多種A型核纖層蛋白突變導致肌失養症的功能和分子分析於斑馬魚模式之研究

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研究生：	潘劭維 Pan, Shao-Wei
論文名稱：	多種A型核纖層蛋白突變導致肌失養症的功能和分子分析於斑馬魚模式之研究 Functional and molecular analysis of LMNA mutations related muscular dystrophy in zebrafish
指導教授：	喻秋華 Yuh, Chiou-Hwa 汪宏達 Wang, Horng-Dar
口試委員:	鐘育志 Jong, Yuh-Jyh 游麗如 You, Li-Ru 紀雅惠 Chi, Ya-Hui
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物科技研究所 Biotechnology
論文出版年：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	93
中文關鍵詞：	斑馬魚、高通量基因克隆技術、A型核纖層蛋白、肌失養症
外文關鍵詞：	zebrafish, Gateway Cloning, LMNA, muscular dystrophy
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LMNA基因表現A型和 C核纖層蛋白(lamin A/C)，LMNA 的突變會導致一系列人類疾病，如發育異常、先天性肌營養不良和早衰，統稱為核纖層蛋白病症(laminopathy)。目前，核纖層蛋白病症無法完全根治，只能舒緩疾病症狀和支持治療。從診斷為先天性肌營養不良症的患者中鑑定出三個新突變 LMNA(L35P)、LMNA(A539V)、LMNA(W520G) 和兩個已知突變 LMNA(E358K) 和 LMNA(R453W)。在該項目中，我們建立了過量表達野生型和5種A型核纖層蛋白突變在骨骼肌的轉基因斑馬魚，並監測了游泳行為、肌肉耐力和組織病理學，並使用這些LMNA斑馬魚模型進行了藥物測試。
首先，我們發現LMNA突變的成魚表現出異常的表型。 LMNA(A539V)魚F0成魚尾部和頭部無異常，F1成魚身體彎曲。 LMNA(R453W) 在 F1 中也表現出彎曲的身體。 LMNA(W520G) TG1的F1魚在3個月內全部死亡。我們通過 DanioVision 測量幼魚游泳速度發現LMNA(L35P)、LMNA(E358K) 和 LMNA(R453W)幼魚游泳明顯慢於野生型斑馬魚和野生型LMNA轉基因斑馬魚。我們通過T-迷宮和游泳隧道測量成魚的游泳速度和肌肉耐力，我們發現除了LMNA(E358K)以外的所有LMNA轉基因成魚的游泳速度和肌肉耐力都比的野生型斑馬魚慢。使用組織化學染色進行肌肉活檢，我們發現 LMNA(L35P)、LMNA(E358K)、LMNA(R453W) 和 LMNA(A539V)轉基因魚表現出先天性纖維類型不對稱和纖維尺寸減小。使用這些LMNA斑馬魚模型進行了藥物測試，發現左旋肉鹼治療1個月拯救LMNA(L35P)肌肉耐力，肌酸治療1個月逆轉LMNA(R453W)肌肉耐力。我們的研究結果為 LMNA 相關先天性肌營養不良症的潛在治療方法的未來發展提供了新的思路。

LMNA encodes nuclear envelope proteins lamin A and C, mutations of LMNA cause a series of human diseases, such as developmental abnormalities, congenital muscular dystrophy, and premature aging, collectively called laminopathies. Currently, there is no cure for laminopathies, only symptomatic treatment and supportive care. Three novel mutations LMNA(L35P), LMNA(A539V), LMNA(W520G) and two known mutations LMNA(E358K) and LMNA(R453W) were identified from patients diagnosed with congenital muscular dystrophy. In this project, we established transgenic fish overexpressing wild-type LMNA and five LMNA mutants in the skeletal muscle, and swimming behavior, muscle endurance and histopathological were monitored, and we also used those LMNA zebrafish model for testing drugs.
Firstly, we found LMNA mutation adult fish show the abnormal phenotype. LMNA(A539V) fish display no tail and head abnormality in F0 adults, and crooked body in F1 adult. LMNA(R453W) also show crooked body in F1. The F1 fish of LMNA(W520G) all dead by 3 months. The LMNA(L35P), LMNA(E358K), and LMNA(R453W) transgenic fish larva dramatically swimming slower than those in AB wild-type and LMNA(WT) measured by DanioVision. We found all LMNA transgenic adult fish except LMNA(E358K) exhibit slower swimming speed and weaker muscle endurance than those in AB(WT) measured by T-maze and swimming tunnel respectively. Using histochemical staining for the muscle biopsy, we found LMNA(L35P), LMNA(E358K), LMNA(R453W) and LMNA(A539V) transgenic fish displays fiber type disproportion and decreased fiber size. Using these LMNA zebrafish models for drug testing, L-carnitine treatment rescue muscle endurance of LMNA(L35P), and creatine treatment reverse muscle endurance of LMNA(R453W). Our results shed a new light in the future development of potential treatment for LMNA-related congenital muscular dystrophy.

