Structure-guided functional analysis of GluA4-NPTX2 interaction during PVIN homeostatic scaling

PVIN 稳态缩放过程中 GluA4-NPTX2 相互作用的结构引导功能分析

• 批准号: 10808384
• 负责人: William Dylan Hale
• 金额: $ 10.3万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-15 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10808384
• 关键词: Adhesions; Affinity; Binding; Binding Sites; Biochemical; Biochemistry; Biology; Biophysics; Brain; Cations; Cell Adhesion Molecules; Cell surface; Cells; Complex; Cryoelectron Microscopy; Data; Disease; Disinhibition; Electrophysiology (science); Excitatory Synapse; Exhibits; Functional disorder; Future; Genetic; Glutamate Receptor; Glutamates; Goals; Health; Human; Interneurons; Learning; Membrane; Memory; Mental disorders; Mentors; Methods; Mission; Modeling; Molecular; Mus; Mutagenesis; N-terminal; Neurons; Neurosciences; Neurotransmitter Receptor; Parvalbumins; Pathology; Physiology; Positioning Attribute; Process; Property; Proteins; Regulation; Research; Role; Schizophrenia; Structural Models; Structure; Surface; Synapses; Synaptic plasticity; Techniques; Testing; Therapeutic; Training; Transgenic Mice; United States National Institutes of Health; Work; antagonist; career; design; excitatory neuron; expectation; extracellular; functional outcomes; human disease; improved; innovation; insight; light microscopy; mouse model; mutant; nervous system disorder; neuronal pentraxin; neuropsychiatric disorder; particle; pharmacologic; postsynaptic; presynaptic; prevent; receptor; response; stoichiometry; structural biology; synaptic function; tenure track; therapeutic development; therapeutic target

PROJECT SUMMARY AMPA-type glutamate receptors (AMPARs) are the major excitatory neurotransmitter receptors in the brain and changes in AMPAR number at synapses underlie learning and memory as well as human disease. A detailed understanding of how AMPARs are organized at synapses is critical to understand how synaptic strength is regulated and for the development of therapeutics to correct circuit imbalances in human disease. The long-term goal of this proposal is to use Cryo-EM to understand how the structural basis of AMPAR N-terminal domain interactions (NTDs) drive functional outcomes such as increased AMPAR accumulation and synaptic strength. The rationale for this approach is twofold 1) it will help resolve long-standing questions about the regulation of key neurotransmitter receptors; and 2) a detailed structural model of AMPARs participating in key regulatory complexes will guide future therapeutic approaches that seek to alter the strength of excitatory input onto neurons implicated in psychiatric illnesses like schizophrenia. The adhesion protein NPTX2 binds to AMPARs, clusters AMPARs at synapses, and is required for homeostatic scaling of interneuron-specific GluA4- containing AMPARs. Therefore, NPTX2-dependent GluA4 scaling is an ideal model for testing the hypothesis that direct extracellular interactions with AMPARs control synaptic strength. This approach is innovative because models of AMPAR plasticity have never been observed in structural detail. This research is significant because it will yield new insights into how AMPAR interactions drive plasticity and how this can be exploited for therapeutic benefit in the future. An example of such an approach would be a structure-guided therapeutic strategy for clustering GluA4 on the surface of Parvalbumin-expressing interneurons (PVINs), which exhibit lowered excitatory drive in models of schizophrenia. The long-term goal of this project will be achieved with the following two specific aims: 1) Determine the structure of the NPTX2/GluA4 complex via single particle Cryo-EM. and 2) Test whether NPTX2 drives GluA4 PVIN scaling through a direct interaction. For the first aim we will employ single-particle Cryo-EM to solve the structure of the activity-regulated synaptic adhesion molecule NPTX2 in complex with the interneuron- specific GluA4 AMPARs. For the second aim, we will employ transgenic mouse models, biochemistry, neuron culture, confocal light microscopy, and electrophysiology to test the hypothesis that direct binding of NPTX2 to the NTD of GluA4 drives homeostatic scaling in disease-associated PVINs. The applicant has proposed this work in part to further their long-term goal of establishing an independent research career connecting the structure of synaptic proteins to their synaptic function. The candidate will undertake extensive training in Cryo-EM and biophysics which will be facilitated by an expert mentoring team composed of an AMPAR Cryo-EM expert, an AMPAR plasticity expert, and an expert in NPTX2, all of whom will mentor the applicant through the transition to a tenure track academic position.

项目摘要 AMPA型谷氨酸受体（AMPARS）是主要兴奋性神经递质受体 大脑和突触中的AMPAR数量的变化是学习和记忆以及人类疾病的基础。一个 详细了解在突触中如何组织AMPAR对于了解突触的方式至关重要 强度受到调节，并用于开发治疗剂以纠正人类疾病中的电路失衡。 该提案的长期目标是使用Cryo-Em了解AMPAR的结构基础 N末端结构域相互作用（NTDS）驱动功能结果，例如增加AMPAR积累和 突触强度。这种方法的理由是双重的1）它将有助于解决有关的长期问题 关键神经递质受体的调节； 2）参与AMPAR的详细结构模型 关键的监管络合物将指导未来的治疗方法，试图改变兴奋的力量 对与精神分裂症等精神病有关的神经元的输入。粘附蛋白NPTX2与 Ampars，簇Ampars在突触下，是间含膜特异性GLUA4-的稳态缩放所必需的 包含AMPAR。因此，NPTX2依赖性GLUA4缩放是检验假设的理想模型 与AMPARS控制突触强度的直接细胞外相互作用。这种方法是创新的，因为 从未在结构细节中观察到AMPAR可塑性的模型。这项研究很重要，因为 它将产生有关AMPAR相互作用如何驱动可塑性以及如何利用治疗的新见解 将来受益。这种方法的一个例子是结构引导的治疗策略 在表达白蛋白的中间神经元（PVIN）的表面上的聚类Glua4，显示出降低 精神分裂症模型中的兴奋性驱动。 以下两个具体目标将实现该项目的长期目标：1）确定 NPTX2/GLUA4复合物通过单个粒子冷冻EM的结构。 2）测试NPTX2是否驱动 GLUA4 PVIN通过直接相互作用进行缩放。对于第一个目的，我们将使用单粒子冷冻EM到 解决活性调节的突触粘附分子NPTX2与中间神经元中的结构 特定的glua4 ampars。为了第二个目标，我们将采用转基因小鼠模型，生物化学，神经元 培养，共聚焦光学显微镜和电生理学，以检验直接结合NPTX2与 GLUA4的NTD驱动与疾病相关的PVIN的稳态缩放。 申请人提出了这项工作，部分是为了进一步建立独立的长期目标 研究职业将突触蛋白与其突触功能联系起来。候选人会 对Cryo-EM和生物物理学进行广泛的培训，这些培训将由专家指导团队促进 由AMPAR Cryo-EM专家，AMPAR可塑性专家和NPTX2专家组成，所有这些都将 通过过渡到任期轨道学术职位，指导申请人。