RESUMO
Human mutations in PQBP1, a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain. Examination of a conditional Pqbp1-knockout (cKO) mouse with microcephaly failed to reveal either abnormal centrosomes or mitotic spindles, increased neurogenesis from the neural stem progenitor cell (NSPC) pool or increased cell death in vivo. Instead, we observed an increase in the length of the cell cycle, particularly for the M phase in NSPCs. Corresponding to the developmental expression of Pqbp1, the stem cell pool in vivo was decreased at E10 and remained at a low level during neurogenesis (E15) in Pqbp1-cKO mice. The expression profiles of NSPCs derived from the cKO mouse revealed significant changes in gene groups that control the M phase, including anaphase-promoting complex genes, via aberrant transcription and RNA splicing. Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO. These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation. Finally, we demonstrated that in utero gene therapy for Pqbp1-cKO mice by intraperitoneal injection of the PQBP1-AAV vector at E10 successfully rescued microcephaly with preserved cortical structures and improved behavioral abnormalities in Pqbp1-cKO mice, opening a new strategy for treating this intractable developmental disorder.
Assuntos
Terapia Genética , Microcefalia/genética , Microcefalia/terapia , Células-Tronco Neurais/fisiologia , Proteínas Nucleares/deficiência , Adenoviridae/genética , Animais , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Apoptose/genética , Encéfalo/patologia , Proteínas de Transporte/genética , Moléculas de Adesão Celular/metabolismo , Ciclo Celular , Proliferação de Células , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Microcefalia/patologia , Nestina/genética , Nestina/metabolismo , Neurogênese , Proteínas Nucleares/genética , Sinapsinas/genética , Sinapsinas/metabolismoRESUMO
Parkinson's disease (PD), a neurological disease suited to gene therapy, is biochemically characterized by a severe decrease in the dopamine content of the striatum. One current strategy for gene therapy of PD involves local production of dopamine in the striatum achieved by inducing the expression of enzymes involved in the biosynthetic pathway for dopamine. We previously showed that the coexpression of tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), using two separate adeno-associated virus (AAV) vectors, resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine-lesioned parkinsonian rats, compared with the expression of TH alone. Not only levels of TH and AADC but also levels of tetrahydrobiopterin (BH4), a cofactor of TH, and GTP cyclohydrolase I (GCH), a rate-limiting enzymes for BH4 biosynthesis, are reduced in parkinsonian striatum. In the present study, we investigated whether transduction with separate AAV vectors expressing TH, AADC, and GCH was effective for gene therapy of PD. In vitro experiments showed that triple transduction with AAV-TH, AAV-AADC, and AAV-GCH resulted in greater dopamine production than double transduction with AAV-TH and AAV-AADC in 293 cells. Furthermore, triple transduction enhanced BH4 and dopamine production in denervated striatum of parkinsonian rats and improved the rotational behavior of the rats more efficiently than did double transduction. Behavioral recovery persisted for at least 12 months after stereotaxic intrastriatal injection. These results suggest that GCH, in addition to TH and AADC, is important for effective gene therapy of PD.
Assuntos
Descarboxilases de Aminoácido-L-Aromático/genética , GTP Cicloidrolase/genética , Terapia Genética/métodos , Doença de Parkinson/terapia , Tirosina 3-Mono-Oxigenase/genética , Animais , Descarboxilases de Aminoácido-L-Aromático/biossíntese , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Linhagem Celular , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Dependovirus , Dopamina/metabolismo , GTP Cicloidrolase/biossíntese , Perfilação da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Injeções , Masculino , Atividade Motora , Oxidopamina , Doença de Parkinson/patologia , Ratos , Ratos Wistar , Fatores de Tempo , Transformação Genética , Transgenes , Tirosina 3-Mono-Oxigenase/biossínteseRESUMO
We describe the unusual case of a 51-year-old woman with spinocerebellar ataxia type 1 (SCA1) who showed choreiform movements in addition to cerebellar ataxia. To date, extrapyramidal signs including involuntary movements have been rarely reported in SCA1. Surface electromyogram in our patient revealed grouped discharges whose duration was longer than that of chorea observed in HD, indicating that the involuntary movements represented choreoathetosis rather than pure chorea. These choreiform movements have not been seen in non-hereditary spinocerebellar ataxia. Therefore, if "sporadic" cases of cerebellar ataxia show such movements, the possibility of genetic origin of the ataxia is high and a surveillance of various forms of hereditary spinocerebellar ataxia including SCA1 is required.
Assuntos
Cerebelo/patologia , Coreia/fisiopatologia , Ponte/patologia , Ataxias Espinocerebelares/fisiopatologia , Cerebelo/fisiopatologia , Coreia/patologia , Análise Mutacional de DNA , Eletromiografia , Feminino , Humanos , Imageamento por Ressonância Magnética , Pessoa de Meia-Idade , Músculo Esquelético/fisiopatologia , Ponte/fisiopatologia , Ataxias Espinocerebelares/patologiaRESUMO
Efficient gene transfer and regulated transgene expression in primate embryonic stem (ES) cells are highly desirable for future applications of the cells. In the present study, we have examined using the nonintegrating Sendai virus (SeV) vector to introduce the green fluorescent protein (GFP) gene into non-human primate cynomolgus ES cells. The GFP gene was vigorously and stably expressed in the cynomolgus ES cells for a year. The cells were able to form fluorescent teratomas when transplanted into immunodeficient mice. They were also able to differentiate into fluorescent embryoid bodies, neurons, and mature blood cells. In addition, the GFP expression levels were reduced dose-dependently by the addition of an anti-RNA virus drug, ribavirin, to the culture. Thus, SeV vector will be a useful tool for efficient gene transfer into primate ES cells and the method of using antiviral drugs should allow further investigation for regulated SeV-mediated gene expression.