RESUMO
The G2019S mutation in the leucine-rich repeat kinase 2 (LRRK2) gene is the most common genetic cause of Parkinson's disease (PD), accounting for a significant proportion of both autosomal dominant familial and sporadic PD cases. Our aim in the present study is to generate a mammalian model of mutant G2019S LRRK2 pathogenesis, which reproduces the robust nigral neurodegeneration characteristic of PD. We developed adenoviral vectors to drive neuron-specific expression of full-length wild-type or mutant G2019S human LRRK2 in the nigrostriatal system of adult rats. Wild-type LRRK2 did not induce any significant neuronal loss. In contrast, under the same conditions and levels of expression, G2019S mutant LRRK2 causes a progressive degeneration of nigral dopaminergic neurons. Our data provide a novel rat model of PD, based on a prevalent genetic cause, that reproduces a cardinal feature of the disease within a rapid time frame suitable for testing of neuroprotective strategies.
Assuntos
Encéfalo/patologia , Modelos Animais de Doenças , Degeneração Neural/patologia , Neurônios/patologia , Doença de Parkinson/patologia , Proteínas Serina-Treonina Quinases/genética , Análise de Variância , Animais , Western Blotting , Encéfalo/metabolismo , Contagem de Células , Dopamina/metabolismo , Feminino , Imuno-Histoquímica , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Mutação , Degeneração Neural/genética , Degeneração Neural/metabolismo , Neurônios/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos WistarRESUMO
The involvement of alpha-synuclein in familial forms of Parkinson's disease suggests a potential causative role in the pathogenesis. We have explored the possibility of generating animal models of Parkinson's disease by overexpressing alpha-synuclein in the nigrostriatal pathway using viral vectors. Both lentiviral and adeno-associated vectors efficiently transduce dopaminergic neurons in the substantia nigra, and transgenic expression of alpha-synuclein leads to the progressive loss of neurons positive for dopaminergic markers, with the formation of intraneuronal alpha-synuclein aggregates. With a high tropism for nigral dopaminergic neurons, adeno-associated vectors allow for the monitoring of dopaminergic function using spontaneous and drug-induced behaviour. We propose that virus-based rodent alpha-synuclein models provide a valuable approach for the preclinical testing of therapeutics.
Assuntos
Terapia Genética/métodos , Vetores Genéticos/fisiologia , Lentivirus/fisiologia , Doença de Parkinson/terapia , Animais , Modelos Animais de Doenças , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Neural stem cells (NSCs) are widely endorsed as a cell source for replacement strategies in neurodegenerative disease. However, their usefulness is currently limited by the inability to induce specific neurotransmitter phenotypes in these cells. In order to direct dopaminergic neuronal fate, we overexpressed Pitx3 in NSCs that were then exposed to E11 developing ventral mesencephalon (VM) in explant culture. This resulted in a significant potentiation of dopaminergic differentiation of the cells. When transplanted into the 6-hydroxydopamine lesioned Parkinsonian rats, these cografts of VM and Pitx3 overexpressing NSCs resulted in a significant restitution of motor function. In addition, there were greater numbers of Girk2 positive A9 neurons in the periphery of the transplants that were NSC derived. This demonstrates that given the correct signals, NSCs can be induced to become dopaminergic neurons that can differentiate into the correct nigrastriatal phenotype required for the treatment of Parkinson's disease.
Assuntos
Transplante de Tecido Encefálico/métodos , Dopamina/biossíntese , Doença de Parkinson/terapia , Transplante de Células-Tronco/métodos , Animais , Diferenciação Celular , Sobrevivência Celular , Técnicas de Cocultura , Modelos Animais de Doenças , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos , Proteínas de Homeodomínio/metabolismo , Lentivirus/genética , Mesencéfalo/transplante , Atividade Motora , Neurônios/patologia , Neurônios/transplante , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Técnicas de Cultura de Tecidos , Fatores de Transcrição/metabolismoRESUMO
CD31(+)CD45RA(+)RO(-) lymphocytes contain high numbers of T cell receptor circle (TREC)-bearing T cells; however, the correlation between CD31(+)CD4(+) lymphocytes and TREC during aging and under lymphopenic conditions has not yet been sufficiently investigated. We analyzed TREC, telomere length and telomerase activity within sorted CD31(+) and CD31(-) CD4(+) lymphocytes in healthy individuals from birth to old age. Sorted CD31(+)CD45RA(+)RO(-) naive CD4(+) lymphocytes contained high TREC numbers, whereas CD31(+)CD45RA(-)RO(+) cells (comprising < or =5% of CD4(+) cells during aging) did not contain TREC. CD31(+) overall CD4(+) cells remained TREC rich despite an age-related tenfold reduction from neonatal (100 : 1000) to old age (10 : 1000). Besides a high TREC content, CD31(+)CD45RA(+)RO(-)CD4(+) cells exhibited significantly longer telomeres and higher telomerase activity than CD31(-)CD45RA(+)RO(-)CD4(+) cells, suggesting that CD31(+)CD45RA(+)RO(-)CD4(+) cells represent a distinct population of naive T cells with particularly low replicative history. To analyze the value of CD31 in lymphopenic conditions, we investigated six children after allogeneic hematopoietic stem cell transplantation (HSCT). Reemerging overall CD4(+) as well as naive CD45RA(+)RO(-)CD4(+) cells predominantly expressed CD31 and correlated well with the recurrence of TREC 5-12 months after HSCT. Irrespective of limitations in the elderly, CD31 is an appropriate marker to monitor TREC-rich lymphocytes essentially in lymphopenic children after HSCT.
