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1.
Biochem Biophys Res Commun ; 482(2): 282-288, 2017 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-27847319

RESUMO

Skeletal muscle insulin resistance is considered to be the primary defect involved in type 2 diabetes mellitus (T2DM). Despite transcriptome studies in limited T2DM human subjects suggesting an association of T2DM with impaired oxidative phosphorylation in muscle, its molecular pathogenesis remains largely unknown. To identify dysregulated genes and gene networks that are associated with T2DM in human skeletal muscle, we examined expression patterns of 56,318 transcribed genes on 92 T2DM cases and 184 gender-, age- and race-matched non-diabetic controls from the Genotype-Tissue Expression (GTEx) database. RNA-Sequencing data suggest that diabetic skeletal muscle is characterized by decreased expression of genes that are related to insulin resistance (IRS2, MTOR, SLC2A4, and PPARA), carbohydrate, energy, and amino acid metabolism pathways (NDUFS1, NDUFA10, NDUFB4, NDUFB5, NDUFA5, NDUFB10, SDHB, SDHC, ATP5H, ATP5A, and ATP5J). Up-regulated genes in T2DM are mainly enriched in apoptosis pathways (TP53, GADD45A, TNFRSF10B, TP53AIP1, and PMAIP1), and notably include immune-related pathways suggestive of a response to various infectious diseases (C2, CFB, C4A, C4B, C1S, C1R, C3, HLA-DRA, HLA-DMA, HLA-DOA, and HLA-DPB1). These results confirm the essential regulation of impaired insulin signaling and oxidative phosphorylation in the muscle of T2DM patients, and provide novel molecular insights into the pathophysiological mechanisms of T2DM.


Assuntos
Apoptose/imunologia , Diabetes Mellitus Tipo 2/imunologia , Resistência à Insulina/imunologia , Proteínas Musculares/imunologia , Músculo Esquelético/imunologia , Transcriptoma/imunologia , Humanos , Infecções/imunologia , Transdução de Sinais/imunologia
2.
Nat Genet ; 37(10): 1099-103, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16142235

RESUMO

Cultured human embryonic stem cell (hESC) lines are an invaluable resource because they provide a uniform and stable genetic system for functional analyses and therapeutic applications. Nevertheless, these dividing cells, like other cells, probably undergo spontaneous mutation at a rate of 10(-9) per nucleotide. Because each mutant has only a few progeny, the overall biological properties of the cell culture are not altered unless a mutation provides a survival or growth advantage. Clonal evolution that leads to emergence of a dominant mutant genotype may potentially affect cellular phenotype as well. We assessed the genomic fidelity of paired early- and late-passage hESC lines in the course of tissue culture. Relative to early-passage lines, eight of nine late-passage hESC lines had one or more genomic alterations commonly observed in human cancers, including aberrations in copy number (45%), mitochondrial DNA sequence (22%) and gene promoter methylation (90%), although the latter was essentially restricted to 2 of 14 promoters examined. The observation that hESC lines maintained in vitro develop genetic and epigenetic alterations implies that periodic monitoring of these lines will be required before they are used in in vivo applications and that some late-passage hESC lines may be unusable for therapeutic purposes.


Assuntos
Embrião de Mamíferos/citologia , Genoma Humano/genética , Mutação , Células-Tronco/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , DNA/genética , DNA/metabolismo , Metilação de DNA , DNA Mitocondrial/química , Humanos , Regiões Promotoras Genéticas
3.
Synapse ; 65(1): 21-34, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20506319

RESUMO

Prenatal cocaine exposure induces cytoarchitectural changes in the embryonic neocortex; however, the biological mechanisms and type of cortical neurons involved in these changes are not known. Previously, we found that neural progenitor proliferation in the neocortical ventricular zone (VZ) is inhibited by cocaine; here, we examine the changes in cortical neurogenesis and migration of glutamate and GABA neurons induced by prenatal cocaine exposure. Pregnant rats received 20 mg/kg of cocaine intraperitoneally twice at an interval of 12 h during three periods of neocortical neurogenesis. Neocortical area and distribution of developing neurons were examined by counting Tuj1+, glutamate+, or GABA+ cells in different areas of the cerebral cortex. Cocaine decreased neocortical area by reducing the size of the Tuj1+ layer, but only when administered during early periods of neocortical neurogenesis. The number of glutamatergic neurons was increased in the VZ but was decreased in the outer cortical laminae. Although the number of GABA+ neurons in the VZ of both the neocortex and ganglionic eminences was unchanged, GABA+ cells decreased in all other neocortical laminae. Tangential migration of GABA+ cells was also disrupted by cocaine. These findings suggest that in utero cocaine exposure disturbs radial migration of neocortical neurons, possibly because of decreased radial glia guiding support through enhanced differentiation of neocortical VZ progenitors. Cocaine interrupts radial migration of both glutamatergic and GABAergic neurons within the neocortex, in addition to the tangential migration of GABAergic neurons from the subcortical telecephalon. This may result in abnormal neocortical cytoarchitecture and concomitant adverse functional effects.


