Dynamic control of synaptic substructure and function by adhesion molecules

粘附分子对突触亚结构和功能的动态控制

• 批准号: 9788754
• 负责人: Austin Michael Ramsey
• 金额: $ 3.71万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-05 至 2020-09-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9788754
• 关键词: 3-Dimensional; AMPA Receptors; Acute; Adhesions; Affect; Affinity; Binding; Binding Proteins; Cell Adhesion Molecules; Cells; Cleaved cell; Communication; Complex; Confocal Microscopy; Data; Diffusion; Disease; Electrophysiology (science); Elements; Engineering; Exhibits; Exocytosis; Extracellular Domain; Family; Financial compensation; Glutamate Receptor; Glutamates; Goals; Image; Investigation; Knock-out; Knowledge; Lateral; Lead; Leucine-Rich Repeat; Maintenance; Measures; Mediating; Mental disorders; Methods; Microscopy; Modeling; Modification; Molecular; Nervous system structure; Neurobiology; Neurons; Neurotransmitters; Peptide Hydrolases; Play; Positioning Attribute; Process; Protein Isoforms; Proteins; RNA Splicing; Receptor Activation; Regulation; Resolution; Retrieval; Role; Series; Site; Slice; Structure; Surface; Synapses; Synaptic Transmission; Tertiary Protein Structure; Testing; Thrombin; Time; Training; Vesicle; Wit; Work; density; design; experimental study; extracellular; improved; in vivo; insight; interest; knock-down; loss of function; molecular imaging; mutant; nanocluster; nanocolumn; nanoscale; nervous system disorder; neural circuit; neurotransmitter release; novel; patch clamp; postsynaptic; presynaptic; receptor; relating to nervous system; single molecule; spatial relationship; stem; synaptic function; synaptogenesis; therapy design; transmission process

The complex neural processes of information encoding, storage, and retrieval are enabled by precise and efficient regulation of synaptic strength. Investigation into mechanisms of synaptic transmission will inform not only how we think about neural circuit functions, but also how we can better treat neurobiological diseases and disorders at a fundamental level. Our lab recently discovered a novel element of subsynaptic structure by which receptor activation may be modulated independent of conventional mechanisms. Proteins that establish presynaptic sites of neurotransmitter exocytosis are tightly aligned across the synapse with postsynaptic nanoclusters of receptors. This “nanocolumn” of trans-synaptic structure is expected to impact synaptic efficacy by controlling the likelihood of receptor activation (Tang et al., 2016). However, despite much detailed examination of how receptors move in and around synapses, we almost completely lack understanding of the mechanisms that determine their positioning within the synapse and across from sites of release. Though many mechanisms may contribute to nanocolumn formation, a particularly attractive model is that synaptic cell adhesion molecules (CAMs) mediate alignment through high affinity trans-synaptic protein binding. My goal is to test this idea. However, distinguishing the ongoing roles of CAMs at synapses following their known roles in synaptogenesis is difficult. Synaptic CAMs undergo extensive splicing and include a large variety of proteins with similar functional domains, provoking widespread mechanistic compensation over the days following knockout or knockdown. To avoid these effects, I have been developing approaches to acutely perturb CAM trans-synaptic binding on the time scale of just minutes. My preliminary data adapts an approach originally developed by Peixoto et al. (2012) by inserting a protease cleavage site into the protein of interest, enabling acute and specific cleavage of desired protein domains. My design includes a knockdown-replacement strategy, permits independent tracking of the cleaved components, and can be expanded to target multiple proteins simultaneously. Here, I propose to apply my approach to test whether the synaptic CAM Leucine-Rich Repeat Transmembrane neuronal 2 (LRRTM2) mediates synaptic nanoalignment. LRRTM2 is a strong candidate to test first because it participates in trans-synaptic binding with key proteins (postsynaptic PSD-95 and presynaptic neurexin), it regulates synaptogenesis, and its knockdown results in decreased evoked EPSCs. Intriguingly, unlike most other CAMs, LRRTM2 also directly binds AMPARs within the postsynaptic density. With patch-clamp electrophysiology, super-resolution microscopy, and single-molecule tracking, I will use acute cleavage to test whether elimination of LRRTM2 extracellular interactions acutely disrupts trans- synaptic protein alignment, AMPA receptor mobility, and synaptic strength. These results will be the first test of an important new synaptic mechanism, and will provide key training establishing the basis for subsequent postdoctoral work.

信息编码、存储和检索的复杂神经过程是通过精确和 突触强度的有效调节。对突触传递机制的研究将不会告诉我们 不仅包括我们如何思考神经回路功能，还包括我们如何更好地治疗神经生物学疾病和 基本水平的紊乱。我们的实验室最近发现了一种新的突触亚结构元素 该受体的激活可以独立于常规机制进行调节。建立的蛋白质 神经递质胞吐作用的突触前位点与突触后位点紧密排列在突触上 受体纳米簇。这种跨突触结构的“纳米柱”预计会影响突触 通过控制受体激活的可能性来提高疗效（Tang 等人，2016）。然而，尽管有很多详细的 在检查受体如何在突触内和突触周围移动时，我们几乎完全缺乏对突触的了解。 决定它们在突触内和释放位点之间定位的机制。尽管 许多机制可能有助于纳米柱的形成，一个特别有吸引力的模型是突触细胞 粘附分子 (CAM) 通过高亲和力跨突触蛋白结合介导排列。我的目标是 来测试这个想法。然而，根据 CAM 在突触中的已知作用来区分其持续作用 突触发生很困难。突触 CAM 经历广泛的剪接并包含多种蛋白质 具有相似的功能域，在接下来的几天内引发广泛的机械补偿 击倒或击倒。为了避免这些影响，我一直在开发剧烈干扰 CAM 的方法 跨突触结合仅需几分钟的时间。我的初步数据采用本方法 由 Peixoto 等人开发。 (2012) 通过将蛋白酶切割位点插入到感兴趣的蛋白质中，使得 所需蛋白质结构域的急性和特异性切割。我的设计包括拆卸替换 策略，允许独立跟踪切割的组件，并且可以扩展到目标多个 同时蛋白质。在这里，我建议应用我的方法来测试突触 CAM Leucine-Rich 是否 重复跨膜神经元 2 (LRRTM2) 介导突触纳米排列。 LRRTM2 是一个强大的 首先测试的候选者，因为它参与与关键蛋白的跨突触结合（突触后 PSD-95 和突触前神经素），它调节突触发生，其敲低会导致诱发的减少 EPSC。有趣的是，与大多数其他 CAM 不同，LRRTM2 还直接结合突触后内的 AMPAR 密度。借助膜片钳电生理学、超分辨率显微镜和单分子追踪，我将 使用急性裂解来测试 LRRTM2 细胞外相互作用的消除是否会急性破坏反式 突触蛋白排列、AMPA 受体流动性和突触强度。这些结果将是第一次测试 一个重要的新突触机制，并将提供关键训练，为后续研究奠定基础 博士后工作。