Modeling schizophrenia gamma deficits using cell-specific RNAi knockdown of GAD67

描述（由申请人提供）：了解精神分裂症（Sz）的病因是NIMH的一个主要重点领域。该R21申请要求两年的支持，以开发一种新颖的，广泛适用的方法来了解Sz和其他精神/神经疾病的皮质电路异常。本申请的总体目标是：(i)建立一种基因、细胞类型和区域特异性方法，在动物模型中模拟Sz的分子死后发现；（ii）用这种方法检测细胞特异性敲低GAD67（Gad1）基因是否会引起典型Sz的γ振荡和皮质回路异常。如果成功，该项目将：(i)验证一种新方法，以研究涉及皮质回路缺陷的Sz等疾病的基因功能；（ii）生成具有良好构效度的Sz动物模型，可用于开发新的治疗方法来改善伽马振荡和认知；（iii）确定GAD67表达下调是否会导致典型的Sz皮质回路缺陷。为了特异性地敲低（KD）基因表达，我们建议使用RNA干扰（RNAi），基于我们之前在睡眠领域使用该技术的经验。我们将首先针对谷氨酸脱羧酶（GAD67），这是抑制性神经递质GABA的主要合成酶，因为临床尸检数据和Sz的动物模型报告了表达钙结合蛋白小白蛋白（PV）的皮质中间神经元中GAD67水平的部分（30-50%）降低。为了实现细胞类型和区域特异性，将在pv阳性神经元（PV-Cre小鼠）中表达Cre重组酶（Cre）的小鼠的前边缘皮层中注射具有Cre重组酶依赖表达靶向GAD67的小发夹RNA （shRNA-GAD67）的病毒载体（腺相关病毒；AAV）。因此，shRNA的重组和表达只会发生在该皮质区域的PV-Pos神经元中，导致GAD67的细胞和区域特异性KD。控制载体将表达不靶向任何已知小鼠基因的打乱shRNA （shRNA- CTRL）。病毒载体也将以cre依赖的方式表达增强的绿色荧光蛋白（GFP），作为病毒表达的标记。病毒表达的选择性将通过PV的荧光免疫组化染色和GFP/PV表达的比较来确定，即GFP是否仅位于PV神经元中。目的基因（GAD67） KD的有效性将通过以下方法进行评估：(i)使用激光捕获显微解剖（LCM）对转染PV-Pos神经元中收获的mRNA进行定量聚合酶链反应（PCR）；（ii） GAD67蛋白免疫组化染色及染色强度亮度测定。对于GAD65和PV，将通过LCM/PCR以及PV的免疫组织化学来证实KD的选择性。我们将使用靶向GAD67和PV-Cre小鼠的RNAi来测试PV-Pos神经元中特异性GAD67的减少是否是导致皮层回路缺陷的关键事件，从而导致体外γ (30-80 Hz)振荡减少，这是由于抑制性突触传递的改变。

DESCRIPTION (provided by applicant): Understanding the cause of schizophrenia (Sz) is a major area of emphasis for NIMH. This R21 submission requests two years of support to develop a novel, widely applicable method to understand cortical circuitry abnormalities in Sz and other psychiatric/neurological disorders. The overall goals of this application are: (i) Establish a gene, cell-type and region-specific method to mimic molecular postmortem findings in Sz in an animal model; (ii) Use this method to test whether cell-specific knockdown of one such gene, GAD67(Gad1), causes gamma oscillation and cortical circuitry abnormalities typical of Sz. If successful, this project will: (i) Validate a novel method to investigate the function of genes implicated in disorders such as Sz involving cortical circuitry deficits; (ii) Generate a new animal model of Sz with good construct validity which can be used to develop new therapeutic approaches to improve gamma oscillations and cognition; and (iii) Determine whether downregulation of GAD67 expression can cause cortical circuitry deficits typical of Sz. In order to specifically knockdown (KD) gene expression we propose to use RNA interference (RNAi), building on our previous experience using this technique in the sleep field. We will initially target glutamic acid decarboxylase (GAD67), the major synthesizing enzyme for the inhibitory neurotransmitter GABA, since clinical postmortem data and animal models of Sz report a partial (30-50%) reduction in GAD67 levels in cortical interneurons expressing the calcium binding protein parvalbumin (PV). To achieve cell-type and region specificity, injections of viral vectors (adeno-associated virus; AAV) with Cre recombinase-dependent expression of small hairpin RNA targeting GAD67 (shRNA-GAD67) will be made in the prelimbic cortex of mice expressing Cre recombinase (Cre) in PV-positive neurons (PV-Cre mice). Thus, recombination and expression of shRNA will only occur in PV-Pos neurons in this cortical area, causing a cell and region specific KD of GAD67. The control vector will express scrambled shRNA not targeting any known mouse gene (shRNA- CTRL). The viral vectors will also express enhanced green fluorescent protein (GFP) in a Cre-dependent fashion as a marker of viral expression. Selectivity of viral expression will be determined by fluorescent immunohistochemical staining for PV and comparison of GFP/PV expression i.e. whether GFP is only located in PV neurons. Effectiveness of the KD of the target gene (GAD67) will be assessed by: (i) Quantitative Polymerase chain reaction (PCR) performed on mRNA harvested from transfected PV-Pos neurons using laser capture microdissection (LCM); (ii) Immunohistochemical staining for GAD67 protein and luminance measures of staining intensity. Selectivity of the KD will be confirmed by LCM/PCR for GAD65 and PV as well as immunohistochemistry for PV. We will use RNAi targeting GAD67 and PV-Cre mice to test whether a reduction in GAD67 specifically in PV-Pos neurons is a key event causing cortical circuitry deficits leading to reduced in vitro gamma (30-80 Hz) oscillations due to altered inhibitory synaptic transmission.