Linking molecular nano-organization of spine synapses and structural plasticity using super-resolution imaging

摘要 突触失效是阿尔茨海默病（AD）中认知下降和记忆功能障碍的原因。AD中的突触功能缺陷和突触失效与树突棘和突触能信号传导的丧失相关。然而，尽管树突棘突触在疾病中的重要性，但棘和突触的小尺寸使我们无法理解突触前和突触后分子如何作为AD发病的标志物以及如何靶向这些结构进行AD干预。我们的建议旨在通过使用超分辨率成像和最先进的方法来理解树突棘突触的纳米分子组织来打破这一障碍，树突棘突触在AD被诊断之前开始消失。我们的初步数据表明，AMPA和NMDA型谷氨酸受体都与AD有关，在不同大小的棘中使用不同的规则进行组织，这表明单个突触可能具有独特的分子特征，可以区分强突触和弱或弱突触。在这里，我们将测试的假设，即脊柱大小的变化决定了组织的AMPAR和NMDAR，脊髓扩大后cLTP将导致特定的AMPAR和NMDAR亚基在突触的差异分布。我们将利用我们的方法结合标记的脊柱和多色STED成像的形态重建。在目标1中，我们将确定谷氨酸受体在单模块和多模块棘的离散纳米模块中的组织。我们将使用PSD-95和巴松管的三色STED成像，以及EGFP标记的棘中的AMPAR或NMDAR亚基。我们将研究不同的亚基是如何相对于对齐的PSD-95/巴松管纳米模块组织的，以及每个纳米模块在谷氨酸受体组成上是否不同。在目标2中，我们将确定结构可塑性诱导后谷氨酸受体在离散纳米模块中的组织。 在这里，我们将用甘氨酸诱导cLTP，并将树突棘形态的实时成像与棘分子组织的事后三色STED分析联合收割机相结合。在实时成像的相同脊柱中，我们将通过PSD-95和巴松管染色来确定纳米模块的数量，并评估AMPAR和NMDAR亚基在增强的、无反应的和对照脊柱的单个纳米模块中的定位。这些高风险/回报的实验将确定谷氨酸受体在突触的分子组织，并可能产生新的途径，在AD的突触失败和功能障碍的调查。我们的研究结果将推动对突触功能变化如何与AD相关的新理解，并可能为早期诊断与AD发病相关的记忆丧失提供变革性工具。

ABSTRACT Synaptic failure is a cause of cognitive decline and memory dysfunction in Alzheimer’s disease (AD). Defective synaptic function and synaptic failure in AD is associated with the loss of dendritic spines and glutamatergic signaling. However, despite the importance of dendritic spine synapses in disease, the small size of spines and synapses has prevented us from understanding how pre- and post-synaptic molecules might serve as markers for the onset of AD and how to target these structures for AD intervention. Our proposal seeks to break down this barrier by using super-resolution imaging and state-of-the-art approaches to understand the nanomolecular organization of dendritic spines synapses, which begin to disappear before AD can be diagnosed. Our preliminary data indicate that AMPA and NMDA type glutamate receptors, which are both linked to AD, are organized using distinct rules in spines of different sizes, suggesting that individual synapses might have unique molecular signatures that can distinguish strong synapses from weak or weakening synapses. Here, we will test the hypothesis that changes in spine size determine the organization of AMPARs and NMDARs and that spine enlargement following cLTP will lead to the differential distribution of particular AMPAR and NMDAR subunits at synapses. We will take advantage of our approach combining morphological reconstructions of labeled spines and multi-color STED imaging. In Aim 1, we will determine the organization of glutamate receptors in discrete nanomodules of single and multi-module spines. We will use three-color STED imaging of PSD-95 and Bassoon, and either AMPAR or NMDAR subunits in EGFP labeled spines. We will examine how different subunits are organized relative to aligned PSD-95/Bassoon nanomodules and whether each nanomodule differs in their glutamate receptor make-up. In Aim 2, we will determine the organization of glutamate receptors in discrete nanomodules after induction of structural plasticity. Here, we will induce cLTP with glycine and combine live imaging of dendritic spine morphology with post-hoc three-color STED analysis of spine molecular organization. In the same spines that were imaged live, we will determine the number of nanomodules by staining for PSD-95 and Bassoon and assess the localization of AMPAR and NMDAR subunits in individual nanomodules of potentiated, unresponsive and control spines. These high risk/reward experiments will determine the molecular organization of glutamate receptors at synapses and will likely generate new avenues for investigation of synaptic failure and dysfunction in AD. Findings from our studies will drive new understanding of how changes in synaptic function are linked to AD and will likely yield transformative tools for the early diagnosis of memory loss that is associated with the onset of AD.