Differential Terminal Expression of GAD67/GAD65 ? Relevance to Schizophrenia

描述(申请人提供)：GABA神经传递的突触前强度部分取决于可供释放的终末GABA的量。GABA末端由GAD67和GAD65蛋白在局部合成。GAD67是GABA的主要合成酶，其转录本的表达减少可能是精神分裂症中复制最多的病理障碍。在精神分裂症中，GAD67基因表达的减少可能导致GABA合成减少，导致GABA神经传递减弱，从而导致振荡障碍。然而，目前尚不清楚GAD67 mRNA的缺失是否伴随着GAD67蛋白的相应下降，特别是在主要功能部位(如末端)。此外，中间神经元，特别是那些GAD67 mRNA被认为在精神分裂症中发生减少的中间神经元，对末端GABA合成的GAD67和GAD65的依赖性尚不清楚。因此，我们开发了一种新的高通量荧光成像方法，该方法可以准确地定量荧光标记的斑点(假定的末端)，不同标记在同一末端的共存，以及这些相同结构中的荧光强度的定量。使用这种方法，我们将进行第一次研究，比较与精神分裂症相关的不同中间神经元终末的GAD67和GAD65蛋白水平，在非人类和人类(精神分裂症和匹配的对照组受试者)灵长类动物背外侧前额叶皮质(DLPFC)中。在拟议的研究完成后，我们将知道：1)在非人灵长类DLPFC中是否存在神经元间亚群之间终末GAD67/GAD65比率的特定差异；2)所观察到的差异是否在非人灵长类与人类灵长类之间保守；3)精神分裂症受试者DLPFC中GABA终末亚群中的GAD67是否减少。这些已获得的知识将使我们能够：1)提出关于GAD67 mRNA减少在精神分裂症中的细胞类型特定后果的假设；2)在非人类灵长类动物存在的丰富的生理、药理学和解剖学数据的背景下解释发现；3)提供必要的数据，以优化未来对人类组织的死后研究的设计。公共卫生相关性：与精神分裂症相关的GABA神经传递缺陷被认为是导致某些认知功能障碍的原因，而认知功能是这种疾病的核心特征。在很大程度上，目前精神分裂症的药物治疗在改善认知功能方面是无效的。这些研究将提供有关关键细胞、潜在药理靶点、参与GABA神经传递以及它们在精神分裂症中如何受到影响的迫切需要的信息。

DESCRIPTION (provided by applicant): The presynaptic strength of GABA neurotransmission is partially determined by the amount of terminal GABA available for release. Terminal GABA is synthesized locally by GAD67 and GAD65 protein. Reduced expression of the transcript for GAD67, the principal synthesizing enzyme for GABA, is perhaps the most replicated pathological disturbance in schizophrenia. Reductions in GAD67 mRNA expression may lead to reduced GABA synthesis, resulting in weaker GABA neurotransmission, and hence oscillatory impairment, in schizophrenia. However, it is unknown if the deficit in GAD67 mRNA is accompanied by a comparable decrease in GAD67 protein, particularly at the major site of function (e.g. the terminal). In addition, the dependency of interneurons, particularly those in which reductions of GAD67 mRNA are proposed to occur in schizophrenia, on GAD67 versus GAD65 for terminal GABA synthesis is unknown. Therefore, we have developed a novel high-throughput fluorescence imaging methodology that allows for the accurate quantification of fluorescently-labeled puncta (putative terminals), the colocalization of different labels in the same terminal, and the quantification of fluorescence intensity in these same structures. Using this methodology, we will perform studies that will be the first to compare the level of GAD67 and GAD65 protein in the terminals of different interneurons that are relevant to schizophrenia in the non-human and human (schizophrenia and matched control subjects) primate dorsolateral prefrontal cortex (DLPFC). At the completion of the proposed studies we will know: 1) if interneuron subpopulation specific differences in terminal GAD67/GAD65 ratios are present in the non-human primate DLPFC; 2) whether observed differences are conserved between non-human and human primates; 3) if GAD67 is reduced in subpopulations of GABA terminals in the DLPFC of subjects with schizophrenia. This acquired knowledge will allow us to: 1) formulate hypotheses about the cell type specific consequences of the GAD67 mRNA reduction in schizophrenia; 2) interpret findings in the context of the rich body of physiologic, pharmacologic, and anatomic data that exists for non-human primate; 3) provide data necessary to optimize the design of future studies in postmortem human tissue. PUBLIC HEALTH RELEVANCE: Deficits in GABA neurotransmission associated with schizophrenia are believed to contribute to the impairments in certain cognitive functions that are core features of the illness. For the most part, current pharmacological treatments for schizophrenia are ineffective at improving cognitive function. These studies will provide much needed information about the key cells, potential pharmacological targets, involved in GABA neurotransmission and how they are affected in schizophrenia.