Assuntos
Movimento Celular/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Cocaína/farmacologia , Ácido Glutâmico/metabolismo , Neurônios/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Animais , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Feminino , Imuno-Histoquímica , Neurônios/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley
4.
Restor Neurol Neurosci ; 39(4): 247-266, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34275915

RESUMO

BACKGROUND: Motor and cognitive decline as part of the normal aging process is linked to alterations in synaptic plasticity and reduction of adult neurogenesis in the dorsal striatum. Neuroinflammation, particularly in the form of microglial activation, is suggested to contribute to these age-associated changes. OBJECTIVE AND METHODS: To explore the molecular basis of alterations in striatal function during aging we analyzed RNA-Seq data for 117 postmortem human dorsal caudate samples and 97 putamen samples acquired through GTEx. RESULTS: Increased expression of neuroinflammatory transcripts including TREM2, MHC II molecules HLA-DMB, HLA-DQA2, HLA-DPA1, HLA-DPB1, HLA-DMA and HLA-DRA, complement genes C1QA, C1QB, CIQC and C3AR1, and MHCI molecules HLA-B and HLA-F was identified. We also identified down-regulation of transcripts involved in neurogenesis, synaptogenesis, and synaptic pruning, including DCX, CX3CL1, and CD200, and the canonical WNTs WNT7A, WNT7B, and WNT8A. The canonical WNT signaling pathway has previously been shown to mediate adult neurogenesis and synapse formation and growth. Recent findings also highlight the link between WNT/ß-catenin signaling and inflammation pathways. CONCLUSIONS: These findings suggest that age-dependent attenuation of canonical WNT signaling plays a pivotal role in regulating striatal plasticity during aging. Dysregulation of WNT/ß-catenin signaling via astrocyte-microglial interactions is suggested to be a novel mechanism that drives the decline of striatal neurogenesis and altered synaptic connectivity and plasticity, leading to a subsequent decrease in motor and cognitive performance with age. These findings may aid in the development of therapies targeting WNT/ß-catenin signaling to combat cognitive and motor impairments associated with aging.


Assuntos
Doenças Neuroinflamatórias , Via de Sinalização Wnt , Perfilação da Expressão Gênica , Humanos , Glicoproteínas de Membrana , Neurogênese/fisiologia , Plasticidade Neuronal/genética , Receptores Imunológicos , Via de Sinalização Wnt/genética
5.
Synapse ; 64(4): 267-73, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19953654

RESUMO

Opioids have been demonstrated to play an important role in CNS development by affecting proliferation and differentiation in various types of neural cells. This study examined the effect of a stable delta opioid peptide [D-Ala(2), D-Leu(5)]-enkephalin (DADLE) on proliferation and differentiation in an AF5 CNS neural progenitor cell line derived from rat mesencephalic cells. DADLE (1 pM, 0.1 nM, or 10 nM) caused a significant growth inhibition on AF5 cells. The opioid antagonist naltrexone at 0.1 nM also caused growth inhibition in the same cells. When DADLE and naltrexone were both added to the AF5 cells, the resultant growth inhibition was apparently additive. DADLE alone or DADLE in combination with naltrexone did not cause apoptosis as evidenced by negative TUNEL staining. The cell-cycle progression analysis indicated that both DADLE (0.1 nM) and naltrexone (0.1 nM) caused an arrest of AF5 cell cycle progression at the G1 checkpoint. Neuronal marker indicated that DADLE- or naltrexone-treated AF5 cells tend to differentiate more when compared to controls. Results demonstrate the nonopioid action of both DADLE and naltrexone on cell cycle arrest and differentiation in a CNS neural progenitor cell line. Results also suggest some potential utilization of DADLE and/or naltrexone in stem cell research.


Assuntos
Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Leucina Encefalina-2-Alanina/farmacologia , Naltrexona/análogos & derivados , Neurônios/fisiologia , Células-Tronco/efeitos dos fármacos , Análise de Variância , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Citometria de Fluxo/métodos , Marcação In Situ das Extremidades Cortadas/métodos , Naltrexona/farmacologia , Neurônios/efeitos dos fármacos , Ratos , Fatores de Tempo , Tubulina (Proteína)/metabolismo
6.
Stem Cells ; 26(6): 1517-25, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18388303

RESUMO

Producing dopaminergic (DA) neurons is a major goal of human embryonic stem cell (hESC) research. DA neurons can be differentiated from hESC by coculture with the mouse PA6 stromal cell line; this differentiation-inducing effect is termed stromal-derived inducing activity (SDIA). The molecular and biochemical nature of SDIA is, however, unknown. Various studies have suggested that SDIA involves either a fixation-resistant component located on the PA6 cell surface or factors secreted into the medium by PA6 cells. To address this question, hESC were cocultured with PA6 cells for 12 days and then further differentiated with sonic hedgehog homolog, fibroblast growth factor-8, and glial cell line-derived neurotrophic factor. After 18 days, 34% of cells were tyrosine hydroxylase (TH)+. When PA6 cells were fixed or irradiated, the number of TH+ cells was decreased by threefold, whereas mitomycin-c treatment of feeder cells decreased the number of TH+ cells by 32%. The neural-inducing effect of PA6 cells, as monitored by beta-III-tubulin expression, was minimally affected by mitomycin-c treatment or fixation but was decreased 50% by irradiation. Medium conditioned by PA6 cells was ineffective in differentiating TH+ cells when used alone. Conditioned medium combined with heparin and/or fixed PA6 cells produced TH+ cell differentiation, although less effectively than PA6 cell coculture. Thus, PA6 cell surface activity is required for neural differentiation of hESC, but secreted factors are required for the specific DA neuron-inducing effect. Disclosure of potential conflicts of interest is found at the end of this article.


