citologia , Oligodendroglia/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Tetraspanina 30/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Animais , Células Cultivadas , Ativação Enzimática , Integrina beta1/metabolismo , Domínios Proteicos , Ratos Sprague-Dawley , Inibidor Tecidual de Metaloproteinase-1/química , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
4.
Proc Natl Acad Sci U S A ; 115(26): E6065-E6074, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29895691

RESUMO

Multiple sclerosis (MS) is a progressive inflammatory demyelinating disease of the CNS of unknown cause that remains incurable. Inflammasome-associated caspases mediate the maturation and release of the proinflammatory cytokines IL-1ß and IL-18 and activate the pore-forming protein gasdermin D (GSDMD). Inflammatory programmed cell death, pyroptosis, was recently shown to be mediated by GSDMD. Here, we report molecular evidence for GSDMD-mediated inflammasome activation and pyroptosis in both myeloid cells (macrophages/microglia) and, unexpectedly, in myelin-forming oligodendrocytes (ODCs) in the CNS of patients with MS and in the MS animal model, experimental autoimmune encephalomyelitis (EAE). We observed inflammasome activation and pyroptosis in human microglia and ODCs in vitro after exposure to inflammatory stimuli and demonstrate caspase-1 inhibition by the small-molecule inhibitor VX-765 in both cell types. GSDMD inhibition by siRNA transduction suppressed pyroptosis in human microglia. VX-765 treatment of EAE animals reduced the expression of inflammasome- and pyroptosis-associated proteins in the CNS, prevented axonal injury, and improved neurobehavioral performance. Thus, GSDMD-mediated pyroptosis in select glia cells is a previously unrecognized mechanism of inflammatory demyelination and represents a unique therapeutic opportunity for mitigating the disease process in MS and other CNS inflammatory diseases.


Assuntos
Caspase 1/metabolismo , Inibidores de Caspase/farmacologia , Dipeptídeos/farmacologia , Modelos Biológicos , Esclerose Múltipla/enzimologia , Oligodendroglia/enzimologia , Piroptose/efeitos dos fármacos , para-Aminobenzoatos/farmacologia , Células Cultivadas , Humanos , Esclerose Múltipla/patologia , Oligodendroglia/patologia
5.
Glia ; 66(9): 1999-2012, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29761559

RESUMO

Apoptosis is recognized as the main mechanism of oligodendrocyte loss in Multiple Sclerosis caused either by immune mediated injury (Barnett & Prineas, ) or a direct degenerative process (oligodendrogliapathy; Lucchinetti et al., ). Cuprizone induced demyelination is the result of non-immune mediated apoptosis of oligodendrocytes (OL) and represents a model of oligodendrogliapathy (Simmons, Pierson, Lee, & Goverman, ). Glycogen Synthase Kinase (GSK) 3b has been shown to be pro-apoptotic for cells other than OL. Here, we sought to investigate whether GSK3b plays a role in cuprizone-induced apoptosis of OL by using a novel inducible conditional knockout (cKO) of GSK3b in mature OL. While depletion of GSK3b has no effect on survival of uninjured OL, it increases survival of mature OL exposed to cuprizone. We show that GSK3b-deficient OLs are protected against caspase-dependent, but not against caspase-independent apoptosis. Active GSK3b is present in the nuclei of OL at peak of caspase-dependent apoptosis. Significant preservation of myelinated axons is associated with GSK3b depletion and glial cell activation is markedly reduced. Collectively, the data show that GSK3b is pro-apoptotic for caspase-dependent cell death, likely through activation of nuclear GSK3b and its depletion promotes survival of oligodendrocytes and attenuates myelin loss.


Assuntos
Apoptose/fisiologia , Doenças Desmielinizantes/enzimologia , Glicogênio Sintase Quinase 3 beta/deficiência , Bainha de Mielina/enzimologia , Oligodendroglia/enzimologia , Animais , Astrócitos/enzimologia , Astrócitos/patologia , Caspases/metabolismo , Núcleo Celular/enzimologia , Núcleo Celular/patologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Cuprizona , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Feminino , Glicogênio Sintase Quinase 3 beta/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/enzimologia , Microglia/patologia , Bainha de Mielina/patologia , Oligodendroglia/patologia
6.
Elife ; 72018 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-29596047

