Glutamate Receptor Trafficking in Visual Development

视觉发育中的谷氨酸受体贩运

• 批准号: 6860982
• 负责人: Martha Na Constantine-Paton
• 金额: $ 16.3万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6860982
• 关键词: NMDA receptors; confocal scanning microscopy; developmental neurobiology; electrophysiology; eye movements; genetically modified animals; glutamate receptor; immunocytochemistry; laboratory mouse; myosins; neural plasticity; protein structure function; retinal ganglion; superior colliculus; synapses; synaptogenesis; visual cortex; visual feedback; visual fields; visual pathways; visual stimulus; voltage /patch clamp; western blottings

DESCRIPTION (provided by applicant): Abnormal activity-mediated developmental regulation of glutamatergic neurotransmission is implicated in amblyopia, disruption of stereopsis and changes in retinal function. Thus, an understanding of molecular bases of synaptic plasticity during visual development is central to the mission of the National Eye Institute. Data suggest that myosin VA (myo VA) moves membrane vesicles on actin and delivers glutamate receptors (GRs) to synapses. Our recent data indicate that this process is rapidly regulated by light-driven synaptic activity following eye-opening. We hope to establish the mouse mutant strain flailer as a model for mechanistic studies of myo VA in synaptogenesis and for visual activity-dependent trafficking of the NMDA receptor and its mature scaffolding complex, the postsynaptic density proteins PSD-95 and GKAP-95. Flailer mice express a fusion gene containing the promoter and first two exons of a brain-specific G protein plus the C-terminal of myo VA (Jones et al. 2000). The flailer protein appears to act as a dominant-negative myo VA in the central nervous system. Flailer mice have neural abnormalities similar to those of myo VA null mutants, but unlike the null mice homozygous flailer mice survive and breed normally. Functional changes caused by the flailer mutation have not yet been explored at synaptic levels. We propose to determine (l) if flailer protein and normal myo VA are present in retina, superficial visual, layers of the superior colliculus (sSC), and visual cortex (VC) of homozygous flailer mice, and (II) if flailer mice show activity-dependent transport of the PSD-95/GKAP NR scaffolding complex to visual synapses within 6 hours of eye-opening. We will perform quantitative immunoblotting of homogenates of these regions and of fractions enriched for dendritic or whole-lysate protein from VC and sSC using antibodies that distinguish normal and flailer myo VA, PSD-95, GKAP-130 and GKAP-95. If flailer protein is present in retina, we will (Ill) determine whether flailer and WT retina are similar in the distribution of PSD-95 after eye-opening and in ganglion cell responses to light. Retinal function will be analyzed by our collaborator Dr. Niang Tian at Yale University. He will use ERG, mutielectrode arrays, and whole-cell patch clamping to study retinal ganglion cell responses to light. We will perform quantitative confocal analyses of retinas processed for PSD-95 immunocytochemistry before and after eye-opening. Finally, (IV) we will determine, central visual pathway glutamate receptor function is normal in flailer mouse brain using whole-cell voltage recordings of glutamate receptor currents in slice preparations of the sSC.

描述（由申请方提供）：活动介导的视神经能神经传递发育调节异常与弱视、立体视觉破坏和视网膜功能变化有关。 因此，了解视觉发育过程中突触可塑性的分子基础是国家眼科研究所的使命的核心。 数据表明，肌球蛋白VA（肌球蛋白VA）移动肌动蛋白上的膜囊泡，并将谷氨酸受体（GR）传递到突触。 我们最近的数据表明，这一过程是迅速调节的光驱动的突触活动后，眼睛睁开。 我们希望建立小鼠突变株flailer作为一个模型的机制研究肌VA在突触和视觉活动依赖的贩运的NMDA受体及其成熟的支架复合物，突触后密度蛋白PSD-95和GKAP-95。 Flailer小鼠表达一种融合基因，该基因含有脑特异性G蛋白的启动子和前两个外显子加上myo VA的C末端（Jones et al. 2000）。Flailer蛋白似乎在中枢神经系统中充当显性负性肌VA。 Flailer小鼠具有类似于myo VA无效突变体的神经异常，但与无效小鼠不同，纯合子Flailer小鼠正常存活和繁殖。 由连枷突变引起的功能变化尚未在突触水平上进行探索。 我们建议确定（1）flailer蛋白和正常肌VA是否存在于纯合子flailer小鼠的视网膜、浅层视觉、上级丘（sSC）层和视觉皮层（VC）中，以及（II）flailer小鼠是否在睁眼后6小时内显示PSD-95/GKAP NR支架复合物向视觉突触的活性依赖性转运。我们将使用区分正常和连枷肌VA、PSD-95、GKAP-130和GKAP-95的抗体，对这些区域的匀浆和富集VC和sSC的树突状或全裂解物蛋白的组分进行定量免疫印迹。 如果flailer蛋白存在于视网膜中，我们将（III）确定flailer和WT视网膜在睁眼后PSD-95的分布和对光的神经节细胞反应中是否相似。 视网膜功能将由我们的合作者耶鲁大学的田娘博士进行分析。 他将使用ERG，多电极阵列和全细胞膜片钳来研究视网膜神经节细胞对光的反应。 我们将在睁眼前后对PSD-95免疫细胞化学处理的视网膜进行定量共聚焦分析。 最后，（四）我们将确定，中央视觉通路谷氨酸受体功能是正常的flailer小鼠大脑使用全细胞电压记录谷氨酸受体电流的切片制备的sSC。

项目成果

A Myosin Va mutant mouse with disruptions in glutamate synaptic development and mature plasticity in visual cortex.

• DOI: 10.1523/jneurosci.4585-12.2013
• 发表时间: 2013-05-08
• 期刊: The Journal of neuroscience : the official journal of the Society for Neuroscience
• 作者: Yoshii A;Zhao JP;Pandian S;van Zundert B;Constantine-Paton M
• 通讯作者: Constantine-Paton M

Deletion of VPS50 protein in mice brain impairs synaptic function and behavior.

小鼠大脑中 VPS50 蛋白的缺失会损害突触功能和行为。

• DOI: 10.1101/2023.07.04.547745
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Ahumada-Marchant,Constanza;Ancatén-Gonzalez,Carlos;Haensgen,Henny;Arancibia,Felipe;Brauer,Bastian;Droste,Rita;Horvitz,HRobert;Constantine-Paton,Martha;Arriagada,Gloria;Chávez,AndrésE;Bustos,FernandoJ
• 通讯作者: Bustos,FernandoJ

Removal of a partial genomic duplication restores synaptic transmission and behavior in the MyosinVA mutant mouse Flailer.

• DOI: 10.1186/s12915-023-01714-y
• 发表时间: 2023-11-14
• 期刊: BMC BIOLOGY
• 影响因子: 5.4
• 作者: Bustos, Fernando J;Pandian, Swarna;Haensgen, Henny;Zhao, Jian-Ping;Strouf, Haley;Heidenreich, Matthias;Swiech, Lukasz;Deverman, Benjamin E;Gradinaru, Viviana;Zhang, Feng;Constantine-Paton, Martha
• 通讯作者: Constantine-Paton, Martha