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）的原因是NIMH的一个主要重点领域。该R21提交文件要求提供两年的支持，以开发一种新的、广泛适用的方法来了解Sz和其他精神/神经系统疾病的皮质回路异常。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，以模拟动物模型中Sz的分子死后发现;（ii）使用该方法测试一种此类基因GAD 67（Gad 1）的细胞特异性敲低是否会导致Sz典型的γ振荡和皮质回路异常。如果成功，该项目将：（i）建立一种新的方法来研究涉及大脑皮层回路缺陷的疾病（如Sz）中涉及的基因功能;（ii）建立一种新的具有良好结构效度的Sz动物模型，可用于开发新的治疗方法，以改善伽马振荡和认知;和（iii）确定GAD 67表达的下调是否可引起Sz典型的皮质回路缺陷。 为了特异性敲低（KD）基因表达，我们建议使用RNA干扰（RNAi），建立在我们以前在睡眠领域使用这种技术的经验。我们将首先靶向谷氨酸脱羧酶（GAD 67），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动物模型报告了表达钙结合蛋白小清蛋白（PV）的皮质中间神经元中GAD 67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阳性神经元中表达Cre重组酶（Cre）的小鼠（PV-Cre小鼠）的前边缘皮层中注射具有Cre重组酶依赖性表达靶向GAD 67的小发夹RNA（shRNA-GAD 67）的病毒载体（腺相关病毒; AAV）。因此，shRNA的重组和表达将仅发生在该皮质区域的PV-Pos神经元中，导致GAD 67的细胞和区域特异性KD。对照载体将表达不靶向任何已知小鼠基因的乱序shRNA（shRNA-CTRL）。病毒载体还将以Cre依赖性方式表达增强的绿色荧光蛋白（GFP）作为病毒表达的标志物。通过PV的荧光免疫组织化学染色和GFP/PV表达的比较（即GFP是否仅位于PV神经元中）来确定病毒表达的选择性。通过以下方法评估靶基因（GAD 67）KD的有效性：（i）使用激光捕获显微切割（LCM）对从转染的PV-Pos神经元收获的mRNA进行定量聚合酶链反应（PCR）;（ii）GAD 67蛋白的免疫组织化学染色和染色强度的亮度测量。将通过LCM/PCR（GAD 65和PV）以及免疫组织化学（PV）确认KD的选择性。我们将使用靶向GAD 67和PV-Cre小鼠的RNAi来测试PV-Pos神经元中GAD 67的特异性减少是否是导致皮质回路缺陷的关键事件，其导致由于抑制性突触传递改变而导致的体外γ（30-80 Hz）振荡减少。 精神分裂症是一种严重的精神疾病，对个人，家庭和医疗保健费用产生负面影响。这种疾病的认知缺陷被认为是由于有缺陷的皮层电路，反映为异常的伽马振荡在脑电图记录。本申请旨在建立一种新的、广泛适用的方法来了解动物模型中精神分裂症的典型皮质伽马异常。

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. PUBLIC HEALTH RELEVANCE: Schizophrenia is a major mental illness with negative impact on the individual, their family and on healthcare costs. Cognitive deficits in this disease are thought to be due to defective cortical circuitry, reflected as abnormal gamma oscillations in electroencephalographic recordings. This application aims to establish a new, broadly applicable method to understand cortical gamma abnormalities typical of schizophrenia in an animal model.