RESUMO

The inwardly rectifying K+ channel Kir4.1 is broadly expressed by CNS glia and deficits in Kir4.1 lead to seizures and myelin vacuolization. However, the role of oligodendrocyte Kir4.1 channels in controlling myelination and K+ clearance in white matter has not been defined. Here, we show that selective deletion of Kir4.1 from oligodendrocyte progenitors (OPCs) or mature oligodendrocytes did not impair their development or disrupt the structure of myelin. However, mice lacking oligodendrocyte Kir4.1 channels exhibited profound functional impairments, including slower clearance of extracellular K+ and delayed recovery of axons from repetitive stimulation in white matter, as well as spontaneous seizures, a lower seizure threshold, and activity-dependent motor deficits. These results indicate that Kir4.1 channels in oligodendrocytes play an important role in extracellular K+ homeostasis in white matter, and that selective loss of this channel from oligodendrocytes is sufficient to impair K+ clearance and promote seizures.


Assuntos
Oligodendroglia/enzimologia , Oligodendroglia/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Potássio/metabolismo , Convulsões/fisiopatologia , Substância Branca/metabolismo , Animais , Deleção de Genes , Homeostase , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Bainha de Mielina/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética
7.
J Neurosci Res ; 96(7): 1265-1276, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29377294

RESUMO

Ceruloplasmin (Cp), an enzyme containing six copper atoms, has important roles in iron homeostasis and antioxidant defense. After spinal cord injury (SCI), the cellular components in the local microenvironment are very complex and include functional changes of resident cells and the infiltration of leukocytes. It has been confirmed that Cp is elevated primarily in astrocytes and to a lesser extent in macrophages following SCI in mice. However, its expression in other cell types is still not very clear. In this manuscript, we provide a sensible extension of these findings by examining this system within a female Sprague-Dawley rat model and expanding the scope of inquiry to include additional cell types. Quantitative reverse transcription polymerase chain reaction and Western blot analysis revealed that the Cp mRNA and protein in SCI tissue homogenates were quite consistent with prior publications. However, we observed that Cp was expressed not only in GFAP+ astrocytes (consistent with prior reports) but also in CD11b+ microglia, CNPase+ oligodendrocytes, NeuN+ neurons, CD45+ leukocytes, and CD68+ activated microglia/macrophages. Quantitative analysis proved that infiltrated leukocytes, activated microglia/macrophages, and astrocytes should be the major sources of increased Cp.


Assuntos
Astrócitos/enzimologia , Ceruloplasmina/biossíntese , Microglia/enzimologia , Traumatismos da Medula Espinal/patologia , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígenos Nucleares/metabolismo , Astrócitos/patologia , Antígeno CD11b/metabolismo , Ceruloplasmina/metabolismo , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Leucócitos/enzimologia , Leucócitos/patologia , Macrófagos/enzimologia , Macrófagos/patologia , Camundongos , Microglia/patologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/enzimologia , Neurônios/fisiologia , Oligodendroglia/enzimologia , Oligodendroglia/patologia , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/induzido quimicamente
8.
J Neurosci ; 38(4): 787-802, 2018 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-29217681

RESUMO

Shp2 is a nonreceptor protein tyrosine phosphatase that has been shown to influence neurogenesis, oligodendrogenesis, and oligodendrocyte differentiation. Furthermore, Shp2 is a known regulator of the Akt/mammalian target of rapamycin and ERK signaling pathways in multiple cellular contexts, including oligodendrocytes. Its role during later postnatal CNS development or in response to demyelination injury has not been examined. Based on the current studies, we hypothesize that Shp2 is a negative regulator of CNS myelination. Using transgenic mouse technology, we show that Shp2 is involved in oligodendrocyte differentiation and early myelination, but is not necessary for myelin maintenance. We also show that Shp2 regulates the timely differentiation of oligodendrocytes following lysolecithin-induced demyelination, although apparently normal remyelination occurs at a delayed time point. These data suggest that Shp2 is a relevant therapeutic target in demyelinating diseases such as multiple sclerosis.SIGNIFICANCE STATEMENT In the present study, we show that the protein phosphatase Shp2 is an important mediator of oligodendrocyte differentiation and myelination, both during developmental myelination as well as during myelin regeneration. We provide important insight into the signaling mechanisms regulating myelination and propose that Shp2 acts as a transient brake to the developmental myelination process. Furthermore, we show that Shp2 regulates oligodendrocyte differentiation following demyelination and therefore has important therapeutic implications in diseases such as multiple sclerosis.


