利用斑馬魚動物模式探討人類聽力損傷

Zebrafish as a powerful platform to study human hearing loss

張簡如(Ju Chang-Chien)

指導教授 ： 徐再靜 ； 楊建洲

中山醫學大學/醫學院/生化微生物免疫研究所/博士(2017年)

摘要

聽力損傷(hearing loss)為一種常見的人類疾病，內耳毛細胞在聽覺形成扮演重要的角色，目前已知遺傳基因變異、環境因素(如：噪音、耳毒性藥物和細菌或病毒性感染)或兩者兼之皆會造成毛細胞損傷而導致永久性的聽力缺失。斑馬魚和人類的基因體具有87 %同源性，且斑馬魚內耳之聽覺毛細胞在發育、型態以及聽覺功能上皆具有高度保留性。本研究利用Tol2基因轉殖系統建立一個毛細胞螢光斑馬魚品系Tg(pvalb3b:TagGFP)，其專一性在內耳聽斑及側線神經叢的毛細胞表現綠色螢光蛋白，同時利用野生型及毛細胞螢光斑馬魚作為模式動物以探討聽力缺失的致病機轉。學習語言前聽力損傷可能由單一基因或多個基因突變所造成，目前已知有59個基因的突變會導致聽力損傷，其主要與內耳中鉀離子及聽覺神經誘發電位之平衡有密切關係，其中連接通道(gap junction)在鉀離子再循環、耳蝸內離子平衡及聽力形成的過程中扮演重要的角色。先前實驗室已利用細胞模式探討人類GJB2基因突變對其通道功能的影響，並已知有兩個同源於人類與老鼠GJB2基因的斑馬魚connexin基因，分別為cx27.5和cx30.3，其會表現於內耳等多種成魚組織中。本研究進一步利用人類HeLa細胞模式分別表現cx27.5和cx30.3基因，結果發現斑馬魚Cx27.5蛋白質會堆積於細胞質中，而Cx30.3蛋白質則是以gap junction斑塊的形式表現於細胞膜上，與人類CX26的表現型態較為相似。此外我們依照先前實驗室所發現之GJB2(CX26)基因突變點分別建構六個相對應的cx30.3突變基因之表現質體(p.G4E、p.G4D、p.R75H、p.R75Q、p.R186K和p.R186Q)，結果顯示六個Cx30.3突變蛋白於HeLa細胞中的表現形態亦與CX26突變蛋白相似。我們利用免疫組織染色法亦發現Cx30.3蛋白質會以斑塊形式表現在成魚內耳毛細胞之細胞膜，同時以morpholino抑制斑馬魚內生性cx30.3基因則發現cx30.3 morphants (wild-type-like和heart edema)於受精後72小時會有耳石縮小、耳石距離縮短以及前聽斑之毛細胞減少等發育異常的現象。因此我們推測斑馬魚cx30.3基因較cx27.5基因更相似於人類GJB2基因，且可能會參與斑馬魚的內耳發育，期盼未來可藉由斑馬魚模式動物來探討人類CX基因突變造成聽障的機制。
胺基酸甘醣類藥物(如：新黴素、鏈黴素和慶大黴素等)則為一種常見的耳毒性藥物，其在臨床用藥上易誘發耳毒性而導致聽力損傷之副作用。目前阿魏酸衍生物在美國臨床試驗上已用於預防及治療聽力相關疾病，因此我們利用毛細胞螢光斑馬魚作為模式動物以評估阿魏酸是否可減緩新黴素的耳毒性傷害，進而探討藥物的作用機轉及其對斑馬魚行為之影響。本研究結果顯示受精後四天大之斑馬魚胚胎經阿魏酸前處理，或在阿魏酸與新黴素共同處理的情況下，其側線毛細胞皆會受到阿魏酸的保護而免受新黴素耳毒性藥物的損傷。我們發現阿魏酸與新黴素間並無直接的交互作用，但阿魏酸可減緩新黴素經由機械性傳導通道快速進入毛細胞，並可作為一種抗氧化物以降低細胞內的活性氧化物質含量進而抑制細胞凋亡現象。同時我們也建立一個斑馬魚行為分析平台藉此探討藥物對斑馬魚的行為影響，結果顯示阿魏酸可有效保護毛細胞免受新黴素的破壞，且斑馬魚對於外界環境的刺激仍具有高度的敏感度。期許未來我們可利用斑馬魚模式動物和行為分析平台更深入瞭解耳毒性藥物的分子致病機制，例如：支持細胞、內耳之離子平衡等，並將其作為一個藥物篩選平台以快速篩選誘發、預防及治療耳毒性聽力缺失的藥物。

