利用Aβ折疊報導細胞篩選黃酮及香豆素衍生物作為促效劑標的BDNF受體TRKB的阿茲海默症治療策略

Abstract

阿茲海默症(AD)是與記憶缺陷和認知功能下降相關的進行性神經退行性疾病。病理學上AD的特徵是大腦中存在澱粉樣蛋白β (Aβ)聚集體(澱粉樣斑塊)和由高磷酸化的Tau蛋白形成的神經原纖維纏結(NFT)。腦源性神經營養因子(BDNF)是神經營養蛋白家族的成員，以高親和力與原肌球蛋白相關的受體激酶B (TRKB)結合，活化下游信號傳導，來促進神經元的存活、分化和神經可塑性。已經發現AD患者的腦中BDNF和TRKB表達水平降低，並且BDNF降低和TRKB受損導致AD中的神經變性。AD小鼠模型的海馬迴中施用TRKB特異性促效劑7,8-二羥基黃酮(7,8-DHF)，可改善了空間記憶並降低樹突損傷，表明TRKB信號傳導增強，為有希望的AD治療策略。本研究使用表達Aβ-GFP的人類神經母細胞瘤SH-SY5Y細胞，對15種黃酮和香豆素衍生物，進行Aβ聚集抑制性及神經保護性的評估。在測試的化合物中，ZN006、ZN013、ZN014、ZN015可抑制Aβ蛋白聚集、降低活性氧化物(ROS)、及抑制乙醯膽鹼脂酶(Acetylcholinesterase)活性，其中ZN014、ZN015並能抑制凋亡蛋白酶-1 (Caspase-1)活性及促進神經突生長(Neurite outgrowth)。在TRKB訊息傳遞路徑上，ZN014、ZN015可透過TRKB受體的Y516、Y817磷酸化，及下游ERK激酶T202/Y204磷酸化、AKT激酶S473磷酸化的活化，進一步促進轉錄因子CREB的S133磷酸化，來增強神經營養因子BDNF及抗凋亡蛋白BCL2的表現。RNA干擾(RNAi)靜默TRKB基因表現的實驗，驗證了這兩種化合物經由TRKB訊息傳遞的神經保護性。總言之，研究結果顯示ZN014和ZN015可抑制氧化壓力及乙醯膽鹼脂酶活性，並增強BDNF-TRKB訊息，來降低Aβ毒性，有潛能應用在AD的治療上。

Alzheimer’s disease (AD) is a progressive neurodegenerative disease associated with memory deficits and cognitive functions 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. Brain-derived neurotrophic factor (BDNF), a member of neurotrophin family, binds to the tropomyosin-related receptor kinase B (TRKB) with high affinity to activate the downstream signaling for neuronal survival, differentiation and plasticity. BDNF and TRKB expression levels have been found to be reduced in brains of AD patients and decreased BDNF and impaired TRKB contribute to neurodegeneration in AD. Administration of 7,8-dihydroxyflavone (7,8-DHF), a TRKB specific agonist, improved spatial memory and minimized dendrite loss in the hippocampus of AD mouse model, indicating enhancement of TRKB signaling for a promising AD treatment strategy. In this study, 15 flavone and coumarin derivatives were evaluated for Aβ aggregation inhibition and neuroprotection using human neuroblastoma SH-SY5Y cells expressing GFP-tagged Aβ folding reporter. Among the tested compounds, ZN006, ZN013, ZN014 and ZN015 reduced Aβ aggregation and reactive oxygen species (ROS), as well as inhibited acetylcholinesterase (AChE) activity. In addition, ZN014 and ZN015 inhibited caspase-1 activity and promoted neurite outgrowth in Aβ-GFP SH-SY5Y cells. Treatment of ZN014 and ZN015 increased Y516 and Y817 phosphorylation of TRKB receptor as well as downstream T202/Y204 phosphorylation of extracellular signal-regulated kinase (ERK) and S473 phosphorylation of AKT serine/threonine kinase 1 (AKT), to up-regulate S133 phosphorylation of cAMP-response-element binding protein (CREB) and its downstream BDNF and anti-apoptotic B-cell lymphoma 2 (BCL2). Knockdown of TRKB by RNA interference (RNAi) counteracted the neuroprotective effects of ZN014 and ZN015. In summary, the studied results suggest that ZN014 and ZN015 reduces Aβ neurotoxicity via inhibition of oxidative stress and AChE as well as activation TRKB signaling, and may provide new strategies in the treatment of AD.