Consequences of In Vivo Neocortical GAD67 Downregulation

描述（由申请人提供）：死后前额叶皮层中谷氨酸脱羧酶67kDa （GAD67）转录物减少是在精神分裂症患者不同队列中最强大和一致的重复发现。这种关键的，脑特异性GABA合成酶在人类皮层不同的神经元间群体中表现出表达缺陷。因此，我们预计在这些不同的神经元间亚表型中，GAD67的皮质定向下调将1)模仿人类死后精神分裂症的发现2)对皮质功能有不同的影响3)导致对皮质抑制的更好理解。在本研究中，我们假设体内含有SST、NPY、CCK和parv的皮质中间神经元中GAD67的下调会导致不同的解剖、神经化学、分子和行为变化，这些变化与人类精神分裂症具有共同的特征。为了验证我们的中心假设，我们提出了4个具体目标，这些目标将集中在四种转基因小鼠系的产生和表征上。在Aim 1中，我们将在SST、NPY、CCK和PARV启动子的控制下，产生4只不同的gfp标记的GAD67亚型小鼠。我们将使用含有SST、NPY、CCK和PARV启动子/增强子区域的小鼠细菌人工染色体（BAC）来实现这一目标，这些区域经过修饰后可表达GFP和GAD67特异性内含子编码的microRNA （miRNA）。在目标2中，我们将对新产生的转基因小鼠株进行解剖/神经化学评估。我们将通过大体解剖测量，一般显微解剖和神经化学方法评估每个小鼠系。目的3将重点关注四种GAD67敲低小鼠品系的行为评估。除了小鼠的基本行为特征（神经学检查和感觉运动评估），我们将评估脉冲前抑制，空间工作记忆和社会行为。Aim 4将建立GAD67 KO小鼠和精神分裂症PFC中共同的新皮质基因表达变化。GAD67转录本在特定神经元亚群中的下调可能会导致中间神经元和锥体细胞的表达变化，我们相信其中一些表达变化将模仿精神分裂症患者死后大脑中的表达变化。为了验证这一点，对于每一只表现出行为/解剖表型的敲低动物，我们将检测已知在人类精神分裂症中被改变的基因的表达（例如SYN2， RGS4， MOG， SST， NPY, GAD67, HSP70， AMPA等）。第一个在PARV+神经元间亚群中GAD67下调的转基因小鼠系已经成功产生，并等待鉴定。公共卫生相关性：新皮质神经元间GAD67表达下调是精神分裂症最一致的重复发现之一。拟议的研究将集中于GAD67在特定皮层间神经元亚群中的体内下调的后果，并将其与精神分裂症受试者大脑的死后发现联系起来。

DESCRIPTION (provided by applicant): Glutamic acid decarboxylase 67kDa (GAD67) transcript reduction in the postmortem prefrontal cortex is the most robust and consistently replicated finding across different cohorts of subjects with schizophrenia. This critical, brain-specific GABA synthesis enzyme is shows expression deficit in distinct interneuronal populations of the human cortex. As a result, we expect that a directed cortical downregulation of GAD67 in these distinct interneuronal subphenotypes will 1) mimic the human postmortem findings in schizophrenia 2) have distinct consequences on cortical functioning and 3) lead to a better understanding of cortical inhibition. In this proposal, we hypothesize that in vivo downregulation of GAD67 in SST, NPY, CCK and PARV-containing cortical interneurons will result in distinct anatomical, neurochemical, molecular and behavioral changes that will share common features with human schizophrenia. To test our central hypothesis we propose 4 specific Aims which will focus on the generation and characterization of four transgenic mouse lines. In Aim 1 we will generate four distinct, GFP-labeled, GAD67 hypomorphic mice under the control of SST, NPY, CCK and PARV promoters. We will achieve this using a mouse bacterial artificial chromosome (BAC) containing SST, NPY, CCK and PARV promoter/enhancer regions modified to express both GFP and an intron-encoded microRNA (miRNA) specific to GAD67. In Aim 2 we will perform an anatomical/neurochemical assessment of the newly generated transgenic mice strains. We will assess each mouse line by gross anatomical measurements, general microanatomy and neurochemical methods. Aim 3 will focus on behavioral assessment of the four GAD67 knockdown mice strains. Beyond the basic behavioral characterization of the mice (neurological exam and sensorymotor assessments) we will assess prepulse inhibition, spatial working memory and social behaviors. Aim 4 will establish the neocortical gene expression changes that are shared between GAD67 KO mice and expression alterations in the PFC of schizophrenia. Downregulation of GAD67 transcripts in specific interneuronal subpopulations will likely lead to expression changes in both interneurons and pyramidal cells, and we believe that some of these expression changes will mimic the expression changes seen in human postmortem brain of subjects with schizophrenia. To test this, for each of the knockdown animals showing a behavioral/anatomical phenotype, we will the expression of genes known to be altered in human schizophrenia (e.g. SYN2, RGS4, MOG, SST, NPY, GAD67, HSP70, AMPA etc). The first transgenic mouse line, with GAD67 downregulation in the PARV+ interneuronal subpopulation has been successfully generated and awaits characterization. PUBLIC HEALTH RELEVANCE: Neocortical interneuronal downregulation of GAD67 expression is one of the most consistently replicated findings of schizophrenia. The proposed studies will focus on the consequences of in vivo downregulation of GAD67 in specific cortical interneuronal subpopulations, and relate them to the postmortem findings in the brains of subjects with schizophrenia.