fmr1基因剔除影響斑馬魚社會行為發育與Omega-3多元不飽和脂肪酸之治療效果

The Developmental Abnormalities in Social Behavior and Therapeutic Effects of Omega-3 Polyunsaturated Fatty Acid in fmr1 Knock-Out Zebrafish (Denio rerio)

徐茂庭(Hsu Mao-Ting)

指導教授 ： 呂國棟

國立臺灣師範大學/理學院/生命科學系/碩士(2016年)

https://doi.org/10.6345/NTNU202204567

摘要

X染色體脆折症(fragile X syndrome, FXS)是最普遍的遺傳性智能障礙症。前人文獻指出FXS在男性及女性的好發比例分別約1/4000及1/8000，而疾病形成原因主要為在表現fmr1基因的啟動子(promoter)上具有過多的CGG重複導致fmr1基因無法正常表現蛋白質(fragile x mental retardation protein, FMRP)與其功能。FXS常見的徵狀包含學習障礙、注意力不集中、過動、焦慮、自閉行為以及社會行為異常等等。而最近斑馬魚由於純熟的基因轉殖技術以及發育學上豐富的研究成果，因此在探討人類疾病中被視為理想的模式動物。而在我們實驗室先前的研究發現fmr1基因剔除斑馬魚會有許多不正常的行為表現，像是過動、不正常的焦慮表現以及恐懼記憶受損。此外，在先前的研究也指出fmr1基因與社會行為的發育有相關。為了探究此議題，我們應用群游行為、群游偏好行為、自發性空間探索行為以及焦慮行為進行相關實驗。此外，我們也利用不飽和脂肪酸的補充來探究其對於在fmr1基因剔除斑馬魚異常行為表現之治療效果。
而我們先前研究發現fmr1基因剔除斑馬魚會有群游偏好行為提早發育的現象，並且其現象與焦慮行為有相關，但在此研究中並沒有針對單純同種魚群以及異種魚群的群游進行研究探討。以外，我們繼續探討此群游行為的差異並利用另一種焦慮行為分析方法(novel tank task)探討兩者的相關性。而結果顯示不論在野生型以及fmr1基因剔除斑馬魚，群游行為會在受精後14天形成，並在基因剔除的動物中的群游程度會比正常野生型來得高。此外，研究也發現基因剔除隻斑馬魚在受精後28天會有過動以及焦慮異常的現象，這些結果指出群游行為的差異與過動及焦慮異常有相關。
另一方面，我們也想探討ω-3不飽和脂肪酸(n-3 polyunsaturated fatty acids, n-3 PUFAs)對於fmr1基因剔除斑馬魚行為異常之治療效果，因為在其中的二十二碳六烯酸(docosahexaenic acid, DHA)以及二十碳五烯酸(eicosapentaenoic acid, EPA)屬於必需的養分，而文獻指出補充DHA以及EPA可以降低過早死亡的風險以及提升心智能力。此外，在給予ω-3不飽和脂肪酸補充後可降低異常行為的發生，其中包含社會行為異常、自閉以及注意力缺陷。而近期的報告指出在fmr1基因剔除小鼠補充ω-3不飽和脂肪酸可以治療異常的行為，例如：情緒變化、社會互動以及非空間性的記憶能力。在我們的研究中，基因剔除斑馬魚端腦中的grin1b表現量顯著的下降，而htr2a以及htr2cl1兩者表現量顯著的提升。而透過氣相色譜法-質譜法聯用分析全魚的脂肪酸比例，發現基因剔除斑馬魚的不飽和脂肪酸比一般野生型低。此外，我們也發現ω-3不飽和脂肪酸的補充可以治療其行為異常，像是不正常的焦慮表現以及恐懼記憶受損。然而，在肝臟中的ω-3不飽和脂肪酸合成相關酵素的基因表現量沒有顯著的差異。統整以上結果，基因剔除斑馬魚之行為異常可能跟不飽和脂肪酸比例較低有關，以及ω-3不飽和脂肪酸補充對於X染色體脆折症是一種具有潛力的治療方法。

