Neuron-Specific Expression of Therapeutic Proteins: Evaluation of Different Cellular Promoters in Recombinant Adenoviral Vectors

• Published in: Molecular and cellular neuroscience 2001-01, Vol.17 (1), p.78-96
• Main Authors: Kügler, S., Meyn, L., Holzmüller, H., Gerhardt, E., Isenmann, S., Schulz, J.B., Bähr, M.
• Format: Article
• Language: English
• Subjects: Adenoviridae - genetics; Animals; Apoptosis - drug effects; bcl-X Protein; Brain - drug effects; Brain - metabolism; Caspase 3; Caspase 9; Caspase Inhibitors; Caspases - metabolism; Cell Differentiation - genetics; Cells, Cultured; Cerebellum - cytology; Cerebellum - drug effects; Cerebellum - metabolism; Coculture Techniques; Cytomegalovirus - genetics; Gene Expression; Genetic Vectors - genetics; Genetic Vectors - metabolism; Hippocampus - cytology; Hippocampus - drug effects; Hippocampus - embryology; Hippocampus - metabolism; Mitochondria - metabolism; Neuroglia - cytology; Neurons - cytology; Neurons - drug effects; Neurons - metabolism; Potassium - metabolism; Promoter Regions, Genetic - genetics; Proto-Oncogene Proteins c-bcl-2 - biosynthesis; Proto-Oncogene Proteins c-bcl-2 - genetics; Proto-Oncogene Proteins c-bcl-2 - pharmacology; Rats; Rats, Sprague-Dawley; synapsin I gene; Synapsins - genetics; Transgenes; Tubulin - genetics; tubulin ^a1 gene
• ISSN: 1044-7431; 1095-9327
• DOI: 10.1006/mcne.2000.0929

In order to achieve neuron-restricted expression of antiapoptotic proteins, cellular promoters were investigated for their expression profiles in the context of adenoviral vectors. Both the synapsin 1 gene and the tubulin α1 gene promoters were strictly neuron specific in cocultures of primary neurons with their essential feeder cells. The neuron-specific enolase gene promoter exhibited only weak activity in cultured hippocampal neurons and was not neuron specific in preparations of cerebellar granule cells. By attaining virtually 100% transduction efficiency we were able to generate “quasi-transgenic” primary neuron cultures using both differentiated and completely undifferentiated hippocampal neurons. In a functional assay, we used the synapsin promoter to evaluate the effect of Bcl-XL overexpression on potassium-withdrawal-induced apoptosis of cerebellar granule neurons. We found nearly complete inhibition of caspase-9 and -3 activation and apoptosis, indicating a major role for mitochondrial pathways in this paradigm of neuronal cell death. The excellent suitability of the synapsin promoter as a strong panneuronal promoter was further demonstrated by its restricted neuronal activity in various brain regions of adult rats in vivo.