Developmental Regulation of Glutamate Receptor Function

描述（由申请人提供）：我们的目标是了解视觉通路中活动依赖性突触发育和可塑性的细胞和分子机制，并促进药物和治疗的开发，以改善早期异常视觉体验、创伤或疾病导致的终身视觉功能障碍。本研究使用啮齿动物的视觉通路，重点是膜相关鸟苷酸激酶（MAGUKS），SAP 102和PSD-95。这些突触支架容纳谷氨酸神经递质受体（NR）的N-甲基-D-天冬氨酸亚型以及它们在视觉驱动的突触发生期间和NR依赖性长时程突触增强和抑制（LTP & LTD）期间通过其发出信号的许多分子。我们假设PSD-95和SAP 102在突触后密度（PSD）处组织单独的NR信号传导模块，在视觉系统PSD处从SAP 102到PSD-95的主要转换发生在睁眼时，并且PSD-95结合的NR驱动突触变化，这也需要突触外SAP 102结合的NR。我们有三个具体目标：1）使用慢病毒介导的PSD-95或SAP 102的过表达或siRNA敲低这些支架的表达来测试SAP 102和PSD-95分别在新生儿和早期睁眼后时期期间在建立和分选突触中具有不同作用的假设。上级丘视层切片的全细胞膜片钳将确定这些操作对突触电流变化和电诱发的LTP和LTD的影响。2）为了验证SAP 102和PSD-95在视觉发育过程中在NR附近保持不同信号模块的假设，我们将检查一系列阶段性的后视觉发育过程。与布罗德研究所蛋白质组学平台主任史蒂文卡尔博士合作，使用液相色谱和串联质谱法，对与每个MAGUKS相关的蛋白质的突触密度分数和免疫沉淀物进行了分析。3)我们有证据表明，NR受体附近的支架和信号模块是由NR亚基与SAP 102和PSD-95之间的特异性关联决定的。因此，将使用分子工程NR受体亚单位，其应保持具有不同离子孔特征的NR邻近SAP 102或PSD-95。在视皮层培养中，我们将描述工程受体的结合特性和靶向。随后，我们将使用慢病毒载体将这些构建体引入缺乏正常NR亚基之一的小鼠品系（NR 2A-/-小鼠）的上级丘（sSC）的浅表视觉层。我们将确定嵌合亚基是否改变或消除我们在这些小鼠睁眼后的sSC中发现的LTP缺陷。我们还将确定正常的、高度定型的谷氨酸电流是否发生变化，我们在正常动物中根据我们对携带工程亚基的NR 2A-/-神经元的预测，在睁眼后对其进行了表征。100名儿童中有2至3名受到一种称为弱视的疾病的损害，这种疾病是指两只眼睛和大脑之间的输入不平衡，一只眼睛失去视觉辨别力。我们对早期视力加强眼睛和大脑之间适当联系的细胞和分子机制的研究将促进这种广泛损害的治疗方法的发展。我们的工作确定视觉发育中的功能连接机制，也将有助于将视网膜功能障碍的视觉修复术整合到产生视觉的大脑回路中。

DESCRIPTION (provided by applicant): Our goal is to understand the cell and molecular mechanisms of activity-dependent synapse development and plasticity in the visual pathway and facilitate the development of pharmaceuticals and therapies to ameliorate life-long visual dysfunctions resulting from early abnormal visual experience, trauma or disease. This research uses rodent visual pathways and focuses on the membrane associated guanylate kinases (MAGUKS), SAP102 and PSD-95. These synaptic scaffolds hold N-methyl-D-aspartate subtypes of glutamate neurotransmitter receptors (NRs) and the many molecules they signal through during visually driven synaptogenesis and during NR-dependent long-term synaptic potentiation and depression (LTP & LTD). We postulate that PSD-95 and SAP102 organize separate NR signaling modules at the post-synaptic density (PSD), that the major switch from SAP102 to PSD-95 at visual system PSD's occurs at eye opening, and that PSD-95 bound NRs drive synaptic change that also requires extrasynaptic SAP102-bound NRs. We have 3 specific aims: 1) To test the hypothesis that SAP102 and PSD-95 have distinct roles in establishing and sorting synapses during the neonate and early post-eye-opening period respectively using lentiviral mediated over- expression of PSD-95 or SAP102 or siRNAs to knock down the expression of these scaffolds. Whole-cell patch-clamping in slices from the visual layers of the superior colliculus will determine the effect of these manipulations on synaptic current changes and on electrically evoked LTP and LTD. 2) To test the hypothesis that SAP102 and PSD-95 hold different signaling modules adjacent to NRs during visual development we will examine a staged series of post-synaptic density fractions and immunoprecipitates of proteins associated with each of the MAGUKS using liquid chromatography and tandem mass spectrometry in collaboration with Dr. Steven Carr, Director the Proteomics Platform at the Broad Institute. 3) We have evidence that the scaffold and signaling modules held near the NR receptor is determined by a specific association between NR subunits and either SAP102 and PSD-95. Therefore, will use molecularly engineered NR receptor subunits that should hold NRs with different ion pore characteristics adjacent to either SAP102 or PSD-95. In visual cortex cultures we will characterize the engineered receptors' binding characteristics and targeting. Subsequently, we will use lentiviral vectors to introduce these constructs into the superficial visual layers of the superior colliculus (sSC) in a mouse strain lacking one of the normal NR subunits (the NR2A-/- mouse). We will determine if the chimeric subunits alter or eliminate a deficit in LTP that we have found in the sSC of these mice after eye opening. We will also determine whether the normal, highly stereotyped glutamate current changes, we have characterized in normal animals after eye-opening are maintained or modified according to our predictions in NR2A-/- neurons carrying the engineered subunits. PUBLIC HEALTH RELEVANCE Two to three in 100 children are impaired by a condition known as amblyopia in which inputs between the two eyes and the brain are imbalanced and one eye loses visual discrimination. Our studies of the cell and molecular mechanisms through which early vision strengthens appropriate connections between the eyes and the brain will facilitate the development of treatments for this wide-spread impairment. Our work identifying mechanisms of functional connectivity in visual development will also help in integrating visual prosthetics for retinal dysfunction into the brain circuits that produce vision.