利用ΔK280 TauRD折疊報導細胞篩選TRKB促效劑作為阿茲海默症治療策略

Using ΔK280 TauRD folding reporter cells to screen TRKB agonists as Alzheimer’s disease treatment strategy

翁鉦逵(Weng Zheng-Kui)

指導教授 ： 李桂楨

國立臺灣師範大學/理學院/生命科學系/碩士(2022年)

https://doi.org/10.6345/NTNU202201750

摘要

阿茲海默症(AD)為一種常見的與年齡相關的神經退化性失智症。其病理特徴包含在大腦中存在澱粉樣蛋白-β (Aβ)斑塊和神經纖維纏結(NFTs)的慢性累積。NFTs是由成對螺旋絲(PHF)在神經元內累積而成，而PHF主要是異常過度磷酸化Tau蛋白組成，會誘導一系列毒性事件，最終導致神經元變性和損傷。腦原性神經營養因子(BDNF)為一種神經營養因子。BDNF與其特異性受體原肌球蛋白相關激酶B (TRKB)結合後，導致TRKB二聚化和活化，隨後活化下游訊號，影響cAMP反應元件結合蛋白1 (CREB)在內的蛋白，來促進神經元存活和增加神經可塑性。證據顯示，在AD患者的大腦中BDNF和TRKB的表達量降低，因此增強TRKB訊號被認為是有希望的治療策略。本研究使用表達ΔK280 TauRD-DsRed促Tau錯誤折疊聚集的人類神經母細胞瘤SH-SY5Y細胞，評估8種化合物抑制Tau錯誤折疊的情形。檢測的化合物中，香豆素衍生物ZN-015和喹啉衍生物VB-030和VB-037可減少細胞內ΔK280 TauRD聚集，並促進神經突生長。通過抑制細菌衍生的ΔK280 TauRD-His在生化分析中的聚集，ZN-015、VB-030和VB-037顯示出化學伴侶活性。此三者雖皆不具DPPH自由基捕捉能力，但ZN-015具氧自由基吸收能力。另外，ZN015、VB-030和VB-037抑制了凋亡蛋白酶-1 (Caspase 1)及/或乙醯膽鹼酯酶(AChE)活性。進一步的TRKB訊息傳遞路徑分析顯示，處理ZN-015、VB-030、VB-037後，細胞內p-TRKB (Y516)、p-TRKB (Y817)、p-AKT (S473)、p-ERK (T202/Y204)、p-RSK (S380)、p-CaMKII (T286)、p-CREB (S133)、BDNF、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的干擾性核糖核酸(RNAi)靜默TRKB基因表現後，可抑制ZN-015、VB-030、VB-037的促進神經突生長。色氨酸螢光猝滅分析(Tryptophan fluorescence quenching assay)顯示，ZN-015、VB-030、VB-037與畢赤酵母表達的TRKB胞外結構域(TRKB-ECD)直接相互作用，支持其透過TRKB訊號發揮作用。這些化合物作為TRKB促效劑的研究，能為AD提供新的治療策略。

關鍵字

阿茲海默症 ； BDNF ； TRKB ； TRKB促效劑 ； Tau細胞模型

並列摘要

Alzheimer’s disease (AD) is a degenerative brain disease and the most common form of age-related dementia. It is well known that patients with AD have a progressive deposition of amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs) in the brain. NFTs are formed by intraneuronal accumulation of paired helical filaments composed of abnormally hyperphosphorylated Tau protein, which induce a series of toxic events to result in neurodegeneration and neuronal loss. Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor. Binding of BDNF to tropomyosin-related kinase B (TRKB) receptor leads to its dimerization and activation and subsequently induces the activation of several intracellular signaling cascades, which ultimately phosphorylates cAMP responsive element binding protein 1 (CREB) and promotes neuronal survival and neuroplasticity. Reduced BDNF and TRKB expression levels have been found in brains of AD patients. Therefore, enhancement of TRKB signaling is a promising AD treatment strategy. In this study, eight compounds were evaluated for inhibiting Tau misfolding in human neuroblastoma SH-SY5Y cells expressing pro-aggregant ΔK280 TauRD-DsRed. Among them, coumarin derivative ZN015 and quinoline derivatives VB-030 and VB-037 reduced ΔK280 TauRD aggregation and promoted neurite outgrowth in these cells. By inhibiting bacterial-derived ΔK280 TauRD-His aggregation in biochemical assay, ZN015, VB-030 and VB-037 displayed chemical chaperone activity. Although these three have no DPPH free radical scavenging ability, but ZN-015 has oxygen free radical antioxidant activity. In addition, ZN015, VB-030 and VB-037 inhibited the caspase 1 and/or acetylcholinesterase (AChE) activity. Studies of TRKB signaling revealed that treatment of ZN-015, VB-030 and VB-037 significantly increased the expression of p-TRKB (Y516), p-TRKB (Y817), p-AKT (S473), p-ERK (T202/Y204), p-RSK (S380), p-CaMKII (T286), p-CREB (S133), BDNF and BCL2, accompanying with reduced BAX expression in ΔK280 TauRD-DsRed cells. Furthermore, the neurite outgrowth promotion effect of ZN-015, VB-030 and VB-037 was counteracted by knockdown of TRKB using RNA interference (RNAi). Tryptophan fluorescence quenching analysis showed that ZN-015, VB-030, and VB-037 interact directly with Pichia pastoris-expressed TRKB extracellular domain (TRKB-ECD), supporting its role through TRKB signaling. The study of these compounds as TRKB agonists may provide new treatment strategies for AD.

並列關鍵字

Alzheimer’s disease ； BDNF ； TRKB ； TRKB agonist ； Tau cell model

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