Assuntos
Diferenciação Celular/fisiologia , Dopamina/fisiologia , Células-Tronco Embrionárias/citologia , Neurônios/citologia , Neurônios/fisiologia , Células Estromais/citologia , Animais , Técnicas de Cultura de Células/métodos , Técnicas de Cocultura , Meios de Cultura , Células-Tronco Embrionárias/fisiologia , Humanos , Imuno-Histoquímica , Camundongos , Células Estromais/fisiologia
7.
Restor Neurol Neurosci ; 27(4): 359-70, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19738328

RESUMO

PURPOSE: Human embryonic stem cells (hESCs) which express a reporter gene consistently during all phases of differentiation would be valuable for basic research on cell transplantation. In this study, we describe karyotypically-abnormal variant hESCs, BGO1V2-EFG, which express hrGFP driven by the EF1 promoter. METHODS: BGO1V2-EFG cells were analyzed by using immunocytochemistry, single cell-based confocal image, and in vitro differentiation, including dopaminergic differentiation. RESULTS: Undifferentiated BGO1V2-EFG cells expressed pluripotent ESC markers and retained the ability to differentiate into cell types of all three germ layers. BGO1V2-EFG cells maintained stable and robust hrGFP expression in vitro in the undifferentiated state and during differentiation. The EF1 promoter retained activity during dopaminergic differentiation, as 76% of tyrosine hydroxlase (TH)-positive cells co-expressed hrGFP by confocal analysis. Treated with sodium butyrate (0.02 mM to 2.0 mM), an inhibitor of histone deacetylase (HDAC), during differentiation did not affect hrGFP expression, although TH expression was reduced by higher concentrations of sodium butyrate. CONCLUSION: BGO1V2-EFG cells maintain stable and robust hrGFP expression in the undifferentiated state and during neural differentiation. Especially, the EF1 promoter was effective in driving hrGFP expression during dopaminergic differentiation. BGO1V2-EFG cells may be useful for transplantation studies in Parkinson disease animal models.


Assuntos
Diferenciação Celular/fisiologia , Dopamina/metabolismo , Células-Tronco Embrionárias/fisiologia , Proteínas de Fluorescência Verde/genética , Butiratos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Dopamina/genética , Relação Dose-Resposta a Droga , Células-Tronco Embrionárias/efeitos dos fármacos , Inibidores Enzimáticos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Microscopia Confocal/métodos , Fator 1 de Elongação de Peptídeos/genética , Regiões Promotoras Genéticas , Transfecção/métodos , Tirosina 3-Mono-Oxigenase/metabolismo
8.
Neurobiol Dis ; 31(3): 342-54, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18632280

RESUMO

Neural transplantation has been investigated experimentally and clinically for the purpose of developing new treatment options for intractable epilepsy. In the present study we assessed the anticonvulsant efficacy and safety of bilateral allotransplantation of genetically engineered striatal GABAergic rat cell lines into the substantia nigra pars reticulata (SNr). Rats with previously-established seizures, induced by amygdala kindling, were used as a model of temporal lobe epilepsy. Three cell lines were transplanted: (1) immortalized GABAergic cells (M213-2O) derived from embryonic rat striatum; (2) M213-2O cells (CL4) transfected with human GAD67 cDNA to obtain higher GABA synthesis than the parent cell line; and (3) control cells (121-1I), also derived from embryonic rat striatum, but which did not show GAD expression. A second control group received injections of medium alone. Transplantation of M213-2O cells into the SNr of kindled rats resulted in significant but transient anticonvulsant effects. Neither control cells nor medium induced anticonvulsant effects. Strong tissue reactions were, however, induced in the host brain of kindled but not of non-kindled rats, and only in animals that received grafts of genetically modified CL4 cells. These tissue reactions included graft rejection, massive infiltration of inflammatory immune cells, and gliosis. The anticonvulsant effect of M213-2O cells emphasizes the feasibility of local manipulations of seizures by intranigral transplantation of GABA-producing cells. On the other hand, the present data suggest that kindling-induced activation of microglia in the SNr can enhance immune reactions to transplanted cells. In this case, under conditions of further immunological stimulation by CL4 cells, transfected with a human cDNA, substantial immune reactions occurred. Thus, it appears that the condition of the host brain and the production of foreign proteins by transplanted cells have to be considered in estimating the risks of rejection of transplants into the brain.