Assuntos
Bainha de Mielina/metabolismo , Neurogênese/fisiologia , Oligodendroglia/citologia , Oligodendroglia/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Animais , Diferenciação Celular/fisiologia , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Oligodendroglia/metabolismo , Peixe-Zebra
9.
J Inherit Metab Dis ; 41(2): 221-229, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29168031

RESUMO

Mucopolysaccharidosis type II (MPSII) is a rare X-linked lysosomal storage disorder caused by mutations in the iduronate-2-sulfatase (IDS) gene (IDS, Xq28). MPSII is characterized by skeletal deformities, hearing loss, airway obstruction, hepatosplenomegaly, cardiac valvular disease, and progressive neurological impairment. At the cellular level, IDS deficiency leads to lysosomal storage of glycosaminoglycans (GAGs), dominated by accumulation of dermatan and heparan sulfates. Human induced pluripotent stem cells (iPSC) represent an alternative system that complements the available MPSII murine model. Herein we report on the reprogramming of peripheral white blood cells from male and female MPSII patients into iPSC using a non-integrating protocol based on the Sendai virus vector system. We differentiated the iPSC lines into IDS deficient and GAG accumulating ß-Tubulin III+ neurons, GFAP+ astrocytes, and CNPase+ oligodendrocytes. The lysosomal system in these cells displayed structural abnormalities reminiscent of those previously found in patient tissues and murine IDS deficient neuronal stem cells. Furthermore, quantitative determination of GAGs revealed a moderate increase in GAG levels in IDS deficient neurons and glia. We also tested the effects of recombinant IDS and found that the exogenous enzyme was internalized from the culture media and partially decreased the intracellular GAG levels in iPSC-derived neural cells; however, it failed to completely prevent accumulation of GAGs. In summary, we demonstrate that this human iPSC based model expresses the cellular and biochemical features of MPSII, and thus represents a useful experimental tool for further pathogenesis studies as well as therapy development and testing.


Assuntos
Glicosaminoglicanos/metabolismo , Iduronato Sulfatase/metabolismo , Células-Tronco Pluripotentes Induzidas/enzimologia , Lisossomos/enzimologia , Mucopolissacaridose II/enzimologia , Células-Tronco Neurais/enzimologia , Neurogênese , Neuroglia/enzimologia , Neurônios/enzimologia , Astrócitos/enzimologia , Astrócitos/patologia , Linhagem da Célula , Células Cultivadas , Feminino , Humanos , Iduronato Sulfatase/genética , Células-Tronco Pluripotentes Induzidas/patologia , Lisossomos/patologia , Masculino , Mucopolissacaridose II/genética , Mucopolissacaridose II/patologia , Células-Tronco Neurais/patologia , Neuroglia/patologia , Neurônios/patologia , Células Precursoras de Oligodendrócitos/enzimologia , Células Precursoras de Oligodendrócitos/patologia , Oligodendroglia/enzimologia , Oligodendroglia/patologia , Fenótipo
10.
Neuropathology ; 37(6): 495-501, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28731225

RESUMO

Protein disulfide isomerase (PDI) is a chaperone protein located in the endoplasmic reticulum (ER). Nitric oxide-induced S-nitrosylation of PDI inhibits its enzymatic activity, leading to protein accumulation and activation of the unfolded protein response. Protein disulfide isomerase P5 (P5) is a member of the PDI family that mostly localizes to the ER lumen. Both S-nitrosylated PDI and S-nitrosylated P5 are found in Alzheimer's disease (AD) brain. Previously, we showed that expression of the ER stress marker, growth arrest, and DNA damage protein (GADD34) was significantly increased in neurons and oligodendrocytes in AD brain. In the present study, we showed that PDI and P5 levels were significantly decreased in oligodendrocytes in the brains of AD patients and an AD mouse model. Interestingly, these decreases were evident before the animals displayed typical AD pathology. Because we previously showed that small short interfering RNA knockdown of PDI or P5 could affect the viability of neuronal cells under ER stress, dysfunction of PDI and P5 under ER stress could cause apoptosis of neuronal cells. In summary, we showed that the levels of PDI and P5 were significantly decreased in the oligodendrocytes of AD patients. This phenomenon was also found in an AD mouse model before the animals displayed AD pathology. The overall findings suggest that oligodendrocytes may play important roles in AD pathogenesis.


