以斑馬魚nicastrin基因異型合子突變種為神經退化模型之研究

Neurodegeneration Study On Zebrafish nicastrin Heterozygous Mutants

陳文傑(Wen-Jie Chen)

指導教授 ： 江運金

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201803306

摘要

目前已知伽馬分泌酶 (γ-secretase)在神經退化疾病中扮演重要角色，而Nicastrin是伽馬分泌酶其中一個次單元，其功能為調控伽馬分泌酶與受質的結合。先前研究報導指出在老鼠神經細胞條件式敲除nicastrin基因會導致神經退化，然而其機制尚不清楚。我們實驗室先前也在一個異型合子nicastrin基因突變(nicastrinhi1384/+) 斑馬魚中發現神經退化的症狀，包括高度磷酸化Tau蛋白增加以及認知能力下降。因此我們希望全盤檢測分析此種斑馬魚的神經退化症狀，並評估其是否能成為神經退化研究的模式生物。我們使用斑馬魚行為軌跡追蹤系統探討nicastrinhi1384/+斑馬魚幼魚行為，發現其在光照刺激期間相較同齡野生種 (wild type) 斑馬魚幼魚有更高的活動力。另一方面，使用T型迷宮行為試驗檢測此nicastrinhi1384/+斑馬魚成魚的認知能力，在排除運動能力差異的影響後，初步結果顯示年齡為12個月及18個月的nicastrinhi1384/+斑馬魚，其認知能力明顯變得比同齡野生種 (wild type) 斑馬魚還要低。然而由於先前在行為實驗中使用的食物被用盡且無法再取得，我們更換了行為實驗中的食物，後續的實驗無法完全重複上述行為實驗結果，但18個月的nicastrinhi1384/+斑馬魚認知能力依然有較差的趨勢。此外，使用基因微陣列分析T型迷宮行為試驗受試班馬魚腦中的轉錄組 (transcriptome)，結果顯示在nicastrinhi1384/+斑馬魚腦中nicastrin訊息RNA的表現量下降，而其餘伽馬分泌酶次單元的訊息RNA表現量並不受影響。上述結果皆在即時聚合酶鏈鎖反應 (quantitative real-time PCR, qPCR) 實驗中得到證實。最後，運用轉錄組功能分析基因微陣列的數據，結果預測在轉錄層級上年長的nicastrinhi1384/+斑馬魚腦中，神經退化症狀受到促進，另一方面，神經存活、神經分化及神經功能的維持受到抑制，說明年長的nicastrinhi1384/+斑馬魚的腦在轉錄的層級上有神經退化的現象。然而使用末端脫氧核苷酸轉移酶脫氧尿苷三磷酸切口末端標記 (Terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL) 檢測腦中細胞凋亡情形，結果顯示18個月的nicastrinhi1384/+與同齡野生種組別並無差異，未來有必要使用其他神經退化的生物標誌來檢測nicastrinhi1384/+斑馬魚的腦中是否出現神經退化的症狀。
總結來說，nicastrinhi1384/+斑馬魚幼魚在光照環境下相較於野生種斑馬魚幼魚有較高的活動力，表示nicastrinhi1384/+斑馬魚與野生種斑馬魚在神經迴路發育過程中可能有些差異。另一方面，我們證實了在nicastrinhi1384/+斑馬魚腦中，nicastrin訊息RNA表現量下降。再者，行為實驗及轉錄組分析結果皆顯示年長nicastrinhi1384/+斑馬魚有神經退化的趨勢。上述結果顯示 nicastrinhi1384/+斑馬魚具有做為神經退化動物模型的潛力。未來我們需要在年長的nicastrinhi1384/+斑馬魚的腦中檢視其他神經退化症狀，例如神經發炎反應、神經細胞數量減少、神經萎縮及突觸減少等等。

關鍵字

斑馬魚 ； 神經退化 ； 行為試驗 ； 伽瑪分泌酶 ； nicastrin基因異型合子突變 ； 基因微陣列

並列摘要

Nicastrin is a subunit of γ-secretase which functions to regulate binding of substrates to γ-secretase and has been implicated in neurodegeneration. However, the mechanism is unclear. Previously, we discovered several neurodegenerative phenotypes, including increased hyperphosphorylated Tau proteins and cognition deficits in aged nicastrinhi1384/+ zebrafish (unpublished data). Accordingly, we intended to comprehensively examine if nicastrinhi1384/+ zebrafish can be a vertebrate model to study neurodegeneration. In the present work, we conducted a visual motor response test on nicastrinhi1384/+ zebrafish larva and uncovered that they were more active under light stimulus than wild type zebrafish larva. Besides, original T-maze behavioral tests on nicastrinhi1384/+ zebrafish demonstrated that memory of 12 and 18 month nicastrinhi1384/+ zebrafish were impaired compared to wild type zebrafish at the same age after excluding the influence of mobility variation. However, as the food which had been used in the previous behavioral tests was run out and unavailable, we switched to a new kind of food and failed to recapitulate the original behavioral tests results in the latter behavioral tests. Nevertheless, memory of nicastrinhi1384/+ zebrafish was still worse than wild type at 18 month of age. On the other hand, transcriptomic analysis on the brains of subject zebrafish of behavioral tests by using microarray showed that nicastrin mRNA level was downregulated in nicastrinhi1384/+ zebrafish, whereas mRNA expression of other γ-secretase subunits was not affected, which were verified by qPCR experiments. Last but not least, disease and functional analysis showed neurodegeneration associated phenotypes were activated, while neuronal survival and functions associated phenotypes were inhibited, implicating that nicastrinhi1384/+ zebrafish have age-dependent neurodegeneration at transcriptional level. However, TUNEL staining displayed no extensive neuronal apoptosis in the telecephalon of 18 month nicastrinhi1384/+ zebrafish.
In conclusion, nicastrinhi1384/+ zebrafish larva displayed higher activity than wild type under light stimulus, and the mechanism needs to be further clarified. On the other hand, we verified the expression of nicastrin was reduced in the brains of adult nicastrinhi1384/+ zebrafish by both microarray and qPCR. Furthermore, both behavioral and transcriptomic analysis demonstrated the trend of age-dependent neurodegeneration in nicastrinhi1384/+ zebrafish, suggesting that nicastrinhi1384/+ zebrafish have potential to be a neurodegeneration model. In the future, we should examine other neurodegeneration phenotypes, such as neuroinflammation, neuronal loss, neuritic dystrophy, and synaptic loss in the brains of aged nicastrinhi1384/+ zebrafish.

並列關鍵字

Zebrafish ； Neurodegeneration ； gamma-secretase ； behavioral tesst ； microarray ； nicastrinhi1384/+

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