Assuntos
Envelhecimento/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfopenia/imunologia , Plasmídeos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Subpopulações de Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Criança , Pré-Escolar , Citometria de Fluxo , Humanos , Lactente , Recém-Nascido , Pessoa de Meia-Idade , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Telomerase/metabolismo , Telômero/metabolismo , Timo/citologia , Timo/crescimento & desenvolvimentoRESUMO
Human neural progenitor cells (hNPC) hold great potential as an ex vivo system for delivery of therapeutic proteins to the central nervous system. When cultured as aggregates, termed neurospheres, hNPC are capable of significant in vitro expansion. In the current study, we present a robust method for lentiviral vector-mediated gene delivery into hNPC that maintains the differentiation and proliferative properties of neurosphere cultures while minimizing the amount of viral vector used and controlling the number of insertion sites per population. This method results in long-term, stable expression even after differentiation of the hNPC to neurons and astrocytes and allows for generation of equivalent transgenic populations of hNPC. In addition, the in vitro analysis presented predicts the behavior of transgenic lines in vivo when transplanted into a rodent model of Parkinson's disease. The methods presented provide a powerful tool for assessing the impact of factors such as promoter systems or different transgenes on the therapeutic utility of these cells.
Assuntos
Técnicas de Transferência de Genes/normas , Terapia Genética/métodos , Vetores Genéticos/genética , Lentivirus/genética , Células-Tronco/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/virologia , Biomarcadores/metabolismo , Transplante de Tecido Encefálico/métodos , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Feto , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Humanos , Neurônios/metabolismo , Neurônios/virologia , Doença de Parkinson/genética , Doença de Parkinson/terapia , Regiões Promotoras Genéticas/genética , Ratos , Ratos Endogâmicos Lew , Esferoides Celulares/citologia , Esferoides Celulares/fisiologia , Esferoides Celulares/virologia , Células-Tronco/virologia , TransgenesRESUMO
Lentiviral vectors have demonstrated great potential as gene therapy vectors mediating efficient ex vivo and in vivo gene delivery and long-term transgene expression in both dividing and nondividing cells. However, for clinical studies it must be demonstrated that lentiviral vector preparations are safe and not contaminated by replication-competent recombinants related to the parental pathogenic virus. Here we describe a sensitive assay for the detection of replication-competent lentiviruses (RCL) in large-scale preparations of HIV-based lentiviral vectors. This RCL assay for lentiviral vectors is based on the principles used for retroviral vectors, using a highly permissive cell line, C8166-45, for RCL amplification and an appropriate positive control virus to establish the assay sensitivity. The assay is capable of detecting 1 RCL infectious unit in a background of 2.5 x 10(8) transducing units of vector in a single test culture. Statistically representative samples from large-scale lentiviral vector productions were assayed using multiple test cultures for each lot. Overall, a total of 1.4 x 10(10) transducing units of vector from 10 independent 14-liter production lots were screened and no RCL was detected. We propose to implement this assay as a release testing for clinical-grade lentiviral vector preparations intended for gene therapy clinical trials.
Assuntos
Vetores Genéticos/genética , Vetores Genéticos/fisiologia , HIV/crescimento & desenvolvimento , HIV/genética , Replicação Viral , Animais , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos/biossíntese , Humanos , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Fatores de TempoRESUMO
We have generated novel lentiviral vectors to integrate various connexin cDNAs into primary, non-dividing cells. We have used these vectors to test whether proper control of insulin secretion depends on a specific connexin isoform and/or on its level of expression. We have observed that transduced connexin32, connexin36 and connexin43 were expressed by primary adult beta-cells at membrane interfaces, were packed into typical gap junction plaques and formed functional channels that allowed a variable coupling, depending on the type and level of connexin expressed. The infected cells spontaneously reaggregated into three-dimensional pseudo-islet organs that could be maintained in culture. We have found that pseudo-islets made by cells transduced with either GFP- or connexin43-expressing lentivirus released insulin in response to various secretagogues similarly to controls. By contrast, pseudo-islets made by cells expressing connexin32, a connexin exogenous to pancreatic islets, or over-expressing connexin36, the endogenous islet connexin, featured a marked decrease in the secretory response to glucose. The data show: (1) that lentiviral vectors allow stable modulation of various connexin in primary, non-proliferating cells; (2) that specific connexin isoforms affect insulin secretion differently; and (3) that adequate levels of coupling via connexin36 channels are required for proper beta-cell function.