RESUMO
Non-human primates are valuable for modelling human disorders and for developing therapeutic strategies; however, little work has been reported in establishing transgenic non-human primate models of human diseases. Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor impairment, cognitive deterioration and psychiatric disturbances followed by death within 10-15 years of the onset of the symptoms. HD is caused by the expansion of cytosine-adenine-guanine (CAG, translated into glutamine) trinucleotide repeats in the first exon of the human huntingtin (HTT) gene. Mutant HTT with expanded polyglutamine (polyQ) is widely expressed in the brain and peripheral tissues, but causes selective neurodegeneration that is most prominent in the striatum and cortex of the brain. Although rodent models of HD have been developed, these models do not satisfactorily parallel the brain changes and behavioural features observed in HD patients. Because of the close physiological, neurological and genetic similarities between humans and higher primates, monkeys can serve as very useful models for understanding human physiology and diseases. Here we report our progress in developing a transgenic model of HD in a rhesus macaque that expresses polyglutamine-expanded HTT. Hallmark features of HD, including nuclear inclusions and neuropil aggregates, were observed in the brains of the HD transgenic monkeys. Additionally, the transgenic monkeys showed important clinical features of HD, including dystonia and chorea. A transgenic HD monkey model may open the way to understanding the underlying biology of HD better, and to the development of potential therapies. Moreover, our data suggest that it will be feasible to generate valuable non-human primate models of HD and possibly other human genetic diseases.
Assuntos
Modelos Animais de Doenças , Doença de Huntington/genética , Doença de Huntington/fisiopatologia , Macaca mulatta/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Encéfalo/metabolismo , Encéfalo/patologia , Coreia/genética , Coreia/fisiopatologia , Distonia/genética , Distonia/fisiopatologia , Éxons/genética , Feminino , Humanos , Proteína Huntingtina , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Masculino , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Gravidez , Análise de SobrevidaRESUMO
BACKGROUND: Huntington's Disease (HD) is a progressive neurodegenerative disorder caused by an expansion in the polyglutamine (polyQ) region of the Huntingtin (HTT) gene. The clinical features of HD are characterized by cognitive, psychological, and motor deficits. Molecular instability, a core component in neurological disease progression, can be comprehensively evaluated through longitudinal transcriptomic profiling. Development of animal models amenable to longitudinal examination enables distinct disease-associated mechanisms to be identified. RESULTS: Here we report the first longitudinal study of transgenic monkeys with genomic integration of various lengths of the human HTT gene and a range of polyQ repeats. With this unique group of transgenic HD nonhuman primates (HD monkeys), we profiled over 47,000 transcripts from peripheral blood collected over a 2 year timespan from HD monkeys and age-matched wild-type control monkeys. CONCLUSIONS: Messenger RNAs with expression patterns which diverged with disease progression in the HD monkeys considerably facilitated our search for transcripts with diagnostic or therapeutic potential in the blood of human HD patients, opening up a new avenue for clinical investigation.
Assuntos
Doença de Huntington/genética , Proteínas do Tecido Nervoso/genética , Transcriptoma , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Proteína Huntingtina , Doença de Huntington/sangue , Estudos Longitudinais , Macaca mulatta , Masculino , RNA Mensageiro/metabolismoRESUMO
The use of bone marrow mesenchymal stem cell- (MSC) transplantation therapy for cardiac diseases is limited due to poor survival of implanted cells. MicroRNAs (miRNAs) have been reported to be involved in regulating almost all cellular processes, including apoptosis. In this study, we found that the miRNA profile was altered during apoptosis induced by hypoxia and serum deprivation (hypoxia/SD). We further revealed that over-expression of miR-21, miR-23a and miR-210 could promote the survival of MSCs exposed to hypoxia/SD. In contrast, down-regulation of miR-21, miR-23a and miR-503 aggravated apoptosis of MSCs. It was indicated that these miRNAs may play important roles during MSC apoptosis induced by hypoxia/SD.
Assuntos
Apoptose , Hipóxia/metabolismo , Células-Tronco Mesenquimais/fisiologia , MicroRNAs/metabolismo , Animais , Regulação para Baixo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Hipóxia/fisiopatologia , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
Induced pluripotent Huntington's disease monkey stem cells (rHD-iPSCs) were established by the overexpression of rhesus macaque transcription factors (Oct4, Sox2, and Klf4) in transgenic Huntington's monkey skin fibroblasts. The rHD-iPSCs were pluripotent and capable of differentiating into neuronal cell types in vitro and developed teratoma in immune compromised mice. We also demonstrated the upregulation of endogenous Oct4 and Sox2 after successful reprogramming to pluripotency in rHD-iPSCs, which was not expressed in skin fibroblasts. rHD-iPSCs also developed cellular features comparable to Huntington's disease (HD), including the accumulation of mutant huntingtin (htt) aggregate and the formation of intranuclear inclusions (NIs) paralleling neural differentiation in vitro. Induced pluripotent stem cells from transgenic HD monkeys open a new era of nonhuman primate modeling of human diseases. rHD-iPSCs that develop key HD cellular features and parallel neural differentiation can be a powerful platform for investigating the developmental impact on HD pathogenesis and developing new therapies, which can be evaluated in HD monkeys from whom the rHD-iPSCs were derived.
Assuntos
Doença de Huntington/genética , Doença de Huntington/patologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Desdiferenciação Celular , Diferenciação Celular , Análise Citogenética , Primers do DNA/genética , Modelos Animais de Doenças , Expressão Gênica , Humanos , Proteína Huntingtina , Doença de Huntington/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Macaca mulatta , Camundongos , Camundongos SCID , Mutação , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/metabolismo , Proteínas Nucleares/genética , Fator 3 de Transcrição de Octâmero/genética , Proteínas Recombinantes/genética , Fatores de Transcrição SOXB1/genética , TransfecçãoRESUMO
We demonstrate enhanced transgenesis in mice by intracytoplasmic injection of envelope-free lentivirus. Envelope-free lentivirus carrying the green fluorescent protein (GFP) gene under the control of the ubiquitin promoter (LVU-GFP) was microinjected into the cytoplasm of mouse zygotes prior to embryo transfer. Ninety-seven percent (31/32) of the adult mice were confirmed transgenic by PCR and Southern blot analysis; all founder mice express GFP when tail snips were examined by fluorescent microscopy prior to genomic DNA extraction. Transgene insertion numbers ranging from 1 to 32 were revealed by Southern blot analysis. Germline transmission was confirmed by the presence of transgene in F1 offspring. As expected, a lower transgenic rate (2.2%; 1/46) resulted when envelope-free LVU-GFP was microinjected into the perivitelline space (PVS) because cell recognition followed by membrane fusion between the viral envelope and the target cell is prerequisite for successful infection by envelope viruses. Here we demonstrate the competence of envelope-free lentivirus in establishing stable gene integration by germline transgenesis in mice at high efficiency, by intracytoplasmic viral injection (INVI) of envelope-free lentivirus into mouse zygotes.