Assuntos
Doença de Alzheimer/enzimologia , Encéfalo/enzimologia , Oligodendroglia/enzimologia , Isomerases de Dissulfetos de Proteínas/biossíntese , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Masculino , Camundongos , Isomerases de Dissulfetos de Proteínas/análise
11.
PLoS One ; 12(6): e0178622, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28582448

RESUMO

The cuprizone animal model, also known as the toxic demyelination model, is a well-reproducible model of demyelination- and remyelination in mice, and has been useful in studying important aspect of human demyelinating diseases, including multiple sclerosis. In this study, we investigated the role of acid sphingomyelinase in demyelination and myelin repair by inducing acute and chronic demyelination with 5- or 12-week cuprizone treatment, followed by a 2-week cuprizone withdrawal phase to allow myelin repair. Sphingolipids, in particular ceramide and the enzyme acid sphingomyelinase, which generates ceramide from sphingomyelin, seem to be involved in astrocyte activation and neuronal damage in multiple sclerosis. We used immunohistochemistry to study glial reaction and oligodendrocyte distribution in acid sphingomyelinase deficient mice and wild-type C57BL/6J littermates at various time intervals after demyelination and remyelination. Axonal injury was quantified using amyloid precursor protein and synaptophysin, and gene expression and protein levels were measured using gene analysis and Western blotting, respectively. Our results show that mice lacking acid sphingomyelinase had a significant increase in myelin recovery and a significantly higher oligodendrocyte cell count after 2 weeks remyelination compared to wild-type littermates. Detrimental astroglial distribution was also significantly reduced in acid sphingomyelinase deficient animals. We obtained similar results in experiments using amitriptyline to inhibit acid sphingomyelinase. These findings suggest that acid sphingomyelinase plays a significant role in myelin repair, and its inhibition by amitriptyline may constitute a novel therapeutic approach for multiple sclerosis patients.


Assuntos
Amitriptilina/farmacologia , Doenças Desmielinizantes/prevenção & controle , Inibidores Enzimáticos/farmacologia , Esclerose Múltipla/prevenção & controle , Oligodendroglia/efeitos dos fármacos , Esfingomielina Fosfodiesterase/genética , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/enzimologia , Astrócitos/patologia , Axônios/efeitos dos fármacos , Axônios/enzimologia , Axônios/patologia , Contagem de Células , Cuprizona , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/enzimologia , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/efeitos dos fármacos , Microglia/enzimologia , Microglia/patologia , Esclerose Múltipla/induzido quimicamente , Esclerose Múltipla/enzimologia , Esclerose Múltipla/patologia , Regeneração Nervosa/efeitos dos fármacos , Oligodendroglia/enzimologia , Oligodendroglia/patologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/deficiência , Sinaptofisina/genética , Sinaptofisina/metabolismo
12.
Sci Rep ; 7(1): 1705, 2017 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-28496133

RESUMO

Folate, an essential micronutrient, is a critical cofactor in one-carbon metabolism for many cellular pathways including DNA synthesis, metabolism and maintenance. Folate deficiency has been associated with an increased risk of neurological disease, cancer and cognitive dysfunction. Dihydrofolate reductase (DHFR) is a key enzyme to regulate folate metabolism, however folate/DHFR activity in oligodendrocyte development has not been fully understood. Here we show that folate enhances oligodendrocyte maturation both in vitro and in vivo, which is accompanied with upregulation of oligodendrocyte-specific DHFR expression. On the other hand, pharmacological inhibition of DHFR by methotrexate (MTX) causes severe defects in oligodendrocyte survival and differentiation, which could be reversed by folate intake. We further demonstrate that folate activates a metabolic regulator AMPKα to promote oligodendrocyte survival and differentiation. Moreover, activation of AMPKα partially rescues oligodendrocyte defects caused by DHFR-inhibition both in vitro and in vivo. Taken together, these findings identify a previously uncharacterized role of folate/DHFR/AMPKα axis in regulating oligodendrocyte survival and myelination during CNS development.


