Developmental Regulation of Glutamate Receptor Function

描述(申请人提供)：我们的目标是了解视觉通路中依赖活动的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，以改善因早期异常视觉体验、创伤或疾病而导致的终身视觉功能障碍。本研究利用啮齿动物的视觉通路，重点研究了膜相关鸟苷晚期蛋白激酶(MAGUKS)、SAP102和PSD-95。这些突触支架含有N-甲基-D-天冬氨酸亚型的谷氨酸神经递质受体(NRs)，以及它们在视觉驱动突触发生和NR依赖的长期突触增强和抑制(LTP&LTD)过程中传递信号的许多分子。我们推测，PSD-95和SAP102在突触后密度(PSD)组织独立的NR信号模块，视觉系统PSD从SAP102到PSD-95的主要转换发生在睁眼时，PSD-95结合的NRS驱动突触变化，这也需要突触外SAP102结合的NRS。我们有三个特定的目的：1)通过慢病毒介导的PSD-95或SAP102或siRNAs的过度表达来下调这些支架的表达，以检验SAP102和PSD-95在新生儿和睁眼后早期分别在建立和分类突触方面具有不同作用的假说。上丘视层全细胞膜片钳制将决定这些操作对突触电流变化和电诱发LTP和LTD的影响。2)为了检验SAP102和PSD-95在视觉发育过程中靠近NRs的不同信号模块的假设，我们将与布罗德研究所蛋白质组学平台主任Steven Carr博士合作，利用高效液相色谱和串联质谱仪检测一系列分阶段的突触后密度组分和与每个MAGUK相关的蛋白质的免疫沉淀。3)我们有证据表明，NR受体附近的支架和信号模块是由NR亚单位与SAP102和PSD-95之间的特定关联决定的。因此，将使用分子工程的NR受体亚基，这些亚基应该包含与SAP102或PSD-95相邻的具有不同离子孔特征的NRs。在视皮层培养中，我们将描述工程受体的结合特性和靶向性。随后，我们将使用慢病毒载体将这些构建物导入缺乏正常NR亚基之一的小鼠上丘(SSC)的浅视觉层(NR2a-/-小鼠)。我们将确定嵌合亚单位是否改变或消除了我们在睁开眼睛后在这些小鼠的SSC中发现的LTP缺陷。我们还将根据我们对携带工程亚基的NR2A-/-神经元的预测，确定我们在正常动物睁开眼睛后所表征的正常的、高度刻板的谷氨酸电流变化是否保持或修改。与公共卫生相关每100名儿童中有2至3人患有弱视，即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能力。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。我们的工作识别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假体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.