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: 10605490
• 负责人: Stephanie Lynn Pollitt
• 金额: $ 6.76万
• 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/10605490
• 关键词: 3-Dimensional; AMPA Receptors; Acute; Architecture; Area; Binding; Biology; Brain; Bypass; CRISPR/Cas technology; Cell Adhesion Molecules; Cell surface; Chronic; Cognition; Collaborations; Complex; Confocal Microscopy; 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; Neuraxis; Neurons; Neurotransmitter Receptor; Peptide Hydrolases; Play; Positioning Attribute; Process; Proteins; Regulation; Resolution; Role; Series; Signal Transduction; Site; Structure; Synapses; Synaptic Membranes; Synaptic Transmission; Synaptic plasticity; Testing; Time; Transcriptional Regulation; Vertebral column; Work; base; 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; 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精确的神经传递和可塑性， 大脑中的突触这些过程需要谷氨酸反应性AMPA受体（AMPAR）， 介导中枢神经系统中的快速突触传递。在许多形式的可塑性，包括 长时程增强（LTP），突触AMPAR水平改变以调节突触强度和调节突触后神经元的活动。 神经回路功能然而，最近的研究表明，突触强度并不是简单地由 突触处AMPAR的数量，以及突触内AMPAR的纳米结构。 在突触下的高密度纳米簇中，通常与突触前神经递质释放对齐 位点，并且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， 控制它们的定位，甚至它们在突触上的保留仍然不清楚。这个项目的首要目标是 该研究的目的是研究AMPAR在突触和突触下尺度上的调节机制。 富含亮氨酸重复的跨膜蛋白2（LRRTM2）已经成为这些的潜在候选者。 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。 关键的是，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LRRTM2作为一种位于突触后致密区的跨膜蛋白，形成多种蛋白 这些相互作用包括与PSD-95、突触前神经毒素以及直接与AMPAR的相互作用。这些相互作用 提出了关于LRRTM2如何保留AMPAR并将其具体定位在 突触，无论是在基础和AMPAR招募后LTP刺激。 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/或突变的形式遗传地替换LRRTM2蛋白。我会用 这种方法进行LRRTM2分布和可塑性依赖性富集的第一次评估， 神经元有了这个工具，我将确定LRRTM2驱动的AMPAR对齐，稳定性， 和丰富。我会设计基因组序列的选择性突变并使用超分辨率 显微镜和单分子跟踪来描述LRRTM2如何与其伴侣特异性相互作用 有助于贩卖AMPAR。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依赖性脊柱生长中的作用是出乎意料的，目前的模型无法解释 LRRTM2功能。我将使用活细胞成像和 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式 粘附分子在成熟的突触，并将提供一个坚实的基础，为我的独立职业生涯。