Assuntos
Adenilato Quinase/metabolismo , Diferenciação Celular , Ácido Fólico/metabolismo , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Animais , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Antagonistas do Ácido Fólico/farmacologia , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismo , Oligodendroglia/enzimologia , Nervo Óptico/patologia , Nervo Óptico/ultraestrutura , Fosforilação/efeitos dos fármacos , Medula Espinal/patologia , Medula Espinal/ultraestrutura , Tetra-Hidrofolato Desidrogenase/metabolismo
13.
Neurobiol Dis ; 97(Pt A): 24-35, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27816769

RESUMO

BACKGROUND: The recently diagnosed leukodystrophy Hypomyelination with Brain stem and Spinal cord involvement and Leg spasticity (HBSL) is caused by mutations of the cytoplasmic aspartyl-tRNA synthetase geneDARS. The physiological role of DARS in translation is to accurately pair aspartate with its cognate tRNA. Clinically, HBSL subjects show a distinct pattern of hypomyelination and develop progressive leg spasticity, variable cognitive impairment and epilepsy. To elucidate the underlying pathomechanism, we comprehensively assessed endogenous DARS expression in mice. Additionally, aiming at creating the first mammalian HBSL model, we genetically engineered and phenotyped mutant mice with a targetedDarslocus. RESULTS: DARS, although expressed in all organs, shows a distinct expression pattern in the adult brain with little immunoreactivity in macroglia but enrichment in neuronal subpopulations of the hippocampus, cerebellum, and cortex. Within neurons, DARS is mainly located in the cell soma where it co-localizes with other components of the translation machinery. Intriguingly, DARS is also present along neurites and at synapses, where it potentially contributes to local protein synthesis.Dars-null mice are not viable and die before embryonic day 11. Heterozygous mice with only one functionalDarsallele display substantially reduced DARS levels in the brain; yet these mutants show no gross abnormalities, including unchanged motor performance. However, we detected reduced pre-pulse inhibition of the acoustic startle response indicating dysfunction of attentional processing inDars+/-mice. CONCLUSIONS: Our results, for the first time, show an in-depth characterization of the DARS tissue distribution in mice, revealing surprisingly little uniformity across brain regions or between the major neural cell types. The complete loss of DARS function is not tolerated in mice suggesting that the identified HBSL mutations in humans retain some residual enzyme activity. The mild phenotype of heterozygousDars-null carriers indicates that even partial restoration of DARS levels would be therapeutically relevant. Despite the fact that they do not resemble the full spectrum of clinical symptoms, the robust pre-pulse inhibition phenotype ofDars+/-mice will be instrumental for future preclinical therapeutic efficacy studies. In summary, our data is an important contribution to a better understanding of DARS function and HBSL pathology.


Assuntos
Aspartato-tRNA Ligase/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/enzimologia , Animais , Aspartato-tRNA Ligase/genética , Astrócitos/enzimologia , Astrócitos/patologia , Atenção/fisiologia , Encéfalo/enzimologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Proteínas de Caenorhabditis elegans/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Comportamento Exploratório/fisiologia , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/fisiologia , Neurônios/enzimologia , Neurônios/patologia , Oligodendroglia/enzimologia , Oligodendroglia/patologia , Fenótipo , Inibição Pré-Pulso/fisiologia , Reflexo de Sobressalto/fisiologia , Medula Espinal/enzimologia , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/patologia , Sinaptossomos/enzimologia , Proteína ran de Ligação ao GTP/metabolismo
14.
Neurobiol Dis ; 96: 323-334, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27717881

RESUMO

Breakdown of neuro-glial N-acetyl-aspartate (NAA) metabolism results in the failure of developmental myelination, manifest in the congenital pediatric leukodystrophy Canavan disease caused by mutations to the sole NAA catabolizing enzyme aspartoacylase. Canavan disease is a major point of focus for efforts to define NAA function, with available evidence suggesting NAA serves as an acetyl donor for fatty acid synthesis during myelination. Elevated NAA is a diagnostic hallmark of Canavan disease, which contrasts with a broad spectrum of alternative neurodegenerative contexts in which levels of NAA are inversely proportional to pathological progression. Recently generated data in the nur7 mouse model of Canavan disease suggests loss of aspartoacylase function results in compromised energetic integrity prior to oligodendrocyte death, abnormalities in myelin content, spongiform degeneration, and motor deficit. The present study utilized a next-generation "oligotropic" adeno-associated virus vector (AAV-Olig001) to quantitatively assess the impact of aspartoacylase reconstitution on developmental myelination. AAV-Olig001-aspartoacylase promoted normalization of NAA, increased bioavailable acetyl-CoA, and restored energetic balance within a window of postnatal development preceding gross histopathology and deteriorating motor function. Long-term effects included increased oligodendrocyte numbers, a global increase in myelination, reversal of vacuolation, and rescue of motor function. Effects on brain energy observed following AAV-Olig001-aspartoacylase gene therapy are shown to be consistent with a metabolic profile observed in mild cases of Canavan disease, implicating NAA in the maintenance of energetic integrity during myelination via oligodendroglial aspartoacylase.


