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

认知、学习和记忆都依赖于谷氨酸能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)，这是 在中枢神经系统中调节快速突触传递。在许多形式的可塑性过程中，包括 长时程增强(LTP)，突触AMPAR水平改变，以调节突触强度和调节 神经回路功能。然而，最近的研究表明，突触的强度不是简单地由 突触处的AMPAR的数量，也取决于它们在突触内的纳米组织。AMPAR得到了丰富 突触下的高密度纳米簇，通常与突触前神经递质的释放相一致 这种分子结构既影响基础突触传递，也影响可塑性。尽管很长时间 认识到AMPAR运输对突触的强度和可塑性至关重要，这些机制 控制它们的位置，甚至它们在突触上的保留仍然不清楚。这件事的首要目标是 建议研究AMPAR在突触和突触下的调节机制。 富含亮氨酸重复序列的跨膜蛋白2(LRRTM2)已成为潜在的候选基因 功能，在多种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干扰LRRTM2，我们的实验室最近发现LRRTM2 调节AMPAR在长期和短期操作后的丰度和纳米定位， 分别进行了分析。作为一种位于突触后密度的跨膜蛋白，LRRTM2形成多种蛋白质 相互作用，包括与PSD-95，突触前Neurexins，以及直接与AMPAR。这些相互作用 提出关于LRRTM2如何保留AMPAR并将其具体定位在 LTP刺激后基础和AMPAR募集期间的突触。 为了可视化和控制内生LRRTM2，我成功地采用了一个基于CRISPR的新工具 用标记的、可剧烈切割的和/或突变的版本从基因上取代LRRTM2蛋白。我会用 这种方法首次评估了LRRTM2的分布和塑性相关的浓缩 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 显微镜和单分子跟踪描绘LRRTM2如何与其合作伙伴特别地相互作用 助长了AMPAR的贩运。最后，我将研究LRRTM2如何支持LTP。我们出人意料的初选 数据表明，LRRTM2在LTP依赖的脊柱生长中发挥了意想不到的作用，目前的模型没有解释这一点 LRRTM2函数。我将结合使用活细胞成像和 电生理学。这些工具和方法为理解细胞的角色建立了新的范式 黏附分子在成熟的突触，将为我的独立职业生涯提供坚实的基础。

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.