關鍵字

聽力損傷 ； 連接蛋白 ； 基因突變 ； 毛細胞 ； 耳毒性 ； 新黴素 ； 阿魏酸 ； 斑馬魚 ； 螢光轉殖斑馬魚

並列摘要

Hair cells in the inner ear play essential roles in hearing. Genetic mutations and many environmental factors, such as acoustic trauma, age and ototoxic drugs, result in hair cell malfunction and then hearing loss, which is a common and irreversible sensory disorder in human populations. Mechanosensory hair cells in the inner ear are highly conserved between zebrafish and mammals in terms of development, morphology, and physiological function so that we created a novel hair cell transgenic reporter zebrafish (pvalb3b:TagGFP) using Tol2 system to study hearing impairment. To date, around 59 auditory genes have been demonstrated, some of which are involved in the recycling potassium and the maintenance of the resting potential. Connexin (CX)-based gap junction (GJ) channels are important for recycling potassium in the inner ear, which are encoded by a family of the CX, such as GJB2. However, the mutational mechanisms of connexin involved in nonsyndromic deafness remain to be elucidated. Previous studies had found that two homologs of GJB2 gene in zebrafish (cx27.5 and cx30.3) expressed during embryogenesis and in multiple adult tissues, including inner ear. In our studies using the connexin-free HeLa cell expression system, Cx27.5 proteins were retained in the cytoplasm, whereas Cx30.3 proteins were targeted to plasma membrane forming GJ plaques as human CX26. In addition, six substitution mutations of zebrafish cx30.3 revealed similar subcellular localization in cells as that of human CX26. IHC staining of tissue sections also showed that Cx30.3 was localized in the cell membrane of hair cells within the inner ear. In functional studies of Cx30.3 using specific morpholino, by 72 hpf, cx30.3 morphants exhibited wild-type-like and heart edema phenotypes with smaller inner ears while the decrease in macular hair cell number was found, which was rescued by cx30.3 cRNA. It is found that zebrafish Cx30.3 and mammalian CX26 may play alike roles in the development and organization of the inner ear; meanwhile, the structure-function relationships in zebrafish Cxs are conserved with that in mammals.
In addition to genetic variations and deafness, ototoxicity is an ignored side effect of aminoglycosides in clinical medicine, but its mechanisms remain largely unknown. Ferulic acid (FA) derivatives have been applied in the clinical trials for preventing and treating auditory dysfunctions in America, and in the present study, effects of FA on neomycin-elicited ototoxicity are demonstrated in the lateral line neuromasts of hair cell reporter transgenic line. In transgenic fish treated with FA before and during neomycin exposure, FA conferred sufficient protection against hair cell damage across a wide range of neomycin concentrations by combining the uptake block of neomycin into sensory hair cells, the antioxidant effects on intracellular ROS, as well as the reduction of TUNAL reactions. No direct dug-drug interaction was found between both. In addition, results of our study regarding zebrafish swimming behavior were consistent with hair cell survival in transgenic fish at 7 dpf. Hence, the protective mechanisms of FA against aminoglycoside ototoxicity were elucidated in this study.
In conclusion, our study provides information for understanding the importance of hair cells in normal hearing function and their possible mechanisms, and that may be of use in the prevention and therapy of hearing impairment caused by aminoglycoside ototoxicity and genetic ablation in connexin genes. Also we established a novel hair cell reporter line and the zebrafish swimming behavior method, and that may be of benefit in hair cell research and screening applications of compounds for inducing, preventing or therapeutic hearing loss.

並列關鍵字

hearing loss ； connexin ； genetic mutations ； hair cell ； ototoxicity ； neomycin ； ferulic acid ； zebrafish ； transgenic zebrafish

參考文獻

136. Huth, M.E., A.J. Ricci, and A.G. Cheng, Mechanisms of aminoglycoside ototoxicity and targets of hair cell protection. Int J Otolaryngol, 2011. 2011: p. 937861.

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