Assuntos
Amidoidrolases/metabolismo , Ácido Aspártico/análogos & derivados , Encéfalo/enzimologia , Doença de Canavan/patologia , Bainha de Mielina/fisiologia , Oligodendroglia/enzimologia , Amidoidrolases/genética , Animais , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Proteínas Relacionadas à Autofagia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Encéfalo/patologia , Doença de Canavan/complicações , Doença de Canavan/diagnóstico por imagem , Doença de Canavan/genética , Criança , Pré-Escolar , Dependovirus/genética , Progressão da Doença , Metabolismo Energético/genética , Feminino , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Lactente , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Transtornos dos Movimentos/etiologia , Proteína Básica da Mielina/metabolismo , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/genética
15.
Int J Dev Neurosci ; 54: 53-61, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27587342

RESUMO

Histone deacetylase (HDAC) inhibition mediated by small molecule HDAC inhibitors (HDACi) has demonstrated divergent effects including toxicity towards transformed cell lines, neuroprotection in neurological disease models, and inhibition of oligodendrocyte precursor cell (OPC) differentiation to mature oligodendrocytes (OL). However, it remains unknown if transient HDAC inhibition may promote OPC survival. Using mouse cortical OPC primary cultures, we investigated the effects of the FDA approved pan-HDACi suberoylanilide hydroxamic acid (SAHA) on OPC survival. Initial studies showed differences in the HDAC expression pattern of multiple HDAC isoforms in OPCs relative to their terminally differentiated progeny cells, OLs and astrocytes. Treatment of OPCs with SAHA for up to 72h using a maximum concentration either at or lower than those necessary for cytotoxicity in most transformed cell lines resulted in over 67% reduction in viability relative to vehicle-treated OPCs. This was at least partly due to increased apoptosis as SAHA-treated cells displayed activated caspase 3 and were protected by the general caspase inhibitor Q-VD-OPH. Additionally, SAHA treatment of whole mice at postnatal day 5 induced apoptosis of cortical OPCs. These results suggest that SAHA negatively impacts OPC survival and may be detrimental to the myelinating brain and spinal cord. Such toxicity may be relevant in a clinical context as SAHA is currently involved in numerous clinical trials and is in consideration for use in the treatment of psychiatric and neurodegenerative conditions.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Histona Desacetilases/metabolismo , Oligodendroglia/enzimologia , Células-Tronco/fisiologia , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/genética , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Animais , Animais Recém-Nascidos , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Ácidos Hidroxâmicos/farmacologia , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oligodendroglia/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Fatores de Tempo , Vorinostat
16.
J Neurosci Res ; 94(11): 1273-83, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27638609

RESUMO

Krabbe's disease (KD) is a degenerative lysosomal storage disease resulting from deficiency of ß-galactocerebrosidase activity. Over 100 mutations are known to cause the disease, and these usually occur in compound heterozygote patterns. In affected patients, nonsense mutations leading to a nonfunctional enzyme are often found associated with other mutations. The twitcher mouse is a naturally occurring model of KD, containing in ß-galactocerebrosidase a premature stop codon, W339X. Recent studies have shown that selected compounds may induce the ribosomal bypass of premature stop codons without affecting the normal termination codons. The rescue of ß-galactocerebrosidase activity induced by treatment with premature termination codon (PTC) 124, a well-characterized compound known to induce ribosomal read-through, was investigated on oligodendrocytes prepared from twitcher mice and on human fibroblasts from patients bearing nonsense mutations. The effectiveness of the nonsense-mediated mRNA decay (NMD) inhibitor 1 (NMDI1), a newly identified inhibitor of NMD, was also tested. Incubation of these cell lines with PTC124 and NMDI1 increased the levels of mRNA and rescued galactocerebrosidase enzymatic activity in a dose-dependent manner. The low but sustained expression of ß-galactocerebrosidase in oligodendrocytes was sufficient to improve the morphology of the differentiated cells. Our in vitro approach provides the basis for further investigation of ribosomal read-through as an alternative therapeutic strategy to ameliorate the quality of life in selected KD patients. © 2016 Wiley Periodicals, Inc.


