The Molecular Virology and Neurogenetics Section was established in FY02. The section merges recent advances in molecular virology with select topics in neurobiology. Neurobiological questions related to neural development, neuronal excitability and transmission as well as neuroprotective or therapeutic approaches against stroke are addressed using gene transfer techniques involving viral vectors. Initial studies focused on the development of state of the art lentiviral vectors to encode and stably transduce neural cells in culture. Vector construct cassettes were designed to allow easy exchange of promoters and transgenes, and the coexpression of single and multiple transgenes. Characterizing the host range and cell type specific expression of the vector in select neural cell types were initial goals. Efficiency and regulation of transgene expression are subsequent goals. Two generations of lentiviral vectors were compared with respect to biosafety and efficacy of cell transduction. The latest, most efficient and safest vector construct was completely sequenced. This vector together with its support plasmids were, for the first time for any lentiviral vector, downgraded by the NIH Institutional Biosafety Committee for use at biosafety level 2, down from biosafety level 2/3. This was a crucial step towards the safe and more flexible future use of these vectors not only in dissociated cell cultures and organotypic brain slices in vitro but also for animal studies in vivo. Pseudotype lentiviral vectors carrying the vesicular stomatitis virus glycoprotein were able to efficiently transduce a wide range of neural cell types, including rat hippocampal neurons and astrocytes, neuronal and oligodendrocyte precursor cells and cortical plate neurons. This demonstrates that the receptor for the vesicular stomatitis virus glycoprotein is ubiquitous and found on most neural cell types. Even multi-potent rat neural stem cells that were selected against the presence of the cell surface apoptotic marker phosphatidyl serine, the putative receptor for vesicular stomatitis virus glycoprotein, were efficiently transduced. This suggests an earlier conclusion that phosphatidyl serine is the vesicular stomatitis virus glycoprotein receptor should be reevaluated. Although infection by the lentiviral vector appeared to be efficient for most cell types, transgene expression from the cytomegalovirus immediate/early promoter differed dramatically between cell types. Expression in astrocytes was generally very strong as compared to the weak expression in neural stem cells or neuronal or oligodendrocyte precursor cells of the developing rat brain. Expression remained low in E18+ hippocampal and E19+ cortical plate neurons. Interestingly, transgene expression could be enhanced dramatically to similar high levels found in astrocytes by a depolarization of neurons in the presence of increased potassium and to a lesser extent by adding forskolin. Transcriptional activation had been found recently to occur via Creb protein phosphorylation mechanisms and binding to the cytomegalovirus immediate/early promoter. Our results also demonstrate that the ability of this promoter to respond to depolarization develops in neurons between early neuronal stages in the ventricular zone and the cortical plate. The efficacy of stable transgene expression of neural cells by third generation lentiviral vectors in vitro encourages their potential use in neural cells and brain regions in vivo. Future studies will now focus on establishing cell type specificity and the control of efficient transgene expression, which are essential to be able to address the specific neurobiological themes described above.
| Mentis, George Z; Gravell, Maneth; Hamilton, Rebecca et al. (2006) Transduction of motor neurons and muscle fibers by intramuscular injection of HIV-1-based vectors pseudotyped with select rabies virus glycoproteins. J Neurosci Methods 157:208-17 |
| 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 |
