Neuron-specific expression of therapeutic proteins: evaluation of different cellular promoters in recombinant adenoviral vectors.
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 alpha1 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-X(L) 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.
Kügler
S
S
Neuro-Regeneration Laboratory, University of Tübingen, Medical School, Verfügungsgebaude, Auf der Morgenstelle 15, Tübingen, 72076, Germany. kuegler@uni-tuebingen.de
Meyn
L
L
Holzmüller
H
H
Gerhardt
E
E
Isenmann
S
S
Schulz
J B
JB
Bähr
M
M
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D053148:Caspase 3; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018920:Coculture Techniques; D003587:Cytomegalovirus / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000196:embryology / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D009457:Neuroglia / Q000166:cytology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D016704:Synapsins / Q000235:genetics*; D019076:Transgenes; D014404:Tubulin / Q000235:genetics; D051020:bcl-X Protein
C494813:Bcl2l1 protein, rat; D061945:Caspase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D016704:Synapsins; D014404:Tubulin; D051020:bcl-X Protein; C505410:Casp3 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; D011188:Potassium