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.

中文翻译：

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PS1/γ-分泌酶和谷氨酸转运蛋白 GLT-1 相互作用位点的鉴定

最近发现的早老素 1 (PS1)（负责生成淀粉样β肽的 γ-分泌酶催化亚基）与 GLT-1（大脑中主要的谷氨酸转运蛋白 (EAAT2)）之间的相互作用，提供了这两个关键之间的机制联系。参与阿尔茨海默病（AD）病理学的因素。调节这种相互作用对于理解 AD 环境及其他环境中这种串扰的后果至关重要。然而，这两种蛋白质之间的相互作用位点尚不清楚。在此，我们利用丙氨酸扫描方法结合基于 FRET 的荧光寿命成像显微镜来识别完整细胞内原生环境中 PS1 和 GLT-1 之间的相互作用位点。我们发现位置276至279的GLT-1残基（TM5）和位置249至252的PS1残基（TM6）对于GLT-1-PS1相互作用至关重要。这些结果已使用 AlphaFold Multimer 预测进行了交叉验证。为了进一步研究是否可以在原代神经元中阻止内源表达的 GLT-1 和 PS1 的这种相互作用，我们设计了针对 PS1 或 GLT-1 结合位点的 PS1/GLT-1 细胞渗透肽（CPP）。我们使用 HIV TAT 结构域来允许细胞渗透，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 的毒性和渗透性。接下来，为了确保 CPP 的效率，我们通过荧光寿命成像显微镜监测了完整神经元中 GLT-1-PS1 相互作用的调节。我们发现 PS1 和 GLT-1 与两种 CPP 之间的相互作用显着减少。我们的研究建立了一种新工具来研究 GLT-1-PS1 相互作用的功能及其在正常生理学和 AD 模型中的相关性。