利用CRE motifs報導細胞和Aβ-GFP細胞篩選BDNF受體TRKB的小分子促效劑做為阿茲海默症治療策略

CRE Motifs Reporter and Aβ-GFP Cellular Models to Screen Therapeutic Small Molecule Agonist Drugs Targeting BDNF Receptor TRKB as Treatment Strategy for Alzheimer's Disease

鄧伃珊(Teng Yu-Shan)

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(2019年)

https://doi.org/10.6345/NTNU201901050

摘要

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，會逐漸破壞認知功能、記憶和思考能力。腦源性神經滋養因子(Brain-derived neurotrophic factor, BDNF)，透過活化高親和力受體--旋轉肌凝蛋白相關蛋白質激酶B (Tropomyosin-related kinase B, TRKB)，並隨後磷酸化環磷腺苷效應元件結合蛋白1 (cAMP responsive element binding protein 1, CREB)，來調節神經元存活(Neuronal survival)、新生(Neurogenesis)和神經可塑性(Neuroplasticity)。前人的研究發現BDNF和TRKB在AD患者腦中的表現量降低，且Aβ顯著下調BDNF、TRKB水平與降低CREB磷酸化。TRKB促效劑7,8-二羥基黃酮(7,8-Dihydroxyflavone, 7,8-DHF)，可改善AD小鼠模型的記憶缺陷。此外，黃芩素(Wogonin)透過增加BDNF和CREB表現，可提升海馬迴神經元缺失和認知功能障礙。這些證據顯示大腦中BDNF-TRKB訊息傳遞與AD的關聯性，因此增強TRKB訊息傳遞是有潛力的AD治療策略。先前本實驗室與本校化學系孫英傑老師合作，透過化合物數據庫的化合物相似性搜索進行虛擬篩選，得到DHFS-1 (即Quercetin)、DHFS-2、Kaempferol、Apigenin等4個7,8-DHF類似物。本研究以7,8-DHF為控制組，檢測Wogonin及這4個類似物作為TRKB促效劑的潛能。首先檢測化合物的溶解度(Solubility)，預測化合物的口服生物利用度(Oral bioavailability)、中樞神經系統有效性(CNS-activity)與血腦屏障通透性(Blood brain barrier permeability)，並檢測化合物捕捉自由基和抑制Aβ聚集的能力。之後建立人類CRE motifs驅動GFP表現的293報導細胞，由上述6個化合物中篩選出能顯著提升CREB轉錄活性的7,8-DHF、DHFS-1、DHFS-2、Apigenin。再利用誘導表現Aβ-GFP的人類SH-SY5Y細胞，檢測所篩選出的潛力TRKB促效劑的神經保護作用。7,8-DHF、DHFS-1、DHFS-2、Apigenin四者皆有抑制Aβ蛋白聚集、降低活性氧化物(ROS)以及促進神經突生長(Neurite outgrowth)之能力。四者中DHFS-1、DHFS-2、Apigenin可抑制凋亡蛋白酶-1 (Caspase-1)的活性，7,8-DHF、Apigenin可抑制乙醯膽鹼脂酶(Acetylcholinesterase)活性。此外，TRKB的RNA干擾(RNAi)作用，會抑制7,8-DHF、DHFS-1、DHFS-2、Apigenin促進神經突生長能力。因此除了已知的7,8-DHF外，DHFS-1、DHFS-2、Apigenin也可能是透過TRKB訊息傳遞來促進神經保護作用。本研究將衍生新穎TRKB促效劑，以期提供阿茲海默症治療策略。

關鍵字

阿茲海默症 ； β-澱粉樣蛋白 ； 腦源性神經滋養因子 ； 受體原肌球蛋白受體激酶B促效劑 ； 細胞模式

並列摘要

Alzheimer's disease (AD) is an age-related progressive degenerative disorder that slowly destroys cognitive functions, memory, and thinking skills. Brain-derived neurotrophic factor (BDNF) is a member of neurotrophin family of growth factor that regulates neuronal survival, neurogenesis and neuroplasticity by activating the high-affinity tropomyosin-related kinase B (TRKB) receptor and subsequently phosphorylating cAMP responsive element binding protein 1 (CREB). BDNF and TRKB expression levels have been found to be reduced in brains of AD patients. Treatment with oligomeric Aβ also significantly down-regulates BDNF and TRKB expression, and decreases CREB phosphorylation. Systemic administration of TRKB agonist 7,8-dihydroxyflavone (7,8-DHF) improves memory deficits in AD mouse model. In addition, wogonin (an O-methylated flavone) attenuates hippocampal neuronal loss and cognitive dysfunction by increasing BDNF and CREB expression. These lines of evidence indicate deterioration of brain BDNF-TRKB signaling and enhancement of TRKB signaling for a promising AD treatment strategy. Previously our laboratory collaborated with Professor Ying-Chieh Sun from Department of Chemistry, NTNU to develop new TRKB agonists. Four 7,8-DHF analogous compounds, DHFS-1 (quercetin), DHFS-2, kaempferol, and apigenin, were found through virtually screened compound databases using compound similarity search engines. In this study, the potential of wogonin and these four analogous compounds as TRKB agonists were examined, using 7,8-DHF as controls. These compounds were firstly tested for solubility, and predicted for oral bioavailability, central nervous system (CNS)-activity, and blood brain barrier permeability. In addition, free radical scavenging and Aβ aggregation inhibitory activities were examined. Then human 293 reporter cells expressing CRE motifs-driving GFP were established to test these six compounds for enhancing CREB-mediated transcription. Among the tested compounds, 7,8-DHF, DHFS-1, DHFS-2, and apigenin significantly increased the transcriptional activity of CREB. Neuroprotective effects of the potential TRKB agonists were further examined in human SH-SY5Y cells expressing Aβ-GFP. 7,8-DHF, DHFS-1, DHFS-2, and apigenin markedly reduced Aβ aggregation and associated reactive oxygen species (ROS), as well as promoted neurite outgrowth in Tet-On Aβ-GFP SH-SY5Y cells. In addition, DHFS-1, DHFS-2, and apigenin inhibited the caspase 1 activity, and 7,8-DHF and apigenin inhibited acetylcholinesterase activity in Aβ-GFP-expressing SH-SY5Y cells. The neurite outgrowth promotion of 7,8-DHF, DHFS-1, DHFS-2, and apigenin was counteracted by knockdown of TRKB. Therefore, in addition to the known 7,8-DHF, the neuroprotective effects of DHFS-1, DHFS-2, and apigenin may also mediate through TRKB signaling. The study could derive novel TRKB agonists to provide therapeutic strategies in AD.

並列關鍵字

Alzheimer’s disease ； Amyloid β ； BDNF ； TRKB agonist ； cell model

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