以促進BDNF-TRKB訊息傳遞路徑作為阿茲海默氏症治療策略的ΔK280 TauRD-DsRed SH-SY5Y細胞模式分析
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2020
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阿茲海默氏症(Alzheimer’s disease, AD)是一種漸進性神經退行性疾病，最常見的臨床症狀是記憶力衰退及認知功能下降。阿茲海默氏症的病理學特徵為大腦中含有Amyloid β (Aβ)聚集形成的類澱粉斑塊，以及過度磷酸化的Tau蛋白形成的神經纖維糾結(Neurofibrillary tangle, NFT)。神經營養因子腦源性神經滋養因子(Brain-derived neurotrophic factor, BDNF)結合在受體型酪胺酸激酶B (Tropomyosin-related kinase B, TRKB)上時，會活化下游訊息傳遞路徑，最終磷酸化環磷腺苷效應元件結合蛋白1 (cAMP responsive element binding protein 1, CREB)，來促進神經元存活和神經可塑性。臨床研究發現阿茲海默氏症病人的海馬迴及皮層中，BDNF蛋白或mRNA的表現量降低。小分子TRKB促效劑7,8-DHF (7,8-二羥基黃酮)可改善AD小鼠認知功能且抑制海馬迴突觸喪失，顯示促進TRKB訊息傳遞，為阿茲海默氏症有潛能的治療策略。本研究使用誘導表現ΔK280 TauRD-DsRed的人類SH-SY5Y細胞，檢測7,8-DHF及Wogonin (黃芩素)、DHFS-1 (即Quercetin槲皮素)、DHFS-2、Kaempferol (山奈酚)、Apigenin (芹菜素)等類似物的神經保護作用。在測試的黃酮類化合物中，7,8-DHF、DHFS-1、Apigenin可增加表現ΔK280 TauRD-DsRed細胞中，DsRed的螢光通量。處理7,8-DHF、DHFS-1、Apigenin可以使細胞內熱休克蛋白HSPB1表現增加，並增加ΔK280 TauRD-DsRed融合蛋白的可溶性，但不影響融合基因的mRNA表現量。另外， 7,8-DHF、DHFS-1、Apigenin的處理，可顯著降低內活性氧化物的生成及凋亡蛋白酶1活性，並增加NRF2轉錄因子的表現及促進神經突生長，但僅Apigenin可降低乙醯膽鹼酯酶活性。進一步的TRKB訊息傳遞路徑分析顯示，處理7,8-DHF、DHFS-1、Apigenin後，ΔK280 TauRD-DsRed細胞內p-TRKB (Y817)、p-ERK (T202/Y204)、p-CREB (S133)、BCL2蛋白表現顯著增加，並伴隨著凋亡調節蛋白BAX的顯著降低。以TRKB的RNA干擾(RNAi)作用靜默TRKB基因表現後，可抑制7,8-DHF、DHFS-1、Apigenin的促進神經突生長。本7,8-DHF及其類似物做為TRKB促效劑的研究，可提供阿茲海默氏症新的治療策略。
Alzheimer’s disease (AD) is a progressive neurodegenerative disease. The most common clinical symptoms of AD are memory loss and cognitive decline. Pathologically AD is characterized by the presence of amyloid β (Aβ) aggregates in the brain (amyloid plaques) and neurofibrillary tangles (NFT) formed by hyperphosphorylated Tau protein. Neurotrophic factor brain-derived neurotrophic factor (BDNF) binds to tropomyosin-related kinase B (TRKB) to activate the downstream signaling pathway, which ultimately phosphorylates cAMP responsive element binding protein 1 (CREB) and promotes neuronal survival and neuroplasticity. Clinical studies have indicated that the expression levels of BDNF protein or mRNA are decreased in the hippocampus and cortex of AD brains. 7,8-Dihydroxyflavone (7,8-DHF), a small-molecule TRKB agonist, improved the cognitive function and inhibited the hippocampal synaptic loss in AD mice. The study demonstrated that promoting TRKB signaling is a potential treatment strategy for AD. In this study, 7,8-DHF and analogues compounds Wogonin, DHFS-1, DHFS-2, Kaempferol, Apigenin were examined for neuroprotective effects using human SH-SY5Y cells expressing ΔK280 TauRD-DsRed. Among the tested flavones, 7,8-DHF, DHFS-1 and Apigenin increased luminous flux of DsRed in ΔK280 TauRD-DsRed-expressing cells. Treatment of 7,8-DHF, DHFS-1 and Apigenin increased heat shock protein family B (small) member 1 (HSPB1) expression and ΔK280 TauRD-DsRed fusion protein solubility, without affecting fusion mRNA expression. In addition, 7,8-DHF, DHFS-1 and Apigenin significantly reduced reactive oxygen species (ROS) production and caspase 1 activity, and increased transcription factor NRF2 expression and promoted neurite outgrowth, whereas only Apigenin reduced acetylcholinesterase