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

使用细胞特异性 RNAi 敲低 GAD67 来模拟精神分裂症 γ 缺陷

• 批准号: 8306722
• 负责人: Ritchie Edward Brown
• 金额: $ 16.09万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-25 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8306722
• 关键词: Agonist; Animal Model; Antibodies; Area; Autopsy; Axon; Brain; Calcium-Binding Proteins; Cells; Chloride Ion; Chlorides; Clinical; Cognition; Cognitive deficits; Custom; Detection; Disease; Down-Regulation; Effectiveness; Enzymes; Event; Family; Frequencies; Gene Expression; Gene Proteins; Gene Targeting; Genes; Genetic Recombination; Glutamate Decarboxylase; Glutamate Receptor; Goals; Green Fluorescent Proteins; Harvest; Health Care Costs; Homologous Gene; Human; Immunohistochemistry; In Vitro; Individual; Injection of therapeutic agent; Interneurons; Lead; Measures; Mental disorders; Messenger RNA; Methods; Modeling; Molecular; Mus; National Institute of Mental Health; Neurons; Neurotransmitters; Operative Surgical Procedures; Parvalbumins; Polymerase Chain Reaction; Prefrontal Cortex; Proteins; RNA Interference; Reporting; Schizophrenia; Sleep; Slice; Specificity; Staining method; Stains; Synaptic Transmission; System; Techniques; Testing; Time; Up-Regulation; Viral; Viral Markers; Viral Vector; adeno-associated viral vector; cell type; data modeling; design; enhanced green fluorescent protein; executive function; experience; gamma-Aminobutyric Acid; gene function; hippocampal pyramidal neuron; improved; kainate; knockout gene; laser capture microdissection; luminance; neocortical; nervous system disorder; neuronal cell body; novel; novel therapeutic intervention; patch clamp; postsynaptic; receptor; recombinase; small hairpin RNA; vector; vector control

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.

描述（由申请人提供）：了解精神分裂症 (Sz) 的病因是 NIMH 重点关注的领域。该 R21 提交申请需要两年的支持，以开发一种新颖的、广泛适用的方法来了解 Sz 和其他精神/神经疾病的皮质回路异常。该应用的总体目标是：（i）建立一种基因、细胞类型和区域特异性方法来模拟动物模型中 Sz 的分子尸检结果； (ii) 使用此方法来测试此类基因 GAD67(Gad1) 的细胞特异性敲低是否会导致 Sz 典型的伽玛振荡和皮质电路异常。如果成功，该项目将： (i) 验证一种新方法来研究与涉及皮质电路缺陷的 Sz 等疾病有关的基因的功能； (ii) 生成具有良好结构有效性的新 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； (iii) 确定 GAD67 表达的下调是否会导致 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)，这是基于我们之前在睡眠领域使用该技术的经验。我们最初的目标是谷氨酸脱羧酶 (GAD67)，它是抑制性神经递质 GABA 的主要合成酶，因为 Sz 的临床尸检数据和动物模型报告表达钙结合蛋白小清蛋白 (PV) 的皮质中间神经元中的 GAD67 水平部分降低 (30-50%)。为了实现细胞类型和区域特异性，将在 PV 阳性神经元表达 Cre 重组酶 (Cre) 的小鼠（PV-Cre 小鼠）的前边缘皮质中注射具有 Cre 重组酶依赖性表达的靶向 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，从而导致 GAD67 的细胞和区域特异性 KD。对照载体将表达不针对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型绿色荧光蛋白（GFP），作为病毒表达的标记。病毒表达的选择性将通过 PV 的荧光免疫组织化学染色和 GFP/PV 表达的比较来确定，即 GFP 是否仅位于 PV 神经元中。靶基因 (GAD67) KD 的有效性将通过以下方式评估： (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 进行定量聚合酶链式反应 (PCR)； (ii) GAD67 蛋白的免疫组织化学染色和染色强度的亮度测量。 KD 的选择性将通过 GAD65 和 PV 的 LCM/PCR 以及 PV 的免疫组织化学来确认。我们将使用针对 GAD67 和 PV-Cre 小鼠的 RNAi 来测试 PV-Pos 神经元中 GAD67 的减少是否是导致皮质电路缺陷的关键事件，从而导致由于抑制性突触传递改变而导致体外伽玛（30-80 Hz）振荡减少。