Assuntos
Transplante de Tecido Encefálico/métodos , Epilepsia/metabolismo , Epilepsia/cirurgia , Substância Negra/metabolismo , Substância Negra/cirurgia , Ácido gama-Aminobutírico/biossíntese , Animais , Transplante de Tecido Encefálico/efeitos adversos , Linhagem Celular Transformada , Modelos Animais de Doenças , Epilepsia/fisiopatologia , Feminino , Terapia Genética/métodos , Glutamato Descarboxilase/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Rejeição de Enxerto/prevenção & controle , Humanos , Excitação Neurológica/metabolismo , Microglia/imunologia , Inibição Neural/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Neurônios/transplante , Ratos , Ratos Wistar , Medição de Risco , Transplante de Células-Tronco/efeitos adversos , Transplante de Células-Tronco/métodos , Substância Negra/fisiopatologia , Transfecção/métodos , Resultado do Tratamento , Regulação para Cima/genética
9.
PLoS Med ; 5(6): e117, 2008 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-18593214

RESUMO

BACKGROUND: Prenatal exposure of the developing brain to cocaine causes morphological and behavioral abnormalities. Recent studies indicate that cocaine-induced proliferation inhibition and/or apoptosis in neural progenitor cells may play a pivotal role in causing these abnormalities. To understand the molecular mechanism through which cocaine inhibits cell proliferation in neural progenitors, we sought to identify the molecules that are responsible for mediating the effect of cocaine on cell cycle regulation. METHODS AND FINDINGS: Microarray analysis followed by quantitative real-time reverse transcription PCR was used to screen cocaine-responsive and cell cycle-related genes in a neural progenitor cell line where cocaine exposure caused a robust anti-proliferative effect by interfering with the G1-to-S transition. Cyclin A2, among genes related to the G1-to-S cell cycle transition, was most strongly down-regulated by cocaine. Down-regulation of cyclin A was also found in cocaine-treated human primary neural and A2B5+ progenitor cells, as well as in rat fetal brains exposed to cocaine in utero. Reversing cyclin A down-regulation by gene transfer counteracted the proliferation inhibition caused by cocaine. Further, we found that cocaine-induced accumulation of reactive oxygen species, which involves N-oxidation of cocaine via cytochrome P450, promotes cyclin A down-regulation by causing an endoplasmic reticulum (ER) stress response, as indicated by increased phosphorylation of eIF2alpha and expression of ATF4. In the developing rat brain, the P450 inhibitor cimetidine counteracted cocaine-induced inhibition of neural progenitor cell proliferation as well as down-regulation of cyclin A. CONCLUSIONS: Our results demonstrate that down-regulation of cyclin A underlies cocaine-induced proliferation inhibition in neural progenitors. The down-regulation of cyclin A is initiated by N-oxidative metabolism of cocaine and consequent ER stress. Inhibition of cocaine N-oxidative metabolism by P450 inhibitors may provide a preventive strategy for counteracting the adverse effects of cocaine on fetal brain development.


Assuntos
Cocaína/farmacologia , Neurônios/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Animais , Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Cimetidina/farmacologia , Ciclina A/genética , Ciclina A/metabolismo , Ciclina A2 , Regulação para Baixo , Feminino , Humanos , Fosforilação , Gravidez , Ratos , Ratos Sprague-Dawley , Transfecção
10.
Restor Neurol Neurosci ; 26(6): 447-58, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19096132

RESUMO

BACKGROUND AND PURPOSE: Human embryonic stem cells (hESC) are considered a renewable source of dopamine producing neurons, and are of particular interest for their potential clinical use in Parkinson's disease. In this study, we characterized human dopaminergic neurons generated by stromal-derived inducing activity (SDIA) from BG01V2, a strain of human embryonic stem cell line, BG01, characterized by a chromosome 17 trisomy. Similar chromosomal changes have been repeatedly observed in hESC cultures in different laboratories, indicating the importance of chromosome 17 for growth and adaptation of hESC to culture. METHODS: We investigated in vitro proliferation of differentiating cells using a BrDU incorporation assay, and monitored the cell population in long term cultures. Despite the cytogenetic abnormality, TH+ neurons were postmitotic at all stages of differentiation. After 30 days of differentiation, cell division ceased in 91% of the overall population of cells in the culture, indicating intact cell cycle regulation. RESULTS: Expression of midbrain specific marker genes (Otx2, Pax5, Msx-1) showed differentiation of hESC-derived neural progenitor cells into midbrain specific dopamine neurons. These neurons expressed the dopamine transporter (DAT), and displayed functional DAT activity and electrical excitability. CONCLUSIONS: TH+ cells derived from the BG01V2 hESC line using SDIA are postmitotic and have functional characteristics of normal dopaminergic neurons.