Assuntos
Códon de Terminação/genética , Galactosilceramidase/deficiência , Galactosilceramidase/genética , Galactosilceramidas/metabolismo , Leucodistrofia de Células Globoides/patologia , Animais , Animais Recém-Nascidos , Linhagem Celular Transformada , Códon de Terminação/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Galactosilceramidas/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Degradação do RNAm Mediada por Códon sem Sentido/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/enzimologia , Oxidiazóis/farmacologia , RNA Mensageiro/metabolismo
17.
Histochem Cell Biol ; 146(1): 45-57, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26921198

RESUMO

We previously demonstrated that NG2-positive oligodendrocyte precursor cells (OPCs) do not express DM-20 mRNA and identified a distinct DM-20 mRNA-positive cell population expressing glutathione-S-transferase pi isoform (GST-pi) in the nucleus (GST-pi(Nuc)) of the adult rat spinal cord. As GST-pi intranuclear localization correlates with progenitor cell properties, we examined the differentiation status of this cell population under the intensive 5-bromo-2'-deoxyuridine (BrdU) administration method, consisting of intraperitoneal BrdU injections every 2 h for 48 h. We observed that a certain population of proliferating/proliferated cells expressed DM-20 mRNA, and sometimes two proliferating/proliferated cells were observed still attached to each other. We performed triple staining for BrdU, DM-20 mRNA, and NG2 and found pairs of neighboring BrdU-positive cells, which were considered to originate from the same progenitor cells and where both cells expressed DM-20 mRNA. Triple staining for BrdU, DM-20 mRNA, and GST-pi detected proliferating/proliferated cells exhibiting the GST-pi(Nuc)/DM-20 mRNA-positive expression pattern. These findings suggested the presence of a GST-pi(Nuc)/DM-20 mRNA-positive oligodendrocyte-lineage progenitor cell population in the adult rat spinal cord. However, we did not find any pair of neighboring BrdU-positive cells with this expression pattern. These observations collectively support the idea that GST-pi(Nuc)/DM-20 mRNA-expressing cells are the progeny of NG2-positive OPCs rather than a novel type of oligodendrocyte-lineage progenitor cells and that DM-20 mRNA expression is dynamically regulated during differentiation of OPCs into oligodendrocytes.


Assuntos
Diferenciação Celular , Glutationa S-Transferase pi/metabolismo , Proteína Proteolipídica de Mielina/genética , Oligodendroglia/citologia , Oligodendroglia/metabolismo , RNA Mensageiro/biossíntese , Medula Espinal/citologia , Animais , Masculino , Oligodendroglia/enzimologia , Ratos , Ratos Wistar , Medula Espinal/metabolismo
18.
Sci Rep ; 6: 20995, 2016 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-26888014

RESUMO

Choline is an essential nutrient for all living cells and is produced extracellularly by sequential degradation of phosphatidylcholine (PC). However, little is known about how choline is produced extracellularly. Here, we report that ENPP6, a choline-specific phosphodiesterase, hydrolyzes glycerophosphocholine (GPC), a degradation product of PC, as a physiological substrate and participates in choline metabolism. ENPP6 is highly expressed in liver sinusoidal endothelial cells and developing oligodendrocytes, which actively incorporate choline and synthesize PC. ENPP6-deficient mice exhibited fatty liver and hypomyelination, well known choline-deficient phenotypes. The choline moiety of GPC was incorporated into PC in an ENPP6-dependent manner both in vivo and in vitro. The crystal structure of ENPP6 in complex with phosphocholine revealed that the choline moiety of the phosphocholine is recognized by a choline-binding pocket formed by conserved aromatic and acidic residues. The present study provides the molecular basis for ENPP6-mediated choline metabolism at atomic, cellular and tissue levels.


