RESUMEN
This study characterized the presynaptic dopaminergic properties of neuronally differentiated mouse embryonic stem (ES) cells. Approximately 30% of the ES cells expressed tyrosine hydroxylase (TH) immunoreactivity when co-cultured with PA6 cells. These cultures expressed high affinity, sodium-dependent dopamine uptake as well as depolarization-induced and calcium-dependent dopamine release of this transmitter. These and other important dopaminergic genes found expressed in these cultures by RT-PCR included Nurr1, vesicular monoamine transporter 2 (VMAT2), TH, dopamine transporter (DAT), and glial cell line-derived neurotrophic factor (GDNF) receptors c-Ret and GFRalpha1. These results demonstrate that differentiated ES cells have the presynaptic functions for maintaining dopaminergic homeostasis, which may be essential for their long-term use in restoring CNS levels of this transmitter.
Asunto(s)
Diferenciación Celular/fisiología , Dopamina/fisiología , Terminales Presinápticos/fisiología , Células Madre/fisiología , Animales , Línea Celular , Dopamina/genética , Dopamina/metabolismo , Embrión de Mamíferos , Ratones , Células Madre/citologíaRESUMEN
Neurodegenerative diseases as a class do not have effective pharmacotherapies. This is due in part to a poor understanding of the pathologies of the disease processes, and the lack of effective medications. Gene delivery is an attractive possibility for treating these diseases. For the paradigm to be effective, efficient, safe and versatile vectors are required. In this study we evaluated three plasmid delivery systems for transgene expression in the rat hippocampus. Two of these systems were designed to have enhanced intracellular biodegradability. It was hypothesized that this system would be less toxic and could increase the free (non-vector) associated plasmids within the cell, leading to increased transgene activity. Polyethylenimine (PEI) and r-AAV-2 (recombinant adeno associated virus-2) were used as positive, non-viral and viral controls respectively, in the in vivo experiments. The results from the studies indicate there is a distinct difference between the various vectors in terms of total cells transfected, type of cell transfected, and toxicity. Non-viral systems were effective at transfecting both neurons and glia cells within the hippocampus, while the r-AAV-2 transfected mainly neurons. In summary, plasmid-mediated systems are effective for transgene expression within the brain and deserve further study.
Asunto(s)
Colesterol/análogos & derivados , Sistemas de Liberación de Medicamentos/métodos , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Hipocampo/metabolismo , Plásmidos/farmacocinética , Animales , Biodegradación Ambiental , Cationes/administración & dosificación , Cationes/farmacocinética , Colesterol/administración & dosificación , Colesterol/farmacocinética , Dependovirus/genética , Evaluación Preclínica de Medicamentos , Humanos , Masculino , Microscopía Confocal , Neuroblastoma/patología , Enfermedades Neurodegenerativas/terapia , Fosfatidiletanolaminas/administración & dosificación , Fosfatidiletanolaminas/farmacocinética , Plásmidos/administración & dosificación , Polietileneimina/administración & dosificación , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción Genética , Transfección , Células Tumorales CultivadasRESUMEN
Administration of nerve growth factor (NGF) by intracerebroventricular infusion or transplantation of NGF-secreting cells to the basal forebrain improves spatial memory in aged animals. Using the adeno-associated virus (AAV) vector system, basal forebrain neurons were transduced to produce NGF ectopically for long intervals (at least 9 months). Rats received intraseptal injections of either the control vector, pTR-UF4, or the pTR-NGFmyc at 3 months of age, prior to testing their performance in the Morris water task. An age-related decrease in the acquisition of the hidden platform location was found at 12 months of age in the pTR-UF4 control group, but not in the pTR-NGFmyc group. Further, when compared to 3 month old untreated animals, the control group, but not the pTR-NGFmyc group, was impaired at 12 months of age. Concomitant to preventing age-related memory deficits, the NGF gene transfer increased cholinergic neuron size by 34% in the medial septum. This approach may therefore represent a viable therapy for age-related dementia involving dysfunction in cholinergic activity and memory, such as Alzheimer's disease.