Novel synthesis and release of GABA in cerebellar granule cell cultures after infection with defective herpes simplex virus vectors expressing glutamic acid decarboxylase.
The inhibitory amino acid neurotransmitter gamma-aminobutyric acid (GABA) is synthesized from glutamate in a single step by the enzyme glutamatic acid decarboxylase (GAD). We sought to determine whether viral vectors containing GAD cDNA could be used to enhance synthesis and stimulation-evoked release of GABA in cultures of CNS neurons. For this purpose, we generated double-cassette defective herpes simplex virus (HSV) vectors that expressed one of the two GAD isoforms (GAD65 or GAD67), and Escherichia coli LacZ. Infection of cerebellar granule cell (CGC) cultures with vectors containing GAD cDNA resulted in a significant increase in isoform-specific expression of GAD, synthesis of GABA, and stimulation-evoked GABA release. GAD65 and GAD67 vector-infected neurons exhibited a comparable profile of GABA levels, synthesis and release, as well as GAD protein distribution. In CGCs cultured for 6 days in vitro (DIV), GABA synthesized after vector-derived GAD expression was released by treatment with glutamate or veratridine, but only in a Ca2+-independent fashion. In more mature (10 DIV) cultures, both Ca2+-dependent, K+ depolarization-induced, as well as Ca2+-independent, veratridine-induced, GABA release was significantly enhanced by GAD vector infection. Treatment of CGCs with kainic acid, which destroys most of the GABAergic neurons (<1% remaining), did not prevent vector-derived expression of GAD nor synthesis of GABA. This suggests that defective HSV vector-derived GAD expression can be used to increase GABA synthesis and release in CNS tissue, even in the relative absence of GABAergic neurons. The use of such GAD vectors in the CNS has potential therapeutic value in neurologic disorders such as epilepsy, chronic pain, Parkinson's and Huntington's disease.
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Departments of Microbiology and Immunology, Georgetown University Medical Center, 3970 Reservoir Road NW, Washington, DC 20007, USA.
Gale
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Martuza
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Rabkin
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D000818:Animals; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology* / Q000187:drug effects; D002522:Chlorocebus aethiops; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000096:biosynthesis; D005968:Glutamate Decarboxylase / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D007608:Kainic Acid / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D018139:Simplexvirus / Q000235:genetics*; D013997:Time Factors; D014162:Transfection / Q000379:methods; D014709:Vero Cells; D005680:gamma-Aminobutyric Acid / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism*
D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase; D007608:Kainic Acid