Assuntos
Diferenciação Celular/fisiologia , Dopamina/metabolismo , Células-Tronco Embrionárias/fisiologia , Neurônios/fisiologia , Actinas/metabolismo , Bromodesoxiuridina/metabolismo , Linhagem Celular , Proliferação de Células , Cromossomos Humanos Par 17 , Cocaína/análogos & derivados , Cocaína/farmacocinética , Técnicas de Cocultura , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Humanos , Potenciais da Membrana/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição Otx/metabolismo , Técnicas de Patch-Clamp/métodos , Ligação Proteica/efeitos dos fármacos , Fatores de Tempo , Trítio/farmacocinética , Tirosina 3-Mono-Oxigenase/metabolismo
11.
Behav Brain Res ; 193(1): 17-27, 2008 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-18571743

RESUMO

We have previously shown that intranigral transplants of immortalized GABAergic cells decrease the number of kainic acid-induced seizures [Castillo CG, Mendoza S, Freed WJ, Giordano M. Intranigral transplants of immortalized GABAergic cells decrease the expression of kainic acid-induced seizures in the rat. Behav Brain Res 2006;171:109-15] in an animal model. In the present study, recurrent spontaneous behavioral seizures were established by repeated systemic injections of this excitotoxin into male Sprague-Dawley rats. After the seizures had been established, cells were transplanted into the substantia nigra. Animals with transplants of control cells (without hGAD67 expression) or with sham transplants showed a death rate of more than 40% over the 12 weeks of observation, whereas in animals with M213-2O CL-4 transplants, the death rate was reduced to less than 20%. The M213-2O CL-4 transplants significantly reduced the percentage of animals showing behavioral seizures; animals with these transplants also showed a lower occurrence of stage V seizures than animals in the other groups. In vivo and in vitro analyses provided evidence that the GABAergic cells show sustained expression of both GAD67 and hGAD67 cDNA, as well as increased gamma-aminobutyric acid (GABA) levels in the ventral mesencephalon of transplanted animals. Therefore, transplantation of GABA-producing cells can produce long-term alleviation of behavioral seizures in an animal model.


Assuntos
Glutamato Descarboxilase/metabolismo , Neurônios/transplante , Convulsões/cirurgia , Substância Negra/cirurgia , Ácido gama-Aminobutírico/biossíntese , Animais , Comportamento Animal/efeitos dos fármacos , Bisbenzimidazol/metabolismo , Linhagem Celular Transformada , Cromatografia Líquida de Alta Pressão , Técnica Indireta de Fluorescência para Anticorpo , Glutamato Descarboxilase/genética , Injeções Intraperitoneais , Ácido Caínico/administração & dosagem , Ácido Caínico/toxicidade , Masculino , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Convulsões/induzido quimicamente , Substância Negra/citologia , Substância Negra/metabolismo , Ácido gama-Aminobutírico/administração & dosagem , Ácido gama-Aminobutírico/farmacologia
12.
Addict Biol ; 13(1): 105-17, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18201295

RESUMO

The present study examines the diagnostic challenges of identifying ante-mortem illicit substance use in human postmortem cases. Substance use, assessed by clinical case history reviews, structured next-of-kin interviews, by general toxicology of blood, urine and/or brain, and by scalp hair testing, identified 33 cocaine, 29 cannabis, 10 phencyclidine and nine opioid cases. Case history identified 42% cocaine, 76% cannabis, 10% phencyclidine and 33% opioid cases. Next-of-kin interviews identified almost twice as many cocaine and cannabis cases as Medical Examiner (ME) case histories, and were crucial in establishing a detailed lifetime substance use history. Toxicology identified 91% cocaine, 68% cannabis, 80% phencyclidine and 100% opioid cases, with hair testing increasing detection for all drug classes. A cocaine or cannabis use history was corroborated by general toxicology with 50% and 32% sensitivity, respectively, and with 82% and 64% sensitivity by hair testing. Hair testing corroborated a positive general toxicology for cocaine and cannabis with 91% and 100% sensitivity, respectively. Case history corroborated hair toxicology with 38% sensitivity for cocaine and 79% sensitivity for cannabis, suggesting that both case history and general toxicology underestimated cocaine use. Identifying ante-mortem substance use in human postmortem cases are key considerations in case diagnosis and for characterization of disorder-specific changes in neurobiology. The sensitivity and specificity of substance use assessments increased when ME case history was supplemented with structured next-of-kin interviews to establish a detailed lifetime substance use history, while comprehensive toxicology, and hair testing in particular, increased detection of recent illicit substance use.


Assuntos
Autopsia , Médicos Legistas , Drogas Ilícitas/análise , Transtornos Relacionados ao Uso de Substâncias/patologia , Líquidos Corporais/química , Cerebelo/química , Cerebelo/patologia , Transtornos Relacionados ao Uso de Cocaína/diagnóstico , Transtornos Relacionados ao Uso de Cocaína/patologia , Cabelo/química , Humanos , Abuso de Maconha/diagnóstico , Abuso de Maconha/patologia , Anamnese , Análise de Sequência com Séries de Oligonucleotídeos , Transtornos Relacionados ao Uso de Opioides/diagnóstico , Transtornos Relacionados ao Uso de Opioides/patologia , Abuso de Fenciclidina/diagnóstico , Abuso de Fenciclidina/patologia , Sensibilidade e Especificidade , Transtornos Relacionados ao Uso de Substâncias/diagnóstico
13.
Neurochem Int ; 50(6): 834-47, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17397968