Abstract .......................................................................................... I
中文摘要 ......................................................................................... II
致謝 ............................................................................................... III
Table of Contents ......................................................................... V
Chapter 1. Introduction ................................................................ 1
1.1 Nuclear envelope proteins lamin A/C ............................................ 1
1.2 Laminopathy is a rare disease cause by LMNA mutation ............ 1
1.3 Mechanisms underneath the Laminopathy muscle disease ......... 2
1.3 Congenital muscular dystrophy ...................................................... 2
1.4 Therapies of congenital muscular dystrophy ................................ 3
1.5 The patients with muscular dystrophy ........................................... 4
1.6 Advantage of zebrafish model ......................................................... 4
1.7 Zebrafish model for study congenital muscular dystrophy and
muscle development ....................................................................... 5
1.8 Establish LMNA transgenic zebrafish lines .................................... 6
Chapter 2. Materials and Methods .............................................. 8
2.1 Tol2 Gateway cloning ....................................................................... 8
2.2 Site-directed mutagenesis ............................................................... 9
2.3 Microinjection ................................................................................. 10
2.4 Selection and confirmation of transgenic zebrafish .................... 10
2.5 Zebrafish care and breed ............................................................... 11
2.6 Adult fish phenotype observation ................................................. 11
2.7 Tracking of fish larvae swimming speed ...................................... 11
2.8 T-maze behavior test for adult fish swimming speed .................. 12
2.9 Swimming tunnel for adult fish muscle endurance ..................... 12
2.10. Chemical treatment for larvae fish ............................................. 13
2.11 Metformin treatment for larvae fish ............................................. 14
2.12 Chemical treatment for 1 month fish ........................................... 14
2.13 Tissue collection ........................................................................... 14

2.14 RNA extraction .............................................................................. 15
2.16 Real time quantitative polymerase chain reaction (QPCR) ....... 16
2.17 Prepare the muscle specimens and frozen section ................... 16
2.19 Hematoxylin and Eosin stain (H&E stain) ................................... 17
2. 20 Gömöri trichrome stain ............................................................... 17
2. 21 Nicotinamide adenine dinucleotide tetrazolium reductase stain
(NADH-TR stain) ............................................................................ 18
2. 22 Statistical analysis ....................................................................... 19
Chapter 3. Result ........................................................................ 20
3.1 Establishment of LMNA wild-type and mutants’ transgenic
zebrafish ......................................................................................... 20
3.2 LMNA(A539V) and LMNA(R453W) transgenic zebrafish display
abnormal appearance .................................................................... 21
3.3 The transgenic larva carried pathogenic LMNA(E358K) and
LMNA(R453W) and novel mutation LMNA(L35P) and
LMNA(A539V) swim slower than LMNA(WT) ............................... 22
3.4 The transgenic adult carried pathogenic LMNA(E358K) and
LMNA(R453W) and novel mutation LMNA(A539V) swim slower
than LMNA(WT) in both F0 and F1 ............................................... 22
3.5 The transgenic fish adult carried pathogenic LMNA(R453W) and
novel mutation LMNA(L35P) and LMNA(A539V) exhibit weaker
muscle endurance than LMNA(WT) ............................................. 24
3.6 The heart beat from 4 dpf LMNA mutants transgenic F1 fish are
increased compared to AB(WT) or LMNA(WT) ............................ 24
3.7 Histopathological examination showed LMNA(L35P),
LMNA(E358K), LMNA(R453W) and LMNA(A539V) transgenic fish
displays fiber type disproportion and decreased fiber size ...... 25
3.8 L-carnitine treatment enhance the swimming speed of
LMNA(L35P), and creatine treatment increased the swimming
speed of LMNA(R453W) larva ....................................................... 26
3.9 L-carnitine treatment rescue muscle endurance of LMNA(L35P),
and creatine treatment reverse muscle endurance of
LMNA(R453W) ................................................................................ 27
3.10 Expression of AMPK and mTOR pathways are rescued by
creatine treatment of LMNA(R453W) fish but not L-Carnitine

