評估TrKB之潛力促效劑於阿茲海默氏症細胞與動物模式之神經保護效果

Evaluation of the neuroprotective effects of potential TrkB agonist in Alzheimer’s disease cell and mouse models

范家豪(Fan Chia-Hao)

指導教授 ： 謝秀梅

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/NTNU201901046

摘要

阿茲海默氏症(Alzheimer’s disease, AD)是目前最常見的神經退化性疾病之一，至今尚無顯著有效的治療方法。這種全球性的健康問題不論是對於個人或社會都有極大的影響。AD在病理學上主要有兩個病徵：類澱粉蛋白(β-amyloid peptides, Aβ)堆積成的斑塊(plaques)以及Tau蛋白過度磷酸化所導致的神經纖維糾結(neuro-fibrillary tangles, NFTs)。許多研究證實，在失智症病患腦中之腦源性神經滋養因子(Brain-derived neurotrophic factor, BDNF)濃度較低，且此現象可令更多的Aβ生成。BDNF及其受體原肌球蛋白相關激酶B(tropo-myosin-related kinase B, TrkB)，可調節長期增益效應(LTP)、長期抑制效應(long-term depre-ssion, LTD)、軸突出芽、樹突增生、突觸可塑性和神經元分化。7,8-二羥基黃酮(7,8-dihydroxyflavon, 7,8-DHF)是目前已知的TrkB促效劑，可與細胞膜上的TrkB受體結合並引發同源二聚化，啟動下游之訊息傳導。本研究是使用結構與7,8-DHF類似之12種新型的TrkB促效劑，經過MTT實驗評估發現12種促效劑對神經細胞都無明顯細胞毒性及神經突生長之影響。接著於小鼠海馬迴細胞初級培養給予Aβ25-35誘導神經損傷作為AD模式篩選平台，結果顯示LMDS-1能有效緩解因Aβ25-35寡聚體所導致的神經突長度及分支數目降低之現象，因此選擇LMDS-1進入動物實驗測試，並以7,8-DHF為正控制組。先利用立體定位注射Aβ25-35至小鼠海馬迴CA1區域以建立AD模式動物，再給予7,8-DHF或LMDS-1之處理，並於期間進行一系列行為實驗，結果顯示LMDS-1可有效改善因Aβ25-35誘導所導致的短期及長期記憶受損。在病理分析上，我們利用免疫組織化學染色和西方墨點法分析小鼠海馬迴組織，結果顯示LMDS-1可有效改善因Aβ25-35誘導所導致的Aβ堆積、成熟神經細胞缺失、pERK及synaptophysin表現量降低。由病理分析結果推論，LMDS-1可能是經由pY516TrkB活化Ras/MAPK路徑來激活CREB轉錄因子，進而上調mBDNF之表現量，改善因Aβ25-35所導致的記憶缺失。綜合上述結果，LMDS-1是具有治療AD潛力的新型TrkB促效劑，能夠改善動物記憶功能，並緩解AD代表性之病理特徵，希望能為日後AD研究提供一些新的方向與目標。

關鍵字

阿茲海默氏症 ； 類澱粉蛋白 ； Tau蛋白 ； 腦源性神經滋養因子 ； TrkB促效劑 ； 7,8-二羥基黃酮

並列摘要

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases that without significant therapeutics currently. This global health problem has a huge impact on individuals and social interactions. The biological disorders of AD includes memory impairment and cognitive dysfunctions. The two main pathological features often identified are extracellular Aβ deposition and hyperphosphorylation of tau protein. Many studies have shown that abnormalities of neurotrophin, brain-derived neurotrophic factor (BDNF), occur in patients with dementia which promote synthesis of A peptides. BDNF and its receptor, tropomyosin-related kinase B (TrkB), regulate long-term depression (LTD) and long-term potentiation (LTP), axonal sprouting, proliferation of dendritic arbor, synaptic plasticity, and neuronal differentiation. It is shown 7,8-dihydroxyflavon (7,8-DHF) can bind to TrkB receptor on the cell membrane and initiate homodimerization, leading to the pathway activation. In this study, 12 novel TrkB agonists candidates similar in structure of 7,8-DHF were used. After evaluation by MTT assay, 12 compounds were found to have no obvious impact in cytotoxicity and neurite outgrowth on SH-SY5Y cells. We further used Aβ25-35 to induce neuronal damage in the mouse hippocampal primary culture and which was used as an AD model to screen the 12 compounds. The results showed that LMDS-1 can effectively alleviate the decrease in neurite length and number of branches caused by Aβ25-35 oligomers. LMDS-1 was further applied to animal experiments and uses 7,8-DHF as a positive control. AD model was generated by stereotactic injection of Aβ25-35 into the hippocampal CA1 region of mice, and then treated with 7,8-DHF and LMDS-1. A series of behavioral experiments were performed and the results showed that LMDS-1 could effectively improve the impairment in short-term and long-term memory caused by Aβ25-35 induction. In pathological analysis, we used immunohistochemical staining and western blotting showed that LMDS-1 can effectively alleviate the symptom of AD, including the accumulation of Aβ and the loss of mature nerve cells. The levels of pERK and synaptophysin were also attenuated by the treatment of LMDS-1.
Inferred from the pathological analysis results, LMDS-1 may activate the Ras/MAPK pathway via pY516TrkB to activate the CREB transcription factor, thereby upregulating the expression of mBDNF.
　　Based on the above results, LMDS-1 is a novel TrkB agonist with the potential to treat AD. It can improve the memory function of animals and alleviate the pathological features of AD. It hopes to provide some new directions and goals for future AD research.

並列關鍵字

Alzheimer's disease ； Aβ ； BDNF ； TrkB ； Tau ； 7,8-DHF

參考文獻

Arancibia S, Silhol M, Moulière F, Meffre J, Höllinger I, Maurice T, Tapia-Arancibia L (2008) Protective effect of BDNF against beta-amyloid induced neurotoxicity in vitro and in vivo in rats. Neurobiology of Disease 31:316-326.

Blurton-Jones M, Kitazawa M, Martinez-Coria H, Castello NA, Müller F-J, Loring JF, Yamasaki TR, Poon WW, Green KN, LaFerla FM (2009) Neural stem cells improve cognition via BDNF in a transgenic model of Alzheimer disease. Proc Natl Acad Sci U S A 106:13594-13599.

Brothers HM, Gosztyla ML, Robinson SR (2018) The Physiological Roles of Amyloid-β Peptide Hint at New Ways to Treat Alzheimer's Disease. Frontiers in Aging Neuroscience 10.

Calissano P, Matrone C, Amadoro G (2010) Nerve growth factor as a paradigm of neurotrophins related to Alzheimer's disease. Developmental Neurobiology 70:372-383.

Castello NA, Nguyen MH, Tran JD, Cheng D, Green KN, LaFerla FM (2014) 7,8-Dihydroxyflavone, a small molecule TrkB agonist, improves spatial memory and increases thin spine density in a mouse model of Alzheimer disease-like neuronal loss. PLoS One 9:e91453-e91453.