Consequences of In Vivo Neocortical GAD67 Downregulation

描述(由申请人提供)：谷氨酸脱羧酶67 kDa(GAD67)在死后前额叶皮质的转录减少是最有力和一致的发现，在不同的精神分裂症受试者队列中。这种关键的、大脑特异的GABA合成酶在人类大脑皮层不同的神经元间群体中显示出表达缺陷。因此，我们预计，在这些不同的神经元间亚型中，GAD67在皮质中的定向下调将1)模仿人类在精神分裂症中的尸检结果；2)对皮质功能有不同的影响；3)更好地理解皮质抑制。在这个提议中，我们假设在体内SST、NPY、CCK和PARV-含PARV的皮质中间神经元中GAD67的下调将导致不同的解剖学、神经化学、分子和行为变化，这些变化将与人类精神分裂症的共同特征相同。为了验证我们的中心假设，我们提出了四个具体目标，重点是四个转基因小鼠品系的产生和特征。在目标1中，我们将在SST、NPY、CCK和PARV启动子的控制下，产生四种不同的GFP标记的GAD67亚型小鼠。我们将使用包含SST、NPY、CCK和PARV启动子/增强子区域的小鼠细菌人工染色体(BAC)来实现这一点，该区域经过修饰后既能表达GFP，又能表达GAD67特异的内含子编码的microRNA(MiRNA)。在目标2中，我们将对新产生的转基因小鼠品系进行解剖学/神经化学评估。我们将通过大体解剖测量、普通显微解剖和神经化学方法来评估每一种小鼠品系。目标3将侧重于对四个GAD67基因敲除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)外，我们还将评估脉冲前抑制、空间工作记忆和社会行为。目的4将建立GAD67 KO小鼠之间共享的新皮质基因表达变化和精神分裂症患者PFC的表达变化。在特定的神经元间亚群中GAD67转录本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脑中的表达变化。为了测试这一点，对于每一种表现出行为/解剖学表型的击倒动物，我们将研究在人类精神分裂症中已知改变的基因(例如SYN2、RGS4、MOG、SST、NPY、GAD67、HSP70、AMPA等)的表达。第一个在PARV+神经元间亚群中GAD67下调的转基因小鼠已经成功产生，并等待鉴定。公共卫生相关性：新皮质神经元间下调GAD67的表达是精神分裂症最一贯的重复发现之一。拟议的研究将集中在体内特定皮质神经元间亚群GAD67下调的后果，并将它们与精神分裂症受试者大脑中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.