Developmental Regulation of Glutamate Receptor Function

谷氨酸受体功能的发育调节

• 批准号: 8268452
• 负责人: Martha Na Constantine-Paton
• 金额: $ 39.22万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-08 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8268452
• 关键词: Adolescent; Adult; Age; Amblyopia; Animals; Antibodies; Appearance; Binding; Brain; Cells; Characteristics; Chemosensitization; Child; Chimera organism; Code; Collaborations; Complex; Confusion; Cytoplasmic Tail; DNA Sequence; Development; Discrimination; Disease; Electric Stimulation; Engineering; Extracellular Domain; Eye; Functional disorder; Glutamate Receptor; Glutamates; Goals; Health; Hour; Impairment; Institutes; Ions; Kinetics; Label; Lateral Geniculate Body; Lentivirus Vector; Life; Liquid Chromatography; Long-Term Potentiation; Mass Spectrum Analysis; Mediating; Mental Depression; Molecular; Mouse Strains; Mus; N-Methylaspartate; Neurons; Neurotransmitter Receptor; Pharmacologic Substance; Prosthesis; Proteins; Proteomics; Rattus; Receptor Signaling; Regulation; Research; Retinal; Rodent; Role; Series; Signal Transduction; Signaling Molecule; Slice; Sorting - Cell Movement; Staging; Stereotyping; Synapses; Tail; Testing; Time; Tissues; Transmembrane Domain; Trauma; Vision; Visual; Visual Accommodation; Visual Acuity; Visual Cortex; Visual Pathways; Visual system structure; Work; density; experience; knock-down; lentiviral-mediated; membrane-associated guanylate kinase; neonate; patch clamp; postnatal; pup; receptor; receptor binding; receptor function; research study; scaffold; superior colliculus Corpora quadrigemina; synaptogenesis; tandem mass spectrometry; therapy development; vision development

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.

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，并促进药物和疗法的开发，以改善因早期异常视觉体验、创伤或疾病导致的终生视觉功能障碍。这项研究使用啮齿动物视觉通路，重点关注膜相关鸟苷酸激酶 (MAGUKS)、SAP102 和 PSD-95。这些突触支架含有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过程中以及 NR 依赖性长期突触增强和抑制 (LTP & LTD) 过程中通过的许多信号分子。我们假设 PSD-95 和 SAP102 在突触后密度 (PSD) 组织单独的 NR 信号模块，视觉系统 PSD 处从 SAP102 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 SAP102 结合的 NR。我们有 3 个具体目标：1) 分别使用慢病毒介导的 PSD-95 或 SAP102 或 siRNA 的过表达来敲低这些支架的表达，测试 SAP102 和 PSD-95 在新生儿和睁眼后早期期间分别在建立和分选突触方面具有不同作用的假设。对上丘视觉层切片进行全细胞膜片钳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) 为了检验 SAP102 和 PSD-95 在视觉发育过程中与 NR 相邻的不同信号模块的假设，我们将与博德研究所蛋白质组学平台主任 Steven Carr 博士合作，使用液相色谱和串联质谱检查一系列分阶段的突触后密度分数和与每个 MAGUKS 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 亚基与 SAP102 和 PSD-95 之间的特定关联决定的。因此，将使用分子工程化的 NR 受体亚基，该亚基应保持与 SAP102 或 PSD-95 相邻的具有不同离子孔特征的 NR。在视觉皮层培养中，我们将表征工程化受体的结合特征和靶向。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（NR2A-/- 小鼠）的上丘（sSC）浅表视觉层。我们将确定嵌合亚基是否会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眼后表征的正常、高度定型的谷氨酸电流变化是否得到维持或改变。公共健康相关性 每 100 名儿童中就有 2 到 3 名患有弱视疾病，这种情况下，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有助于开发针对这种广泛存在的损伤的治疗方法。我们的工作确定了视觉发育中的功能连接机制，也将有助于将治疗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。