RESUMO

Oxidative stress and apoptotic cell death have been implicated in the dopaminergic cell loss that characterizes Parkinson's disease. While factors contributing to apoptotic cell death are not well characterized, oxidative stress is known to activate an array of cell signaling molecules that participate in apoptotic cell death mechanisms. We investigated oxidative stress-induced cytotoxicity of hydrogen peroxide (H2O2) in three cell lines, the dopaminergic mesencephalon-derived N27 cell line, the GABAergic striatum-derived M213-20 cell line, and the hippocampal HN2-5 cell line. N27 cells were more sensitive to H2O2-induced cell death than M213-20 and HN2-5 cells. H2O2 induced significantly greater increases in caspase-3 activity in N27 cells than in M213-20 cells. H2O2-induced apoptotic cell death in N27 cells was mediated by caspase-3-dependent proteolytic activation of PKCdelta. Gene expression microarrays were employed to examine the specific transcriptional changes in N27 cells exposed to 100 microM H2O2 for 4 h. Changes in genes encoding pro- or anti-apoptotic proteins included up-regulation of BIK, PAWR, STAT5B, NPAS2, Jun B, MEK4, CCT7, PPP3CC, and PSDM3, while key down-regulated genes included BNIP3, NPTXR, RAGA, STK6, YWHAH, and MAP2K1. Overall, the changes indicate a modulation of transcriptional activity, chaperone activity, kinase activity, and apoptotic activity that appears highly specific, coordinated and relevant to cell survival. Utilizing this in vitro model to identify novel oxidative stress-regulated genes may be useful in unraveling the molecular mechanisms underlying dopaminergic degeneration in Parkinson's disease.


Assuntos
Dopamina/fisiologia , Mesencéfalo/metabolismo , Neurônios/metabolismo , Estresse Oxidativo/genética , Doença de Parkinson/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA , Regulação da Expressão Gênica , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Peróxido de Hidrogênio/farmacologia , Mesencéfalo/citologia , Neostriado/citologia , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Neurônios/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Doença de Parkinson/patologia , Proteína Quinase C-delta/metabolismo , RNA/biossíntese , RNA/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Brain Res ; 1147: 77-88, 2007 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-17368428

RESUMO

The developmental biology of the dopamine (DA) system may hold important clues to its reconstruction. We hypothesized that factors highly expressed during nigrostriatal development and re-expressed after injury and disease may play a role in protection and reconstruction of the nigrostriatal system. Examination of gene expression in the developing striatum suggested an important role for the heparin binding growth factor family at time points relevant to establishment of dopaminergic innervation. Midkine, pleiotrophin (PTN), and their receptors syndecan-3 and receptor protein tyrosine phosphatase beta/zeta, were highly expressed in the striatum during development. Furthermore, PTN was up-regulated in the degenerating substantia nigra of Parkinson's patients. The addition of PTN to ventral mesencephalic cultures augmented DA neuron survival and neurite outgrowth. Thus, PTN was identified as a factor that plays a role in the nigrostriatal system during development and in response to disease, and may therefore be useful for neuroprotection or reconstruction of the DA system.


Assuntos
Proteínas de Transporte/metabolismo , Citocinas/metabolismo , Dopamina/metabolismo , Neostriado/metabolismo , Doença de Parkinson/metabolismo , Substância Negra/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Animais , Proteínas de Transporte/genética , Estudos de Casos e Controles , Citocinas/genética , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Midkina , Neostriado/citologia , Neostriado/crescimento & desenvolvimento , Ratos , Ratos Endogâmicos F344 , Receptores Proteína Tirosina Quinases/metabolismo , Valores de Referência , Substância Negra/citologia , Substância Negra/crescimento & desenvolvimento , Paralisia Supranuclear Progressiva/metabolismo , Sindecana-3/metabolismo
15.
Neuropsychopharmacology ; 42(3): 774-784, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27534267

RESUMO

Because of unavoidable confounding variables in the direct study of human subjects, it has been difficult to unravel the effects of prenatal cocaine exposure on the human fetal brain, as well as the cellular and biochemical mechanisms involved. Here, we propose a novel approach using a human pluripotent stem cell (hPSC)-based 3D neocortical organoid model. This model retains essential features of human neocortical development by encompassing a single self-organized neocortical structure, without including an animal-derived gelatinous matrix. We reported previously that prenatal cocaine exposure to rats during the most active period of neural progenitor proliferation induces cytoarchitectural changes in the embryonic neocortex. We also identified a role of CYP450 and consequent oxidative ER stress signaling in these effects. However, because of differences between humans and rodents in neocorticogenesis and brain CYP metabolism, translation of the research findings from the rodent model to human brain development is uncertain. Using hPSC 3D neocortical organoids, we demonstrate that the effects of cocaine are mediated through CYP3A5-induced generation of reactive oxygen species, inhibition of neocortical progenitor cell proliferation, induction of premature neuronal differentiation, and interruption of neural tissue development. Furthermore, knockdown of CYP3A5 reversed these cocaine-induced pathological phenotypes, suggesting CYP3A5 as a therapeutic target to mitigate the deleterious neurodevelopmental effects of prenatal cocaine exposure in humans. Moreover, 3D organoid methodology provides an innovative platform for identifying adverse effects of abused psychostimulants and pharmaceutical agents, and can be adapted for use in neurodevelopmental disorders with genetic etiologies.


