Enhancement of neuronal protection from oxidative stress by glutamic acid decarboxylase delivery with a defective herpes simplex virus vector.
We have developed defective herpes simplex virus 1 (HSV-1) vectors, based on amplicon plasmids with a replication-deficient mutant, as helper for the transfer of the glutamic acid decarboxylase (GAD67) or beta-galactosidase (beta-gal) gene as control directed by HCMV promoter into neuronal-like cells (PC12) and primary neurons. GAD67 protein was detected immunochemically, while GAD67 activity in virus-producing and nonproducing cell lines was detected enzymatically or by GABA release. Infection with GAD67-expressing amplicon vectors enhanced the resistance of PC12 cells to H(2)O(2). This protection was related to increased energy metabolism, as shown by MTT reduction and ATP level, and involved the GABA shunt, as shown by the reduction in ATP level seen in the presence of gamma-vinyl GABA (GVG), a specific GABA transaminase inhibitor. Level of glutathione (GSH), which requires ATP for its synthesis, was increased by the GAD67 transgene. The activity of glucose-6-phosphate dehydrogenase involved in the maintenance of the NADPH that can be used for the regeneration of the GSH pool, was increased by infection with amplicon vectors. Thus, replication-deficient HSV-1 and the GAD67 transgene have complementary neuroprotective effects and infection with GAD67-expressing amplicon vectors was able to protect nondifferentiated cortical neurons from glutamate toxicity mediated by oxidative stress. Such defective GAD67-expressing HSV-1, as neurotropic vector, should be helpful in neurodegenerative diseases implicating alterations of energy metabolism and oxidative stress in neuronal cells expressing GABA transaminase.
Lamigeon
C
C
Laboratoire de Neurovirologie Moléculaire, INSERM U433, Faculté de Médecine R.T.H. Laënnec, Lyon, France.
Prod'Hon
C
C
De Frias
V
V
Michoudet
C
C
Jacquemont
B
B
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000502:physiology*; D018698:Glutamic Acid / Q000633:toxicity; D005978:Glutathione / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006579:Heterozygote; D006861:Hydrogen Peroxide / Q000633:toxicity; D007527:Isoenzymes / Q000235:genetics / Q000502:physiology*; D009474:Neurons / Q000502:physiology*; D018384:Oxidative Stress / Q000502:physiology*; D016716:PC12 Cells; D010957:Plasmids / Q000235:genetics; D051381:Rats; D019076:Transgenes / Q000235:genetics; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D007527:Isoenzymes; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D001616:beta-Galactosidase; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; D005978:Glutathione