關鍵字

斑馬魚 ； X染色體脆折症 ； 自閉症 ； fmr1 ； 社會行為 ； ω-3不飽和脂肪酸

並列摘要

Fragile X syndrome (FXS) is the most generally hereditary form of human mental retardation. Previous researches showed that the onset ratio of FXS is approximately 1/4000 in male and 1/8000 in female. It frequently induced by triplet repeat expansion (CGG) mutation in fragile X mental retardation 1 (fmr1) gene promoter, and resulted in absence of the fragile x mental retardation protein (FMRP) expression. The common symptoms of fragile X patients include learning disabilities, inattention, hyperactivity, anxiety, autistic behaviors, social impairments, as well as other behavioral abnormalities. Recently, zebrafish is considered as an ideal animal model for studying human neurological disorder, due to the progression of genetic techniques and accumulated knowledge on the developmental biology of zebrafish. Our previous results demonstrated the behavioral abnormalities in fmr1 knock out zebrafish such as hyperactivity, abnormal anxiety level, fear memory impairment and autism-like behavior. The present study was aimed to study the functional role of fmr1 gene on the development of social behavior. For achieving this goal, behavioral experiment including shoaling behavior, shoaling preference, locomotor activity monitoring and novel tank task were applied. In addition, we also evaluated the possible therapeutic effect of dietary supplement with polyunsaturated fatty acid on the behavioral abnormalities in fmr1 KO zebrafish.
Our results demonstrated the precocious development of shoaling preference behavior is found in fmr1 KO zebrafish which might be resulted from the elevated anxiety level in fmr1 KO zebrafish, but do not affect the development of shoaling preference on conspecific zebrafish. We determined the relation between shoaling preference behavior and anxiety level by novel tank test, a well-established behavioral test for anxiety-like behavior in zebrafish. Results indicated the shoaling behavior appeared after 14 dpf, and the level of shoaling in fmr1 KO zebrafish is higher than the wildtype control. Furthermore, the locomotor activity was elevated in fmr1 KO zebrafish at 28 dpf, and they expressed higher anxiety level in novel tank test. These results suggest that the change of shoaling behavior in fmr1 KO zebrafish may be resulted from hyperactivity and increase of anxiety.
We also evaluated the possible therapeutic effects of omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as ALA, EPA, and DHA, on behavioral abnormalities in fmr1 KO fish. It is well-known that DHA and EPA are essential nutrients which can reduce the mortality of premature born infants, and they have been proved to enhance mental function in both aging and Alzheimer patients. Studies also suggest the dietary supplement of n-3 PUFAs can reduce the behavioral abnormalities including social-relative problems, autistic, and attention deficit. Recently, n-3 PUFAs supplementation was proved to rescue the behavioral abnormalities, such as alterations in emotionality, social interaction and non-spatial memory in fmr1 KO mice. In our experiments, it indicated that the telencephalic gene expression of grin1b declined in fmr1 KO zebrafish, but htr2a and htr2cl1 elevated after fmr1 loss-off-function. According to our gas chromatography-mass spectrometry (GC-MS) results, a reduction in total PUFAs of the fmr1 KO zebrafish body was found which raised the possibility of using n-3 PUFAs as an adjunctive therapy for FXS. Our results demonstrated that after 4 weeks of n-3 PUFAs dietary treatment can partially rescue abnormal behaviors, such as elevated anxiety level and avoidance learning impairment. However, in the liver gene expression of omega-3 synthesis enzymes, there was no significantly difference between wild-type and fmr1 KO zebrafish. We suggested that the lack of PUFAs may account for the abnormal behaviors in fmr1 KO zebrafish, and the n-3 PUFAs supplementation is a potential therapy agent for FXS patients.

並列關鍵字

zebrafish ； fragile X syndrome ； autism ； fragile X mental retardation 1 ； social behavior ； omega-3 polyunsaturated fatty acids

參考文獻

Abitbol M, Menini C, Delezoide AL, Rhyner T, Vekemans M, Mallet J (1993) Nucleus basalis magnocellularis and hippocampus are the major sites of FMR-1 expression in the human fetal brain. Nat Genet 4:147-153.

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