淀粉样β肽通过 FRET/FLIM 测定破坏神经元细胞中 VAMP-2 和 SNAP-25 之间的相互作用。

Amyloid-β Peptides Disrupt Interactions Between VAMP-2 and SNAP-25 in Neuronal Cells as Determined by FRET/FLIM.

机构信息

Department of Pharmaceutics and the Brain Barriers Research Center, College of Pharmacy, University of Minnesota, Minneapolis, MN, USA.

University of Minnesota Informatics Institute, University Imaging Center, University of Minnesota, Minneapolis, MN, USA.

出版信息

J Alzheimers Dis. 2020;77(1):423-435. doi: 10.3233/JAD-200065.

DOI:10.3233/JAD-200065

PMID:32675412

Abstract

BACKGROUND

Synaptic dysfunction prevalent in Alzheimer's disease (AD) brain is closely associated with increased accumulation of amyloid-β (Aβ) peptides in the brain parenchyma. It is widely believed that Aβ peptides trigger synaptic dysfunction by interfering with the synaptic vesicular fusion and the release of neurotransmitters, primarily facilitated by the SNARE protein complexes formed by VAMP-2, SNAP-25, and syntaxin-1. However, Aβ interactions with SNARE proteins to ultimately disrupt synaptic vesicular fusion are not well understood.

OBJECTIVE

Our objective is to elucidate mechanisms by which Aβ peptides perturb SNARE complexes.

METHODS

Intensity (qualitative) and lifetime (quantitative) based measurements involving Forster (fluorescence) resonance energy transfer (FRET) followed by fluorescence lifetime imaging microscopy (FLIM) were employed to investigate the effect of Aβ peptides on dynamic interactions between VAMP-2, labeled with cerulean (Cer) at the N-terminus (FRET donor), and SNAP-25 labeled with citrine (Cit) on the N-terminus (FRET acceptor). The FRET and FLIM interactions at the exocytosis locations on the pre-synaptic membrane were recorded under spontaneous and high potassium evoked conditions. Moreover, cellular accumulation of fluorescein labeled Aβ (F-Aβ) peptides and their co-localization with Cer-VAMP2 was investigated by confocal microscopy.

RESULTS

The F-Aβ40 and F-Aβ42 are internalized by differentiated N2A cells, where they colocalize with Cer-VAMP2. Both Aβ40 and Aβ42 decrease interactions between the N-termini of Cer-VAMP2 and Cit-SNAP25 in N2A cells, as determined by FRET/FLIM.

CONCLUSION

By perturbing the N-terminal interactions between VAMP-2 and SNAP-25, Aβ40 and Aβ42, can directly interfere with the SNARE complex formation, which is critical for the docking and fusion of synaptic vesicles.

摘要

背景

阿尔茨海默病（AD）大脑中普遍存在的突触功能障碍与脑实质中淀粉样β（Aβ）肽的积累增加密切相关。人们普遍认为，Aβ 肽通过干扰突触小泡融合和神经递质的释放而触发突触功能障碍，主要由 VAMP-2、SNAP-25 和 syntaxin-1 形成的 SNARE 蛋白复合物来促进。然而，Aβ 与 SNARE 蛋白相互作用最终破坏突触小泡融合的机制尚不清楚。

目的

我们的目的是阐明 Aβ 肽扰乱 SNARE 复合物的机制。

方法

使用基于强度（定性）和寿命（定量）的测量方法，包括荧光共振能量转移（FRET）后荧光寿命成像显微镜（FLIM），研究 Aβ 肽对 Cerulean（Cer）标记在 N 端（FRET 供体）的 VAMP-2 与 Citrine（Cit）标记在 N 端（FRET 受体）的 SNAP-25 之间动态相互作用的影响。在自发和高钾诱发条件下，记录前突触膜上的 exocytosis 位置的 FRET 和 FLIM 相互作用。此外，通过共聚焦显微镜研究了荧光素标记的 Aβ（F-Aβ）肽的细胞内积累及其与 Cer-VAMP2 的共定位。