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（AMPAR）是脑内主要的兴奋性神经递质受体， 突触处AMPAR数量的变化是学习和记忆以及人类疾病的基础。一 详细了解AMPAR在突触中的组织方式对于理解突触如何在突触中形成是至关重要的。 强度的调节和治疗的发展，以纠正人类疾病的电路失衡。 该提案的长期目标是使用Cryo-EM来了解AMPAR的结构基础 N-末端结构域相互作用（NTD）驱动功能结果，如增加AMPAR积累和 突触强度这种方法的理由有两个：1）它将有助于解决长期存在的问题， 关键神经递质受体的调节;和2）AMPAR参与的详细结构模型 关键的调节复合物将指导未来的治疗方法，寻求改变兴奋性的强度， 输入到与精神分裂症等精神疾病有关的神经元上。粘附蛋白NPTX 2结合到 AMPAR，在突触处聚集AMPAR，并且是神经元间特异性GluA 4- 含有AMPARs。因此，NPTX 2依赖性GluA 4缩放是检验假设的理想模型 指导细胞外与AMPAR的相互作用控制突触强度。这种方法是创新的，因为 AMPAR塑性模型从未在结构细节中观察到。这项研究意义重大，因为 它将为AMPAR相互作用如何驱动可塑性以及如何利用这一点进行治疗提供新的见解。 在未来受益。这种方法的一个例子是结构指导的治疗策略， 在表达小清蛋白的中间神经元（PVIN）表面聚集GluA 4， 精神分裂症模型中的兴奋性驱力 该项目的长期目标将通过以下两个具体目标实现：1）确定 通过单颗粒Cryo-EM的NPTX 2/GluA 4复合物的结构。2）测试NPTX 2是否驱动 GluA 4 PVIN通过直接相互作用缩放。对于第一个目标，我们将采用单粒子冷冻EM， 解决了与中间神经元复合的活性调节突触粘附分子NPTX 2的结构， 特异性GluA 4 AMPAR。对于第二个目标，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，以测试的假设，直接结合NPTX 2， GluA 4的NTD驱动疾病相关PVIN中的稳态缩放。 申请人提出这项工作的部分原因是为了进一步实现他们建立一个独立的 将突触蛋白的结构与其突触功能联系起来的研究生涯。候选人将 在冷冻EM和生物物理学方面进行广泛的培训，这将由专家指导小组提供帮助 由一位AMPAR Cryo-EM专家、一位AMPAR可塑性专家和一位NPTX 2专家组成，他们都将 指导申请人通过过渡到终身职位跟踪学术职位。