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

项目总结 AMPA型谷氨酸受体(AMPAR)是中枢神经系统的主要兴奋性神经递质受体。 大脑和突触上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 力量受到调节，并用于治疗方法的发展，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来了解AMPAR的结构基础 N-末端结构域相互作用(NTD)推动功能结果，如增加AMPAR积累和 突触的力量。这种方法的理由有两个：1)它将有助于解决长期存在的以下问题 关键神经递质受体的调节；2)AMPAR参与的详细结构模型。 关键的调节复合体将指导未来寻求改变兴奋性强度的治疗方法 对神经元的输入与精神分裂症等精神疾病有关。黏附蛋白NPTX2结合于 AMPAR，聚集在突触上的AMPAR，是神经元间特异性GluA4-的动态平衡调节所必需的。 含有AMPAR。因此，依赖于NPTX2的GluA4比例是检验该假设的理想模型 直接与AMPAR细胞外相互作用控制突触强度。这种方法是创新的，因为 AMPAR的塑性模型从未在结构细节中观察到过。这项研究具有重要意义，因为 它将为AMPAR相互作用如何推动可塑性以及如何将其用于治疗提供新的见解 在未来受益。这种方法的一个例子是结构指导的治疗策略 在小白蛋白表达的中间神经元(PVIN)表面聚集GluA4，表现为降低 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 单粒子冷冻EM法研究NPTX2/GluA4复合体的结构2)测试NPTX2是否驱动 GluA4 PVIN通过直接相互作用进行伸缩。对于第一个目标，我们将使用单粒子低温EM来 解决活性调节的突触黏附分子NPTX2与中间神经元的复合体结构- 特异性GluA4 AMPAR。对于第二个目标，我们将使用转基因小鼠模型，生物化学，神经元 培养、共聚焦光学显微镜和电生理学来测试NPTX2直接结合到 GluA4的NTD驱动疾病相关PVIN的动态平衡缩放。 申请人提出这项工作的部分原因是为了推进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。候选人将会 在专家指导小组的协助下，进行广泛的低温EM和生物物理学培训 由AMPAR冷冻-EM专家，AMPAR塑性专家和NPTX2专家组成，他们都将 指导申请者过渡到终身教职轨道的学术职位。

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.