Assuntos
Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Animais , Cristalografia por Raios X , Células Endoteliais/enzimologia , Fígado Gorduroso/enzimologia , Fígado Gorduroso/genética , Fígado/enzimologia , Camundongos , Camundongos Knockout , Oligodendroglia/enzimologia , Especificidade de Órgãos , Fosfatidilcolinas/genética , Relação Estrutura-Atividade , Especificidade por Substrato
19.
Apoptosis ; 21(1): 25-35, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26459425

RESUMO

Krabbe disease is a genetic demyelinating syndrome characterized by deficiency of the enzyme ß-galactosylceramidase, lysosomal psychosine accumulation, and loss of myelin-forming cells. In this study, some apoptotic markers such as apoptotic index (AI), DNA fragmentation, caspase-3, PTEN, Bad, and PI3K were determined in oligodendrocyte precursors from wild type or twitcher mice untreated or treated with psychosine. Twitcher is a natural mouse model of Krabbe disease containing a premature stop codon (W339X) in the ß-galactosylceramidase gene. Moreover, a possible involvement of connexin (Cx)43 in cell death of oligodendrocyte precursors induced by psychosine was investigated with the final aim to provide a contribution to the knowledge of the molecular mechanisms and pathophysiological events that occur in Krabbe disease. Connexins are a multigene family of structurally related trans-membrane proteins able to modulate essential cellular processes such as proliferation, differentiation and migration. Among these, Cx43 is the predominant isoform in many cell types, including neural progenitor cells. Our results showed an increase of AI, DNA fragmentation, caspase-3, PTEN, Bad, and Cx43 associated to a decrease of PI3K, pAKT and pBad. Taken together, these findings suggest an involvement of Cx43 in the psychosine-mediated apoptosis of primary oligodendrocyte progenitors from wild type or twitcher mice, used for the first time as cell models in comparison. It could open unexplored perspective also for other demyelinating diseases.


Assuntos
Encéfalo/efeitos dos fármacos , Conexina 43/genética , Galactosilceramidase/deficiência , Leucodistrofia de Células Globoides/genética , Oligodendroglia/efeitos dos fármacos , Psicosina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Encéfalo/enzimologia , Encéfalo/patologia , Caspase 3/genética , Caspase 3/metabolismo , Diferenciação Celular/efeitos dos fármacos , Conexina 43/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Galactosilceramidase/genética , Regulação da Expressão Gênica , Humanos , Leucodistrofia de Células Globoides/enzimologia , Leucodistrofia de Células Globoides/patologia , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Lisossomos/patologia , Camundongos , Camundongos Knockout , Oligodendroglia/enzimologia , Oligodendroglia/patologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Psicosina/metabolismo , Transdução de Sinais , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismo
20.
EMBO J ; 34(19): 2465-80, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26290337

RESUMO

Polynucleotide kinase-phosphatase (PNKP) is a DNA repair factor possessing both 5'-kinase and 3'-phosphatase activities to modify ends of a DNA break prior to ligation. Recently, decreased PNKP levels were identified as the cause of severe neuropathology present in the human microcephaly with seizures (MCSZ) syndrome. Utilizing novel murine Pnkp alleles that attenuate expression and a T424GfsX48 frame-shift allele identified in MCSZ individuals, we determined how PNKP inactivation impacts neurogenesis. Mice with PNKP inactivation in neural progenitors manifest neurodevelopmental abnormalities and postnatal death. This severe phenotype involved defective base excision repair and non-homologous end-joining, pathways required for repair of both DNA single- and double-strand breaks. Although mice homozygous for the T424GfsX48 allele were lethal embryonically, attenuated PNKP levels (akin to MCSZ) showed general neurodevelopmental defects, including microcephaly, indicating a critical developmental PNKP threshold. Directed postnatal neural inactivation of PNKP affected specific subpopulations including oligodendrocytes, indicating a broad requirement for genome maintenance, both during and after neurogenesis. These data illuminate the basis for selective neural vulnerability in DNA repair deficiency disease.


Assuntos
Reparo do DNA , Mutação da Fase de Leitura , Instabilidade Genômica , Células-Tronco Neurais/enzimologia , Oligodendroglia/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Humanos , Camundongos , Camundongos Mutantes , Microcefalia/enzimologia , Microcefalia/genética , Microcefalia/patologia , Células-Tronco Neurais/patologia , Oligodendroglia/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética
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