使用Aβ折疊報告細胞篩選靶向ERK及AMPK/EEF2K訊息的合成化合物作為新穎阿茲海默症治療策略

Abstract

阿茲海默症(Alzheimer’s disease, AD)是一種與年齡相關的神經退化性疾病，會逐漸的破壞記憶力以及學習能力，其主要病理特徵有大腦中β-澱粉樣蛋白(Aβ)堆積形成的斑塊，以及過度磷酸化tau蛋白所形成的神經纖維纏結。其中Aβ的堆積會損害神經元的突觸可塑性、增加細胞內的活性氧物質，並對神經元造成損害，因此開發AD藥物的其中一種方法，便是防止Aβ的堆積或阻斷Aβ寡聚體的神經毒性。研究有發現，在AD患者的CA1腦區中，編碼核糖體蛋白的基因受到異常的調節，其中與蛋白質的合成有關的真核延伸因子2 (Eukaryotic elongation factor 2, EEF2)在AD疾病中出現下調。EEF2在神經元的存活中起到重要的作用，可維持突觸可塑性。在真核細胞轉譯的過程中，EEF2的活性受到嚴格的控管。真核延伸因子2激酶(Eukaryotic elongation factor 2 kinase, EEF2K)藉磷酸化EEF2的T56位點抑制其活性，來調控轉譯的過程。EEF2K的活性受多種訊息路徑調控，如AMP活化蛋白激酶(AMP-activated protein kinase, AMPK)及細胞外訊息調節激酶(Extracellular-signal regulated kinase, ERK)。AMPK的訊息傳導可透過磷酸化S398位點來活化EEF2K，促使EEF2磷酸化失活，從而減少蛋白質合成；而ERK可透過磷酸化S366位點來抑制EEF2K的活性，使EEF2去磷酸化活化，來促進蛋白質的合成，如：腦源性神經營養因子(Brain-derived neurotrophic factor, BDNF)。本論文利用Aβ折疊報導細胞Aβ-GFP SH-SY5Y細胞，探究吲哚衍生物(Indole derivatives: NC009-1, -3, -6, -11)及香豆素衍生物(Coumarin derivatives: LM-016, -021, -022, -036)，改善Aβ錯誤折疊的情形及神經保護作用。在八種測試的化合物中，除NC009-3外，所有化合物均符合Lipinski預測口服生物利用度的標準。根據計算出的極性表面積和血腦屏障滲透評分，八種化合物皆被預測為可滲透血腦屏障。所檢測的化合物皆可改善Aβ的錯誤折疊以及相關的氧化壓力。此外，前處理NC009-1、NC009-6及LM-021、LM-036可有效降低凋亡蛋白酶-3/6以及抑制乙醯膽鹼酯酶(Acetylcholinesterase, AChE)的活性，並促進神經突生長。接下來，為了解化合物促進細胞中蛋白質合成之作用機制，使用嘌呤酶素標定的轉譯的表面傳感(Surface sensing of translation, SUnSET)分析法，發現NC009-1、LM-021及LM-036的處理可增加細胞中新合成的蛋白質。此外，NC009-1增加了p-ERK (T202/Y204)和p-EEF2K (S366)的表達，LM-021降低了p-AMPK (T172)和p-EEF2K (S398)的表達，而LM-036同時作用於兩條途徑，增加EEF2K的S366磷酸化並減少S398磷酸化，從而導致EEF2K活性降低。隨後對應的p-EEF2 (T56)抑制活性的降低，增強了p-CREB (S133)及其下游BDNF、BCL2蛋白的表達，並促進神經突生長。研究結果可作為開發新穎且有潛能的阿茲海默症治療策略的參考。

Alzheimer’s disease (AD) is an age-related degenerative disease that gradually destroy memory and learning ability. Its main pathological hallmarks are β-amyloid (Aβ) plaques and neurofibrillary tangles in the brain of subjects with AD. Aβ accumulation causes increased oxidative stress, neuronal dysfunction and neurodegeneration. Therefore, preventing Aβ accumulation may block Aβ neurotoxicity. In AD brains, genes encoding ribosomal proteins and eukaryotic elongation factor 2 (EEF2) are abnormally regulated. EEF2, which is related to de novo protein synthesis, plays an important role in neuronal survival and synaptic plasticity. T56 phosphorylation by EEF2 kinase (EEF2K) inhibits EEF2 activity, thereby reducing synthesis of brain-derived neurotrophic factor (BDNF), a key molecule involved in learning and memory. For EEF2K, S398 phosphorylation by AMP-activated protein kinase (AMPK) increases EEF2K activity, whereas S366 phosphorylation by extracellular-signal regulated kinase (ERK) decreases EEF2K activity. In this study, the effects of indole (NC009-1, -3, -6, -11) and coumarin (LM-016, -021, -022, -036) derivatives on Aβ folding and neuroprotection were examined using SH-SY5Y cells expressing Aβ-GFP. Among the eight tested compounds, six met Lipinski’s criteria in predicting oral bioavailability. In accordance with calculated polar surface area and blood-brain barrier (BBB) permeation score, all were predicted to be BBB-permeable. When tested in Aβ-GFP SH-SY5Y cells, all eight compounds ameliorated Aβ misfolding and associated reactive oxygen species (ROS). Among them, NC009-1, NC009-6, LM-021, LM-036 reduced the caspase-3/-6 and acetylcholinesterase activity, as well as promoted neurite outgrowth, including neurite length, process and branch in Aβ-GFP-expressing SH-SY5Y cells. To investigate if these four compounds targeting ERK/EEF2K or AMPK/EEF2K pathway for neuroprotection, puromycin-labeled surface sensing of translation assay was set up to explore the capacity of test compounds to enhance de novo protein synthesis in Aβ-GFP SH-SY5Y cells. It was found that the treatment of NC009-1, LM-021 and LM-036 can increase the newly synthesized proteins in cells. In addition, NC009-1 increased the expression of p-ERK (T202/Y204) and p-EEF2K (S366), LM-021 decreased the expression of p-AMPK (T172) and p-EEF2K (S398), whereas LM-036 acted on both pathways simultaneously to increase S366 phosphorylation and decrease S398 phosphorylation of EEF2K, leading to decreased EEF2K activity. Subsequent corresponding reduction in p-EEF2 (T56) inhibitory activity enhanced the expression of p-CREB (S133) and its downstream BDNF, BCL2 protein expression, and promoted neurite outgrowth. The study results can serve as a reference for the development of novel and potential Alzheimer’s disease treatment strategies.