Assuntos
Cocaína/farmacologia , Citocromo P-450 CYP3A/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Neocórtex/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Linhagem Celular , Humanos
16.
Neuropsychopharmacology ; 31(12): 2708-15, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17109014

RESUMO

Human embryonic stem cells (hESCs) can proliferate indefinitely yet also differentiate in vitro, allowing normal human neurons to be generated in unlimited numbers. Here, we describe the development of an in vitro neurotoxicity assay using human dopaminergic neurons derived from hESCs. We showed that the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)), which produces features of Parkinson's disease in humans, was toxic for hESC-derived dopaminergic neurons. Treatment with glial cell line-derived neurotrophic factor protected tyrosine hydroxylase-positive neurons against MPP(+)-induced apoptotic cell death and loss of neuronal processes as well as against the formation of intracellular reactive oxygen species. The availability of human dopaminergic neurons, derived from hESCs, therefore allows for the possibility of directly examining the unique features of human dopaminergic neurons with respect to their responses to pharmacological agents as well as environmental and chemical toxins.


Assuntos
1-Metil-4-fenilpiridínio/antagonistas & inibidores , 1-Metil-4-fenilpiridínio/toxicidade , Ensaio de Unidades Formadoras de Colônias/métodos , Células-Tronco Embrionárias/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Modelos Biológicos , Neurônios/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular , Citoproteção/efeitos dos fármacos , Citoproteção/fisiologia , Dopamina/metabolismo , Células-Tronco Embrionárias/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/uso terapêutico , Humanos , Intoxicação por MPTP/tratamento farmacológico , Intoxicação por MPTP/metabolismo , Intoxicação por MPTP/fisiopatologia , Camundongos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Neurotoxinas/antagonistas & inibidores , Neurotoxinas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , Substância Negra/fisiopatologia , Toxicologia/métodos
17.
J Mass Spectrom ; 41(2): 175-84, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16382483

RESUMO

A sensitive and specific method for the simultaneous detection and quantification of amphetamine, opiates, and cocaine and metabolites in human postmortem brain was developed and validated. Analytes of interest included amphetamine, morphine, codeine, 6-acetylmorphine, cocaine, benzoylecgonine, ecgonine methyl ester, ecgonine ethyl ester, cocaethylene, and anhydroecgonine methyl ester. The method employed ultrasonic homogenization of brain tissue in pH 4.0 sodium acetate buffer and solid phase extraction. Extracts were derivatized with N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide and N,O-bis(trimethylsilyl) trifluoroacetamide. Separation and quantification were accomplished on a bench-top positive chemical ionization capillary gas chromatograph/mass spectrometer with selected ion monitoring. Eight deuterated analogs were used as internal standards. Limits of quantification were 50 ng/g of brain. Calibration curves were linear to 1000 ng/g for anhydroecgonine methyl ester and 6-acetylmorphine, and to 2000 ng/g for all other analytes. Accuracy across the linear range of the assay ranged from 90.2 to 112.2%, and precision, as percent relative standard deviation, was less than 16.6%. Quantification of drug concentrations in brain is a useful research tool in neurobiology and in forensic and postmortem toxicology, identifying the type, relative magnitude, and recency of abused drug exposure. This method will be employed to quantify drug concentrations in human postmortem brain in support of basic and clinical research on the physiologic, biochemical, and behavioral effects of drugs in humans.


Assuntos
Anfetamina/análise , Química Encefálica , Encéfalo/metabolismo , Cocaína/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Entorpecentes/análise , Anfetamina/química , Calibragem , Cocaína/química , Humanos , Entorpecentes/química , Sensibilidade e Especificidade
18.
Behav Brain Res ; 171(1): 109-15, 2006 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-16677720

RESUMO

Repeated systemic administration of low doses of kainic acid (KA) induces spontaneous convulsive seizures [Hellier JL, Patrylo PR, Buckmaster PS, Dudek FE. Recurrent spontaneous motor seizures after repeated low-dose systemic treatment with kainate: assessment of a rat model of temporal lobe epilepsy. Epilepsy Res 1998;31:73-84]. In this study, male Sprague-Dawley animals received intranigral transplants of a control cell line M213-2O, or a cell line transfected with human GAD67 cDNA (M213-2O CL4) [Conejero-Goldberg C, Tornatore C, Abi-Saab W, Monaco MC, Dillon-Carter O, Vawter M, et al. Transduction of human GAD67 cDNA into immortalized striatal cell lines using an Epstein-Barr virus-based plasmid vector increases GABA content. Exp Neurol 2000;161:453-61], or no transplant. Eight weeks after transplantation surgery, KA was administered (5 mg/kg/h) until animals reached stage V seizures as described by Racine [Racine RJ. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 1972;32:281-94]. The group transplanted with CL4 required a larger dose of KA and a longer latency to reach a stage V seizure. In addition, this group exhibited significantly fewer stage III and IV seizures. These results indicate that intranigral transplants of a GABA-producing cell line can decrease the number of kainic acid-induced seizures.


