The role of gephyrin translation in inhibitory synaptic plasticity

gephyrin 翻译在抑制性突触可塑性中的作用

• 批准号: 10629239
• 负责人: Theresa Marie Welle
• 金额: $ 3.72万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10629239
• 关键词: Affect; Automobile Driving; Biological Assay; Chemosensitization; Cognition; Data; Dendrites; Electrophysiology (science); Epilepsy; Equilibrium; Excitatory Synapse; Functional disorder; Genes; Glutamates; Goals; Image; In Situ; Inhibitory Synapse; Knowledge; Label; Learning; Ligation; Long-Term Depression; Long-Term Potentiation; Maintenance; Measures; Mediating; Memory; Messenger RNA; MicroRNAs; Molecular; N-Methyl-D-Aspartate Receptors; Neurons; Neurophysiology - biologic function; Pathology; Play; Protein Biosynthesis; Proteins; Proteome; Puromycin; Regulation; Research; Role; Scaffolding Protein; Schizophrenia; Shapes; Site; Synapses; Synaptic Transmission; Synaptic plasticity; Testing; Transcript; Translating; Translational Regulation; Translations; Untranslated RNA; Visualization; autism spectrum disorder; confocal imaging; experimental study; gephyrin; immunocytochemistry; insight; mRNA Translation; neural; neural circuit; neuronal cell body; neuronal excitability; overexpression; postsynaptic; receptor; scaffold; single molecule; synaptic function; synaptic inhibition; translational impact

PROJECT SUMMARY Synaptic plasticity, the activity-dependent alteration in the strength of neuronal connections, forms the molecular basis of learning, memory, and cognition. Both excitatory and inhibitory connections undergo bidirectional synaptic plasticity to tune neuronal excitability, sculpt neural circuits, and coordinate the balance of excitation and inhibition (E/I balance). GABAergic synaptic plasticity is crucial for maintaining E/I balance, and its dysfunction is implicated in pathologies such as epilepsy, schizophrenia, and autism spectrum disorders. However, inhibitory synaptic plasticity and its molecular underpinnings are critically understudied. A crucial regulator of inhibitory synaptic plasticity is gephyrin, a synaptic scaffolding protein essential for GABAA receptor clustering and inhibitory synaptic transmission. In one form of inhibitory synaptic plasticity, inhibitory long-term potentiation (iLTP), gephyrin clustering increases rapidly at dendritic inhibitory synapses to strengthen GABAergic synapses exclusively in dendrites by 20 min post-stimulation. Early mechanisms of iLTP are independent of translation, but much less is known about iLTP persistence and how translation of key proteins maintains iLTP. Answering these questions will provide insights into mechanisms of synaptic plasticity and how changes in GABAergic synaptic strength shape neural circuits long-term. In this proposal, I will address this knowledge gap by focusing on the role of gephyrin translation for maintaining iLTP and more specifically where it occurs and how it is regulated to enact precise and persistent potentiation of inhibitory synaptic connections. I hypothesize that local gephyrin translation supports iLTP exclusively in the dendrites and that translational regulation of gephyrin influences gephyrin synaptic clustering and inhibitory synaptic strength. I will utilize fluorescent in situ labeling of mRNA and visualization of actively translating proteins to determine localization and translation of gephyrin transcripts in dendrites following iLTP (Aim 1). miR153 is a short, non-coding transcript identified as a key regulator of gephyrin translation and implicated in learning, memory, and cognition. By modulating its expression levels in neurons and employing a combination of immunocytochemistry and electrophysiology, I will determine the impact of miR153 function on gephyrin clustering and inhibitory synaptic strength during iLTP (Aim 2). Together, this approach will elucidate how the localization and regulation of gephyrin translation impact specific and lasting changes to inhibitory synaptic strength during iLTP and ultimately reveal the mechanisms driving inhibitory synaptic plasticity to maintain E/I balance for proper neural function.

项目概要 突触可塑性，即神经元连接强度的活动依赖性改变，形成了分子 学习、记忆和认知的基础。兴奋性和抑制性连接都是双向的 突触可塑性调节神经元兴奋性、塑造神经回路并协调兴奋平衡 和抑制（E/I 平衡）。 GABA 突触可塑性对于维持 E/I 平衡至关重要，其 功能障碍与癫痫、精神分裂症和自闭症谱系障碍等疾病有关。 然而，抑制性突触可塑性及其分子基础尚未得到充分研究。一个至关重要的 抑制性突触可塑性的调节剂是 gephyrin，一种 GABAA 受体必需的突触支架蛋白 聚类和抑制性突触传递。在抑制性突触可塑性的一种形式中，抑制性长期 增强作用（iLTP），gephyrin 簇在树突抑制性突触处迅速增加以加强 刺激后 20 分钟，GABA 能突触仅存在于树突中。 iLTP 的早期机制是 独立于翻译，但对于 iLTP 持久性以及关键蛋白的翻译方式知之甚少 维持 iLTP。回答这些问题将有助于深入了解突触可塑性的机制以及如何 GABA能突触强度的变化长期塑造神经回路。在这个提案中，我将解决这个问题 通过关注 gephyrin 翻译在维持 iLTP 中的作用，更具体地说，来弥补知识差距 它发生在哪里以及如何调节它以实现抑制性突触的精确和持久的增强 连接。我假设局部 gephyrin 翻译仅在树突中支持 iLTP，并且 gephyrin 的翻译调节影响 gephyrin 突触聚类和抑制性突触 力量。我将利用 mRNA 的荧光原位标记和主动翻译蛋白质的可视化 确定 iLTP 后树突中 gephyrin 转录本的定位和翻译（目标 1）。 miR153 是一个 短的非编码转录物被确定为 gephyrin 翻译的关键调节因子并与学习有关， 记忆、认知。通过调节其在神经元中的表达水平并采用以下组合 免疫细胞化学和电生理学，我将确定miR153功能对gephyrin的影响 iLTP 期间的聚类和抑制性突触强度（目标 2）。总之，这种方法将阐明如何 gephyrin翻译的定位和调节影响抑制性突触的特定和持久的变化 iLTP 期间的强度，并最终揭示驱动抑制性突触可塑性以维持 E/I 的机制 平衡适当的神经功能。