Perrin Florian, Sinha Priyanka, Mitchell Shane Patrick Clancy, Sadek Michael, Maesako Masato, Berezovska Oksana
MassGeneral Institute for Neurodegenerative Disease, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA.
MassGeneral Institute for Neurodegenerative Disease, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA.
J Biol Chem. 2024 Apr;300(4):107172. doi: 10.1016/j.jbc.2024.107172. Epub 2024 Mar 16.
The recently discovered interaction between Presenilin 1 (PS1), a catalytic subunit of γ-secretase responsible for generating amyloid-β peptides, and GLT-1, a major glutamate transporter in the brain (EAAT2), provides a mechanistic link between these two key factors involved in Alzheimer's disease (AD) pathology. Modulating this interaction can be crucial to understand the consequence of such crosstalk in AD context and beyond. However, the interaction sites between these two proteins are unknown. Herein, we utilized an alanine scanning approach coupled with FRET-based fluorescence lifetime imaging microscopy to identify the interaction sites between PS1 and GLT-1 in their native environment within intact cells. We found that GLT-1 residues at position 276 to 279 (TM5) and PS1 residues at position 249 to 252 (TM6) are crucial for GLT-1-PS1 interaction. These results have been cross validated using AlphaFold Multimer prediction. To further investigate whether this interaction of endogenously expressed GLT-1 and PS1 can be prevented in primary neurons, we designed PS1/GLT-1 cell-permeable peptides (CPPs) targeting the PS1 or GLT-1 binding site. We used HIV TAT domain to allow for cell penetration which was assayed in neurons. First, we assessed the toxicity and penetration of CPPs by confocal microscopy. Next, to ensure the efficiency of CPPs, we monitored the modulation of GLT-1-PS1 interaction in intact neurons by fluorescence lifetime imaging microscopy. We saw significantly less interaction between PS1 and GLT-1 with both CPPs. Our study establishes a new tool to study the functional aspect of GLT-1-PS1 interaction and its relevance in normal physiology and AD models.
早老素1（PS1）是γ-分泌酶的催化亚基，负责生成淀粉样β肽，它与大脑中的主要谷氨酸转运体GLT-1（EAAT2）之间最近发现的相互作用，为阿尔茨海默病（AD）病理过程中涉及的这两个关键因素提供了一种机制联系。调节这种相互作用对于理解这种串扰在AD及其他情况下的后果可能至关重要。然而，这两种蛋白质之间的相互作用位点尚不清楚。在此，我们采用丙氨酸扫描方法并结合基于荧光共振能量转移（FRET）的荧光寿命成像显微镜，来确定完整细胞内天然环境中PS1和GLT-1之间的相互作用位点。我们发现，GLT-1第276至279位残基（跨膜区5，TM5）和PS1第249至252位残基（跨膜区6，TM6）对于GLT-1与PS1的相互作用至关重要。这些结果已通过AlphaFold Multimer预测进行了交叉验证。为了进一步研究内源性表达的GLT-1和PS1之间的这种相互作用在原代神经元中是否可以被阻断，我们设计了靶向PS1或GLT-1结合位点的PS1/GLT-1细胞穿透肽（CPP）。我们使用HIV TAT结构域来实现细胞穿透，并在神经元中进行了检测。首先，我们通过共聚焦显微镜评估了CPP的毒性和穿透能力。接下来，为了确保CPP的有效性，我们通过荧光寿命成像显微镜监测完整神经元中GLT-1与PS1相互作用的调节情况。我们发现，两种CPP都使PS1与GLT-1之间的相互作用显著减少。我们的研究建立了一种新工具，用于研究GLT-1与PS1相互作用的功能方面及其在正常生理学和AD模型中的相关性。
