Mechanisms of cell adhesion molecule LRRTM2 in basal and potentiated synaptic signaling

细胞粘附分子LRRTM2在基础和增强突触信号传导中的机制

• 批准号: 10753395
• 负责人: Stephanie Lynn Pollitt
• 金额: $ 7.18万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10753395
• 关键词: 3-Dimensional; AMPA Receptors; Acute; Architecture; Area; Binding; Biology; Brain; Bypass; CRISPR/Cas technology; Cell Adhesion Molecules; Cell surface; Central Nervous System; Chronic; Cognition; Collaborations; Complex; Confocal Microscopy; DLG4 gene; Data; Dendritic Spines; Electrophysiology (science); Engineering; Evaluation; Family; Foundations; Genetic; Genomics; Glutamates; Goals; Growth; Image; Individual; Integral Membrane Protein; Knock-out; Learning; Leucine-Rich Repeat; Long-Term Potentiation; Measures; Mediating; Memory; Methods; Microscopy; Modeling; Molecular; Mutate; Mutation; Neurons; Neurotransmitter Receptor; Peptide Hydrolases; Play; Positioning Attribute; Process; Proteins; Regulation; Role; Series; Signal Transduction; Site; Structure; Synapses; Synaptic Membranes; Synaptic Transmission; Synaptic plasticity; Testing; Time; Transcriptional Regulation; Vertebral column; Visualization; Work; career; density; experimental study; extracellular; fascinate; live cell imaging; loss of function; nano; nanocluster; nanoscale; neural circuit; neurotransmission; neurotransmitter release; novel; novel strategies; operation; postsynaptic; presynaptic; prevent; recruit; scaffold; single molecule; superresolution microscopy; synaptic function; temporal measurement; tool; trafficking; transmission process

Cognition, learning, and memory all rely on precise neurotransmission and plasticity at glutamatergic synapses in the brain. These processes require glutamate-responsive AMPA receptors (AMPARs), which mediate fast synaptic transmission in the central nervous system. During many forms of plasticity, including Long-Term Potentiation (LTP), synaptic AMPAR levels are altered to modulate synaptic strength and regulate neural circuit function. However, recent studies have shown that synaptic strength is not determined simply by the number of AMPARs at the synapse, but also by their nanoorganization within it. AMPARs are enriched subsynaptically in high-density nanoclusters that are often aligned with presynaptic neurotransmitter release sites, and this molecular architecture impacts both basal synaptic transmission and plasticity. Despite long acknowledgement that AMPAR trafficking is critical for synaptic strength and plasticity, the mechanisms controlling their positioning and even their retention at synapses remain unclear. The overarching goal of this proposal is to investigate mechanisms by which AMPARs are regulated at both synaptic and subsynaptic scales. Leucine-Rich Repeat Transmembrane protein 2 (LRRTM2) has emerged as potential candidate for these functions, with essential roles in multiple synaptic processes including AMPAR-mediated transmission and LTP. Critically, by using an acute method rather than knockout to disrupt LRRTM2, our lab recently found that LRRTM2 regulates both the abundance and nanopositioning of AMPARs after long and short-term manipulations, respectively. As a transmembrane protein located in the postsynaptic density, LRRTM2 forms multiple protein interactions, including with PSD-95, presynaptic Neurexins, and directly with AMPARs. These interactions suggest fascinating hypotheses about how LRRTM2 might retain AMPARs and position them specifically within the synapse, both basally and during AMPAR recruitment post-LTP stimulation. To visualize and control endogenous LRRTM2, I have successfully adapted a new CRISPR-based tool to genetically replace the LRRTM2 protein with a tagged, acutely cleavable, and/or mutated version. I will use this approach to perform the first evaluation of LRRTM2 distribution and plasticity-dependent enrichment in neurons. With this tool, I will then determine the mechanisms of LRRTM2-driven AMPAR alignment, stability, and enrichment. I will engineer selective mutations of the genomic sequence and use super resolution microscopy and single-molecule tracking to delineate how LRRTM2 interactions with its partners specifically contribute to AMPAR trafficking. Finally, I will examine how LRRTM2 supports LTP. Our surprising preliminary data suggests an unexpected role for LRRTM2 in LTP-dependent spine growth not explained by current models of LRRTM2 function. I will investigate these mechanisms using a combination of live cell imaging and electrophysiology. These tools and approaches establish new paradigms for understanding the roles of cell adhesion molecules at mature synapses, and will provide a firm foundation for my independent career.

认知、学习和记忆都依赖于谷氨酸能的精确神经传递和可塑性 大脑中的突触。这些过程需要谷氨酸响应性 AMPA 受体 (AMPAR)， 介导中枢神经系统的快速突触传递。在许多形式的可塑性过程中，包括 长时程增强 (LTP)，改变突触 AMPAR 水平以调节突触强度并调节 神经回路功能。然而，最近的研究表明，突触强度并不仅仅取决于 突触处 AMPAR 的数量，还取决于突触内的纳米组织。 AMPAR 丰富 突触下的高密度纳米团簇通常与突触前神经递质释放一致 位点，这种分子结构影响基础突触传递和可塑性。尽管长 承认 AMPAR 运输对于突触强度和可塑性至关重要，其机制 控制它们的位置，甚至它们在突触上的保留仍不清楚。本次活动的总体目标是 该提案的目的是研究 AMPAR 在突触和突触亚尺度上的调节机制。 富含亮氨酸的重复跨膜蛋白 2 (LRRTM2) 已成为这些药物的潜在候选者 功能，在多个突触过程中发挥重要作用，包括 AMPAR 介导的传递和 LTP。 重要的是，通过使用急性方法而不是敲除来破坏 LRRTM2，我们的实验室最近发现 LRRTM2 在长期和短期操作后调节 AMPAR 的丰度和纳米定位， 分别。作为一种位于突触后密度的跨膜蛋白，LRRTM2形成多种蛋白 相互作用，包括与 PSD-95、突触前神经素以及直接与 AMPAR 的相互作用。这些互动 提出关于 LRRTM2 如何保留 AMPAR 并将它们专门定位在 突触，无论是在基底还是在 LTP 刺激后的 AMPAR 募集过程中。 为了可视化和控制内源性 LRRTM2，我成功地采用了一种新的基于 CRISPR 的工具 用标记的、可急性切割的和/或突变的版本从基因上替换LRRTM2蛋白。我会用 这种方法对 LRRTM2 分布和可塑性依赖性富集进行首次评估 神经元。有了这个工具，我将确定 LRRTM2 驱动的 AMPAR 对齐、稳定性、 和充实。我将设计基因组序列的选择性突变并使用超分辨率 显微镜和单分子追踪来描述 LRRTM2 如何与其伙伴相互作用 有助于 AMPAR 贩运。最后，我将研究 LRRTM2 如何支持 LTP。我们令人惊讶的初步 数据表明 LRRTM2 在 LTP 依赖性脊柱生长中发挥着意想不到的作用，目前的模型无法解释 LRRTM2 功能。我将结合活细胞成像和 电生理学。这些工具和方法为理解细胞的作用建立了新的范例 成熟突触的粘附分子，将为我的独立职业生涯提供坚实的基础。