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，促进药物和治疗的发展，以改善由早期异常视觉经验，创伤或疾病引起的终身视觉功能障碍。本研究利用啮齿类动物的视觉通路，重点研究了膜相关鸟苷酸激酶（MAGUKS）、SAP102和PSD-95。这些突触支架包含谷氨酸神经递质受体（NRs）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和NRs依赖的长期突触增强和抑制（LTP & LTD）期间发出信号的许多分子。我们假设PSD-95和SAP102在突触后密度（PSD）处组织独立的NR信号模块，从SAP102到PSD-95在视觉系统PSD的主要转换发生在睁眼时，PSD-95结合的NR驱动突触变化，这也需要突触外SAP102结合的NR。我们有三个具体目的：1)通过慢病毒介导的PSD-95或SAP102或sirna的过表达来敲低这些支架的表达，验证SAP102和PSD-95在新生儿和睁眼后早期突触的建立和分类中分别具有不同的作用。在上丘视觉层切片中进行全细胞膜片夹紧将确定这些操作对突触电流变化以及电诱发LTP和LTD的影响。2)为了验证SAP102和PSD-95在视觉发育过程中与NRs相邻的信号模块不同的假设，我们将使用液相色谱法和液相色谱法检查与每个MAGUKS相关的蛋白质的分阶段的突触后密度分数和免疫沉淀3)我们有证据表明NR受体附近的支架和信号模块是由NR亚基与SAP102和PSD-95之间的特定关联决定的。因此，我们将使用分子工程NR受体亚基，以容纳SAP102或PSD-95附近具有不同离子孔特征的NR。在视觉皮层培养中，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结构引入缺乏正常NR亚基之一的小鼠品系（NR2A-/-小鼠）的上丘（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。我们还将根据NR2A-/-神经元携带工程亚基的预测，确定正常动物睁眼后正常的、高度定型的谷氨酸电流变化是否维持或改变。每100名儿童中就有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连接的细胞和分子机制的研究将促进这种广泛损害的治疗发展。我们对视觉发育中功能连接机制的研究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.