Super-Resolution Microscopy of Small Quantum Dots to Elucidate the Mechanisms of Alzheimer's Disease

项目总结/摘要 阿尔茨海默病（AD）困扰着500多万美国人，但没有已知的药物能够预防或停止 这种疾病在AD完全发展为不溶性淀粉样蛋白-β斑块沉积和神经变性之前， 一种与作为学习基础的突触可塑性受损相关的进行性认知衰退， 记忆这种异常的突触可塑性可能是由可溶性β淀粉样蛋白寡聚体影响突触可塑性引起的。 AMPA和NMDA受体的水平，两种介导诱导和表达的谷氨酸能受体， 突触可塑性然而，其潜在的详细机制尚不清楚， 由于这些受体的复杂行为和纳米尺度的小尺寸， 它们所处的突触区域。 这项建议的目标是了解早期脑缺血中异常突触可塑性的分子细节， AD通过开发基于小纳米颗粒的光学探针和新的显微镜技术来分析 AMPA和NMDA受体在正常和AD脑中的位置和动力学。这一目标将得以实现 通过三位主要研究人员的个人和集体努力，保罗·塞尔文（显微镜），安德鲁· Smith（量子点）和Hee Jung Chung（神经生物学）。他们以前曾作为一个团队， 两篇关于生成可以进入神经元的小量子点（sQD）（直径< 10 nm）的手稿 突触，并准确地跟踪受体数量和动态放置在分离培养的神经元。 为了实现这一目标，Aim 1将优化用于解离的sQD的超分辨率成像技术。 海马培养物和具有完整电路的厚海马切片，特别关注1-和2-光子 激发与FIONA和PALM/STORM显微镜。这将允许在所有三个维度上的< 20 nm分辨率。 目标2将开发一组新的sQD，它们更小，稳定，单价，具有最小的非特异性 与组织的相互作用。目标3将应用量子点和超分辨光学方法进行单分子 海马培养物和野生型急性切片中突触可塑性过程中谷氨酸受体的成像 和AD转基因模型小鼠。由于我们正在进行的成功合作，我们能够与 AD模型，而新的显微镜和量子点正在生成。这项研究将 增加我们对AD早期发病机制的了解，从而促进新的 可以特异性抑制这种疾病的认知下降的进展的治疗策略。

PROJECT SUMMARY / ABSTRACT Alzheimer's disease (AD) afflicts more than 5 million Americans, yet no known drug is able to prevent or stop the disease. Before AD fully develops with insoluble amyloid-β plaque deposits and neurodegeneration, there is a progressive cognitive decline associated with the impairment of synaptic plasticity that underlies learning and memory. This abnormal synaptic plasticity is likely caused by soluble amyloid-β oligomers affecting the synaptic levels of AMPA and NMDA receptors, two glutamatergic receptors that mediate induction and expression of synaptic plasticity. However, the underlying detailed mechanisms are not known and are exceptionally challenging to study due to the complex behavior of these receptors and the small nanometer-scale dimensions of the synaptic domains in which they reside. The goal of this proposal is to understand the molecular details of abnormal synaptic plasticity present in early AD by developing small nanoparticle-based optical probes and new microscopy techniques to analyze the position and dynamics of AMPA and NMDA receptors in normal and AD brains. This goal will be accomplished through the individual and collective efforts of three principle investigators, Paul Selvin (microscopy), Andrew Smith (quantum dots) and Hee Jung Chung (neurobiology). They have previously worked as a team to publish two manuscripts on generating small quantum dots (sQD) (< 10 nm diameter) that can enter the neuronal synapse and accurately follow the receptor number and dynamic placement in dissociated cultured neurons. To achieve this goal, Aim 1 will optimize super-resolution imaging techniques for sQDs in dissociated hippocampal culture and thick hippocampal slices with intact circuitry, specifically focusing on 1- and 2-photon excitation with FIONA and PALM/STORM microscopy. This will allow < 20 nm resolution in all three dimensions. Aim 2 will develop a novel set of sQDs that are smaller, stable, and monovalent with minimal non-specific interaction with tissue. Aim 3 will apply sQDs and super-resolution optical methods to perform single-molecule imaging of glutamate receptors during synaptic plasticity in hippocampal culture and acute slices from wild-type and AD transgenic model mice. Because of our on-going successful collaboration, we are able to work with the AD model immediately, while new microscopy and quantum dots are being generated. This research will increase our understanding of the early pathogenesis of AD and therefore foster the development of new therapeutic strategies that could specifically inhibit the progression of cognitive decline of this disease.