The Molecular Virology and Neurogenetics Section focuses on the development of viral vectors for the nervous system. Regulatable lentiviral vectors were designed to alter neuronal excitability and to protect or treat neural cells against neurological diseases such as epilepsy, Alzheimer's disease, stroke and others. Current experimental and potential future therapeutic control of neuronal excitability and neuroprotection are primary goals. Membrane hyperpolarization by regulated expression of the inwardly rectifying potassium channel Kir2.1 is our initial approach to control neuronal excitability. Kir2.1 plays an important role in determining the resting membrane potential and modulating the excitability of many neurons in the forebrain. It is also expressed in some glial cells, muscle and endothelial cells. In normal heart muscle, Kir2.1 shapes the cardiac action potential. Point mutations in Kir2.1 cause Andersen's syndrome, a channelopathy, which manifests itself in periodic paralysis, cardiac arrhythmias and skeletal alterations. Lowering the membrane potential of neurons by Kir2.1 alters neuronal excitability (silence) and affects neuronal transmission. Regulated, cell type specific expression of Kir2.1 is essential to establish expression levels that are tolerated by targeted neurons. Cortical plate neurons and hippocampal neurons and glia were used as target cells. Lentiviral vectors encoding the bicistronic messenger RNA for the EGFP reporter and Kir2.1 were generated in Hek293T cells. Vector production was severely reduced by expression of Kir2.1, with the production limited primarily to the first day post-transfection, while cytopathology increased with this kidney derived Hek293T cell line. Infection of neurons with the vector expressing Kir2.1 from the cytomegalovirus major immediate early (CMV) promoter showed no detrimental effect to neurons over a five days period. This contrasts the reported neuronal apoptosis caused by expression of the kidney channel Kir1.1. Cell type specificity of expression, promoter strength and regulation, transport of Kir2.1 to the postsynaptic density as well as Kir2.1 turnover rates are currently under investigation. In cortical plate neurons and hippocampal neurons, EGFP expression from the CMV promoter is augmented by depolarization after an increase in extracellular K+ resulting in Ca2+-influx. Modification of CMV promoter activity in neurons, after addition of K+ and other specific reagents to the culture medium, indicate that this promoter contains regulatory elements that could potentially be used to autoregulate Kir2.1 expression. This may be beneficial at times of hyperexcitability during epileptic seizures and ischemic insults during stroke. One of the goals is therefore to define the promoter elements responsible for specific expression in neurons and glia to create novel protective viral vectors that are induced by neuronal injury. Alternate approaches to modify neuronal excitability and transmission were addressed by two collaborations. A lentiviral vector was assembled, which encodes EGFP-syntaphilin, a novel presynaptic protein that affects synaptic vesicle release. It could potentially be used to alter neuronal transmission (Sheng, NINDS). A recently completed collaboration focuses on a truncated NMDA-receptor fusion construct, which may be used to alter receptor trafficking to the postsynaptic density and thereby reduce neuronal excitability and transmission (Wenthold, NIDCD). A report of these studies has recently been submitted for publication. Earlier studies demonstrated that HIV-1 Env expressing cells could be targeted, specifically infected and stably transduced by our novel lentiviral vectors carrying the HIV-1 receptors, CD4 and CXCR4, in their membrane envelope. CD4 on the surface of the lentiviral envelope, however, unexpectedly diverted these particles also to other cells without causing stable transduction. These results indicate that cell type specific transgene expression by lentiviral vectors is better achieved via cellular promoters rather than by surface glycoproteins. These studies are complete and will shortly be submitted for publication. To achieve a more controlled cell type specific transgene expression, we have therefore isolated and successfully evaluated several neuron and glia specifc promoters. In addition, our lentiviral vector constructs were used in collaboration on Alzheimer's disease (Pant, NINDS). These studies were recently completed and yielded significant results that have important implications for the disease and a potential treatment for Alzheimer's disease. These results will be submitted for publication within a few weeks. The therapeutic potential of these vectors for Alzheimer's disease will subsequently be evaluated in animal models. The studies also clearly show that we can stably and efficiently transduce neural stem cells from rodents. We, therefore, anticipate that besides being tools to identify underlying molecular mechanisms of neuropathogenesis and opportunities for intervention, we will be able to use lentiviral vectors in the future to directly deliver therapeutic transgenes to the nervous system in vivo or indirectly via stably transduced neural stem cells.
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| Ye, Zhiping; Harmison, George G; Ragheb, Jack A et al. (2005) Targeted infection of HIV-1 Env expressing cells by HIV(CD4/CXCR4) vectors reveals a potential new rationale for HIV-1 mediated down-modulation of CD4. Retrovirology 2:80 |
| Zheng, Ya-Li; Kesavapany, Sashi; Gravell, Maneth et al. (2005) A Cdk5 inhibitory peptide reduces tau hyperphosphorylation and apoptosis in neurons. EMBO J 24:209-20 |
