New K C, Rabkin S D
Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20007, USA.
J Neurochem. 1998 Dec;71(6):2304-12. doi: 10.1046/j.1471-4159.1998.71062304.x.
Defective herpes simplex virus (HSV) vectors containing glutamic acid decarboxylase (GAD) cDNAs, either GAD65 or GAD67, were used to examine GAD function and GABA synthesis in rat cortical astrocytes, CNS cells that do not endogenously synthesize GABA. GAD vector infection resulted in isoform-specific expression of GAD as determined by western blotting and immunohistochemistry. Astrocytes infected with a beta-galactosidase vector or uninfected expressed no GAD and contained no detectable GABA. GABA was detected in glial fibrillary acid protein-expressing cells after GAD65 vector infection. Significant amounts of GABA, as determined by HPLC, were synthesized in cultures infected with either GAD vector. The levels of GABA in GAD67 vector-infected cells were almost twofold higher than in GAD65 vector-infected cells. Vector infection did not alter levels of other intracellular amino acids. GABA was tonically released from astrocytes infected with the GAD67 vector, but no increase in release could be detected after treatment of the cells with K+, veratridine, glutamate, or bradykinin. The ability to transduce astrocytes so that they express GAD and thereby increase GABA levels provides a potential strategy for the treatment of neurologic disorders associated with hyperexcitable or diminished inhibitory activity.
含有谷氨酸脱羧酶（GAD）cDNA（GAD65或GAD67）的缺陷型单纯疱疹病毒（HSV）载体，被用于检测大鼠皮质星形胶质细胞（一种内源性不合成γ-氨基丁酸（GABA）的中枢神经系统细胞）中的GAD功能和GABA合成。通过蛋白质免疫印迹法和免疫组织化学法确定，GAD载体感染导致了GAD的亚型特异性表达。感染β-半乳糖苷酶载体或未感染的星形胶质细胞不表达GAD，也检测不到GABA。GAD65载体感染后，在表达胶质纤维酸性蛋白的细胞中检测到了GABA。通过高效液相色谱法测定，在感染任一GAD载体的培养物中均合成了大量GABA。GAD67载体感染细胞中的GABA水平几乎比GAD65载体感染细胞中的高两倍。载体感染未改变其他细胞内氨基酸的水平。GABA从感染GAD67载体的星形胶质细胞中持续释放，但在用钾离子、藜芦碱、谷氨酸或缓激肽处理细胞后，未检测到释放增加。转导星形胶质细胞使其表达GAD从而提高GABA水平的能力，为治疗与过度兴奋或抑制活性降低相关的神经系统疾病提供了一种潜在策略。
