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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要 AMPA 型谷氨酸受体 (AMPAR) 是大脑中主要的兴奋性神经递质受体大脑和突触 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 AMPAR 在突触中的组织方式对于了解突触如何组织至关重要强度得到调节，并用于开发治疗方法以纠正人类疾病中的回路不平衡。该提案的长期目标是使用 Cryo-EM 来了解 AMPAR 的结构基础如何 N 端结构域相互作用 (NTD) 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； 2）参与的AMPAR的详细结构模型关键的调节复合物将指导未来寻求改变兴奋强度的治疗方法输入到与精神分裂症等精神疾病有关的神经元。粘附蛋白 NPTX2 结合 AMPAR，在突触处聚集 AMPAR，是中间神经元特异性 GluA4- 稳态缩放所必需的含有 AMPAR。因此，NPTX2 依赖性 GluA4 缩放是检验假设的理想模型直接细胞外与 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被观察到。这项研究意义重大，因为它将产生关于 AMPAR 相互作用如何驱动可塑性以及如何将其用于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将 GluA4 聚集在表达小清蛋白的中间神经元 (PVIN) 表面，其表现出降低的精神分裂症模型中的兴奋性驱动。本项目的长期目标将通过以下两个具体目标来实现： 1）确定通过单颗粒冷冻电镜观察 NPTX2/GluA4 复合物的结构。 2）测试NPTX2是否驱动 GluA4 PVIN 通过直接相互作用进行缩放。对于第一个目标，我们将采用单粒子冷冻电镜解决了活性调节突触粘附分子 NPTX2 与中间神经元复合物的结构特定的 GluA4 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、共焦光学显微镜和电生理学来测试 NPTX2 直接结合的假设 GluA4 的 NTD 驱动疾病相关 PVIN 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推进他们建立独立的长期目标将突触蛋白的结构与其突触功能联系起来的研究生涯。候选人将接受冷冻电镜和生物物理学方面的广泛培训，并由专家指导团队提供协助由 AMPAR 冷冻电镜专家、AMPAR 塑性专家和 NPTX2 专家组成，他们都将指导申请人过渡到终身教职。