activity in ΔK280 TauRD-DsRed-expressing SH-SY5Y cells. Studies of TRKB signaling revealed that treatment of 7,8-DHF, DHFS-1 and Apigenin significantly increased the expression of p-TRKB (Y817), p-ERK (T202/Y204), p-CREB (S133) and BCL2, accompanying with reduced BAX expression in ΔK280 TauRD-DsRed cells. Furthermore, the neurite outgrowth promotion effect of 7,8-DHF, DHFS-1 and Apigenin was counteracted by knockdown of TRKB using RNA interference (RNAi). The study of 7,8-DHF and analogous compounds as TRKB agonists may provide new treatment strategies for AD.
Alzheimer’s disease (AD) is a progressive neurodegenerative disease. The most common clinical symptoms of AD are memory loss and cognitive decline. Pathologically AD is characterized by the presence of amyloid β (Aβ) aggregates in the brain (amyloid plaques) and neurofibrillary tangles (NFT) formed by hyperphosphorylated Tau protein. Neurotrophic factor brain-derived neurotrophic factor (BDNF) binds to tropomyosin-related kinase B (TRKB) to activate the downstream signaling pathway, which ultimately phosphorylates cAMP responsive element binding protein 1 (CREB) and promotes neuronal survival and neuroplasticity. Clinical studies have indicated that the expression levels of BDNF protein or mRNA are decreased in the hippocampus and cortex of AD brains. 7,8-Dihydroxyflavone (7,8-DHF), a small-molecule TRKB agonist, improved the cognitive function and inhibited the hippocampal synaptic loss in AD mice. The study demonstrated that promoting TRKB signaling is a potential treatment strategy for AD. In this study, 7,8-DHF and analogues compounds Wogonin, DHFS-1, DHFS-2, Kaempferol, Apigenin were examined for neuroprotective effects using human SH-SY5Y cells expressing ΔK280 TauRD-DsRed. Among the tested flavones, 7,8-DHF, DHFS-1 and Apigenin increased luminous flux of DsRed in ΔK280 TauRD-DsRed-expressing cells. Treatment of 7,8-DHF, DHFS-1 and Apigenin increased heat shock protein family B (small) member 1 (HSPB1) expression and ΔK280 TauRD-DsRed fusion protein solubility, without affecting fusion mRNA expression. In addition, 7,8-DHF, DHFS-1 and Apigenin significantly reduced reactive oxygen species (ROS) production and caspase 1 activity, and increased transcription factor NRF2 expression and promoted neurite outgrowth, whereas only Apigenin reduced acetylcholinesterase activity in ΔK280 TauRD-DsRed-expressing SH-SY5Y cells. Studies of TRKB signaling revealed that treatment of 7,8-DHF, DHFS-1 and Apigenin significantly increased the expression of p-TRKB (Y817), p-ERK (T202/Y204), p-CREB (S133) and BCL2, accompanying with reduced BAX expression in ΔK280 TauRD-DsRed cells. Furthermore, the neurite outgrowth promotion effect of 7,8-DHF, DHFS-1 and Apigenin was counteracted by knockdown of TRKB using RNA interference (RNAi). The study of 7,8-DHF and analogous compounds as TRKB agonists may provide new treatment strategies for AD.
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Keywords
阿茲海默氏症, 腦源性神經滋養因子, 受體型酪胺酸激酶B, TRKB促效劑, Tau細胞模式, Alzheimer’s disease, BDNF, TRKB, TRKB agonist, Tau cell model