The role of gephyrin translation in inhibitory synaptic plasticity

项目总结 突触可塑性，神经元连接强度的活性依赖的变化，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经历双向 突触可塑性调节神经元兴奋性，塑造神经回路，协调兴奋平衡 和抑制(E/I平衡)。GABA能突触可塑性是维持E/I平衡的关键，其 功能障碍与癫痫、精神分裂症和自闭症谱系障碍等病理疾病有关。 然而，抑制性突触可塑性及其分子基础的研究还很少。一个至关重要的 抑制突触可塑性的调节剂是Gen，一种GABAA受体必需的突触支架蛋白 聚集和抑制突触传递。在一种形式的抑制性突触可塑性中，抑制性长期 增强作用(ILTP)，在树突状抑制性突触上迅速增加Gen簇以加强 刺激后20分钟，GABA能突触仅见于树突。ILTP的早期机制是 独立于翻译，但关于iLTP持久性和关键蛋白如何翻译的了解要少得多 维护iLTP。回答这些问题将提供对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。在这项提案中，我将解决这一问题 知识鸿沟--侧重于地理卟啉翻译在维持iLTP中的作用，更具体地说 它在哪里发生以及如何调节以实现抑制性突触的精确和持久的增强 关系。我假设局部地理卟啉翻译只支持树突中的iLTP，并且 Gen的翻译调节影响Ge-Ph突触聚集和抑制性突触 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来 确定iLTP后树突状细胞中的Geporrin转录本的定位和翻译(目标1)。MiR153是一种 短的、非编码的转录本被认为是地理卟啉翻译的关键调节因子，并与学习有关， 记忆和认知。通过调节其在神经元中的表达水平，并结合使用 免疫细胞化学和电生理学，我将确定miR153功能对吉普林的影响 ILTP期间的聚集和抑制性突触强度(目标2)。总而言之，这种方法将阐明 地理卟啉翻译的定位和调节影响抑制性突触的特异性和持续性变化 ILTP过程中的强度，并最终揭示驱动抑制突触可塑性以维持E/I的机制 平衡以保证正常的神经功能。

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.