在神经元表面表达的AMPA受体的相对量可以使用在其N-末端的超抑制剂pHluorin(SEP)标记来测量。然而,神经元中蛋白质过表达导致的高信号变异性和细胞内囊泡中观察到的低信号使受体运输的定量表征变得困难。这里,我们建立了基于荧光寿命成像(FLIM)的AMPAR贩运的实时活细胞测定,这允许同时可视化表面和细胞内受体。使用这种方法,我们发现,升高β淀粉样蛋白(Aβ)水平导致细胞内GluA1和GluA2受体的强烈增加,表明Aβ触发了这些AMPAR的内吞作用。在APP/PS1阿尔茨海默病模型小鼠神经元,FLIM揭示了含有GluA1-和GluA3的受体的AMPAR运输特性显着不同,表明慢性Aβ暴露会引发表面和细胞内含GluA3受体的丢失。有趣的是,蛋白磷酸酶1(PP1)的过表达也导致GluA1内吞作用以及突触传递抑制,证实磷酸化在调节AMPAR运输中的重要作用。这种新方法允许细胞外pH的定量测量,受体贩运的微小变化,以及同时测量活神经元中的表面和内化AMPAR,因此可以应用于未来的几个不同的研究。
The relative amount of AMPA receptors expressed at the surface of neurons can be measured using superecliptic pHluorin (SEP) labeling at their N-terminus. However, the high signal variability resulting from protein overexpression in neurons and the low signal observed in intracellular vesicles make quantitative characterization of receptor trafficking difficult. Here, we establish a real-time live-cell assay of AMPAR trafficking based on fluorescence lifetime imaging (
FLIM), which allows for simultaneous visualization of both surface and intracellular receptors. Using this assay, we found that elevating amyloid-beta (Aβ) levels leads to a strong increase in intracellular GluA1 and GluA2-containing receptors, indicating that Aβ triggers the endocytosis of these AMPARs. In APP/PS1 Alzheimer\'s disease model mouse neurons,
FLIM revealed strikingly different AMPAR trafficking properties for GluA1- and GluA3-containing receptors, suggesting that chronic Aβ exposure triggered the loss of both surface and intracellular GluA3-containing receptors. Interestingly, overexpression of protein phosphatase 1 (PP1) also resulted in GluA1 endocytosis as well as depressed synaptic transmission, confirming the important role of phosphorylation in regulating AMPAR trafficking. This new approach allows for the quantitative measurement of extracellular pH, small changes in receptor trafficking, as well as simultaneous measurement of surface and internalized AMPARs in living neurons, and could therefore be applied to several different studies in the future.