New K C, Gale K, Martuza R L, Rabkin S D
Departments of Microbiology and Immunology, Georgetown University Medical Center, 3970 Reservoir Road NW, Washington, DC 20007, USA.
Brain Res Mol Brain Res. 1998 Oct 30;61(1-2):121-35. doi: 10.1016/s0169-328x(98)00203-4.
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.
抑制性氨基酸神经递质γ-氨基丁酸（GABA）由谷氨酸通过谷氨酸脱羧酶（GAD）一步合成。我们试图确定携带GAD cDNA的病毒载体是否可用于增强中枢神经系统神经元培养物中GABA的合成及刺激诱发释放。为此，我们构建了表达两种GAD同工型之一（GAD65或GAD67）和大肠杆菌LacZ的双盒式缺陷单纯疱疹病毒（HSV）载体。用携带GAD cDNA的载体感染小脑颗粒细胞（CGC）培养物，导致GAD同工型特异性表达、GABA合成及刺激诱发的GABA释放显著增加。感染GAD65和GAD67载体的神经元在GABA水平、合成及释放以及GAD蛋白分布方面表现出相似的特征。在体外培养6天（DIV）的CGC中，载体衍生的GAD表达后合成的GABA可通过谷氨酸或藜芦碱处理释放，但仅以不依赖Ca2+的方式释放。在更成熟（10 DIV）的培养物中，GAD载体感染显著增强了依赖Ca2+的、K+去极化诱导的以及不依赖Ca2+的、藜芦碱诱导的GABA释放。用破坏大多数GABA能神经元（剩余<1%）的海人酸处理CGC，并未阻止载体衍生的GAD表达及GABA合成。这表明缺陷型HSV载体衍生的GAD表达可用于增加中枢神经系统组织中GABA的合成及释放，即使在相对缺乏GABA能神经元的情况下也是如此。这种GAD载体在中枢神经系统中的应用在癫痫、慢性疼痛、帕金森病和亨廷顿病等神经系统疾病中具有潜在治疗价值。