Assuntos
Glutamato Descarboxilase/metabolismo , Isoenzimas/metabolismo , Neurônios/transplante , Convulsões/enzimologia , Substância Negra/enzimologia , Ácido gama-Aminobutírico/metabolismo , Animais , Linhagem Celular Transformada , Glutamato Descarboxilase/genética , Humanos , Isoenzimas/genética , Ácido Caínico , Masculino , Neostriado/citologia , Neostriado/enzimologia , Neostriado/transplante , Neurônios/citologia , Neurônios/enzimologia , Ratos , Ratos Sprague-Dawley , Convulsões/induzido quimicamente , Convulsões/prevenção & controle , Substância Negra/citologia , Substância Negra/cirurgia , Transgenes
19.
Brain Res ; 1112(1): 1-15, 2006 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-16901471

RESUMO

AF5 neural cells derived from fetal rat mesencephalic tissue were immortalized with a truncated SV40 LT vector lacking the p53-inactivating domain to maintain long-term cultures with a p53-responsive phenotype. This study examined p53 function in producing programmed cell death in propagating AF5 neural cells after exposure to hydrogen peroxide (H2O2) and the kinase inhibitor staurosporine (STSP). Concentration-dependent exposure of AF5 cells to 0-800 mM H2O2 and STSP at 0-1000 nM revealed increasing cytotoxicity from MTS cell viability assays. Apoptosis occurred at 400 mM H2O2 as evidenced by subG1 DNA and Annexin V flow cytometry analyses and cellular immunofluorescence staining with propidium iodide, anti-Annexin V and DAPI. DNA fragmentation, caspase-3/7 activity and cytochrome c release into cytosol also confirmed H2O2-mediated apoptotic events. p53 protein levels were increased over 24 h by H2O2 in a coordinated fashion with mdm2 expression. p53 activation by H2O2 was evidenced by elevated Ser15 phosphorylation, increased luciferase p53 reporter activity and upregulation of the downstream p53 targets p21(waf1) and apoptotic proteins, bax, Noxa and PUMA. STSP exposure produced apoptosis demonstrated by DNA fragmentation, caspase-3/7 activity, cytochrome c release and over 24 h was accompanied by sustained increase in p53 and Ser15 phosphorylation, rise in p21(waf1) and bax and a transient increase in p53 reporter activity but without Annexin V binding. These findings demonstrate that AF5 cells undergo apoptosis in response to H2O2-mediated oxidative stress and signal pathway disruption by STSP that therefore would be useful in studies related to p53-dependent neuronal cell death and neurodegeneration.


Assuntos
Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Peróxido de Hidrogênio/farmacologia , Neurônios/efeitos dos fármacos , Oxidantes/farmacologia , Estaurosporina/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Análise de Variância , Animais , Anexina A5/metabolismo , Western Blotting/métodos , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo/métodos , Imunofluorescência/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Marcação In Situ das Extremidades Cortadas/métodos , Proteínas do Tecido Nervoso/metabolismo , Ratos , Fatores de Tempo
20.
Restor Neurol Neurosci ; 34(6): 965-976, 2016 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-27834787

RESUMO

PURPOSE: Astrocytes perform a plethora of important functions in the central nervous system (CNS) and are involved in cocaine-evoked synaptic plasticity. Previously, we showed that while cocaine decreased cyclin A2 expression in primary human neural progenitor cells, it increased cyclin A2 expression in human astrocytes. Since cyclin A2 is an essential regulator of the cell cycle, the aim of the present study is to clarify the effect of cocaine on proliferation of human astrocytes and elucidate the underlying molecular mechanisms. METHODS: Primary human astrocytes were treated with either 1, 10, or 100 µM cocaine for 48 hr, and cell proliferation was measured using the CyQUANT cell proliferation assay. To elucidate the molecular mechanisms through which cocaine affects the proliferation of astrocytes, we analyzed gene expression profiles in cocaine-treated primary human astrocytes using a human focused cDNA array. Gene ontology/pathway enrichment analysis, STRING protein-protein interaction analysis, RT-qPCR, and western blotting were used to identify signal transduction pathways that are involved in cocaine-induced astrocyte dysfunction. RESULTS: Cocaine at 10 and 100 µM significantly increased human astrocyte proliferation. Gene expression profiling revealed the JNK MAP kinase pathway as a driver of cell proliferation affected by cocaine in human astrocytes. Further experiments showed that cocaine-induced JNK activation induced up-regulation of cyclin A2, leading to enhanced proliferation of human astrocytes. CONCLUSION: Cocaine-induced abnormal increases in the number of astrocytes may cause disruption in neuron-glia signaling and contribute to synaptic impairment in the CNS. Understanding the mechanisms of cocaine's effects on human astrocytes may help to reveal the involvement of glial cells in addictive behaviors.


Assuntos
Anestésicos Locais/farmacologia , Astrócitos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cocaína/farmacologia , Ciclina A2/metabolismo , MAP Quinase Quinase 4/metabolismo , Regulação para Cima/efeitos dos fármacos , Análise de Variância , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro , Transdução de Sinais/efeitos dos fármacos
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