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

摘要 突触衰竭是阿尔茨海默病(AD)认知功能下降和记忆功能障碍的原因之一。阿尔茨海默病突触功能缺陷和突触衰竭与树突棘和谷氨酸能信号的丢失有关。然而，尽管树突棘突触在疾病中很重要，但棘突和突触的小尺寸阻碍了我们理解突触前和突触后分子如何作为AD发病的标志，以及如何针对这些结构进行AD干预。我们的建议试图通过使用超分辨率成像和最先进的方法来了解树突突触的纳米分子组织来打破这一障碍，树突突触在诊断AD之前就开始消失。我们的初步数据表明，与AD相关的AMPA和NMDA型谷氨酸受体在不同大小的脊椎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突触或弱突触。在这里，我们将检验一种假设，即脊柱大小的变化决定了AMPAR和NMDAR的组织，cLTP后脊柱增大将导致特定的AMPAR和NMDAR亚单位在突触上的不同分布。我们将利用我们的方法结合标记脊椎的形态重建和多色STED成像。在目标1中，我们将确定单个和多个模块脊柱的离散纳米模块中谷氨酸受体的组织。我们将使用PSD-95和巴松管的三色STED成像，以及EGFP标记的脊柱中的AMPAR或NMDAR亚基。我们将研究不同的亚基相对于排列的PSD-95/巴松管纳米模块是如何组织的，以及每个纳米模块在其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离散纳米模块中谷氨酸受体的组织。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脊柱分子组织的后三色STED分析相结合。在同样被实时成像的脊柱中，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亚单位在增强、无反应和对照脊柱的单个纳米模块中的定位。这些高风险/高回报的实验将确定突触上谷氨酸受体的分子组织，并可能为AD突触衰竭和功能障碍的研究提供新的途径。我们的研究结果将推动对突触功能变化如何与AD有关的新理解，并可能为与AD发病相关的记忆丧失的早期诊断提供变革性的工具。

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.