treated LMNA(L35P) ...................................................................... 27
3.11 Metformin cannot rescue the swimming velocity of LMNA
mutant fish ..................................................................................... 28
Chapter 4. Discussion ................................................................ 29
4.1 Memory and anxiety factor in adult fish swimming speed test by
using T-maze behavior. ................................................................. 29
4.2 Drug screening platform was established by testing larvae
swimming speed. ........................................................................... 30
4.3 L-Carnitine improve LMNA (L35P) swimming speed and muscle
endurance in larvae and 1 month fish. ........................................ 30
4.4 Creatine significantly rescued LMNA(R453W) fish muscle defect.
........................................................................................................ 31
4.5 AMPKα and mTOR pathway gene are biomarker of laminopathy
........................................................................................................ 32
4.6 Future perspective .......................................................................... 32
Figures and Tables ..................................................................... 34
Figure 1. Establishment of LMNA over-expression in skeletal muscle
transgenic zebrafish ...................................................................... 34
Figure 2. Confirmation the sequence of LMNA transgenic zebrafish
........................................................................................................ 37
Figure 3. LMNA mRNA levels from AB(WT) and LMNA transgenic
zebrafish ......................................................................................... 39
Figure 4. Appearance of adult LMNA transgenic zebrafish ............... 41
Figure 5. LMNA transgenic zebrafish larvae TG1 swimming speed in
6-8 dpf. ............................................................................................ 43
Figure 6. T- maze behavior swimming speed test for F0 adult LMNA
transgenic zebrafish ...................................................................... 45
Figure 7. T- maze behavior swimming speed test for F1 TG1 adult
LMNA transgenic zebrafish........................................................... 47
Figure 8. Muscle endurance test for F1_TG1 adult LMNA transgenic
zebrafish ......................................................................................... 49
Figure 9. The heartbeat from 4 dpf LMNA mutants transgenic F1 fish
are increased compared to AB(WT) or LMNA(WT) ..................... 51
Figure 10. 50X histopathological of F1 adult LMNA transgenic

zebrafish ......................................................................................... 53
Gömöri trichrome stain ........................................................................ 53
Figure 11. 200X histopathological of F1 adult LMNA transgenic
zebrafish ......................................................................................... 55
Gömöri trichrome stain ........................................................................ 55
Figure 12. 400X histopathological of F1 adult LMNA transgenic
zebrafish ......................................................................................... 57
Gömöri trichrome stain ........................................................................ 57
Figure 13. Chemical treatment for F2 heterozygous larvae LMNA
transgenic zebrafish ...................................................................... 59
Figure 14. Chemical treatment for 1 month F2 heterozygous LMNA
transgenic zebrafish ...................................................................... 65
Figure 15. Expression of AMPK and mTOR pathway for 7 day larvae
and 1 month F2 heterozygous LMNA transgenic zebrafish ....... 67
Figure 16. Metformin treatment for F2 heterozygous larvae LMNA
transgenic zebrafish ...................................................................... 71
Figure 17. Expression of AMPK and mTOR pathway for 7 day larvae
treat metformin. ............................................................................. 73
Table 1. The primer information for generated pME-LMNA ............... 75
Table 2. The primer information for LR reaction confirmation ......... 75
Table 3. The primer information for Site-directed mutagenesis ....... 75
Table 4. The primer information for confirm LMNA transgenic
zebrafish ......................................................................................... 76
Table 5. The primer information for QPCR analysis .......................... 76
Supplementary Information ....................................................... 77
Supplementary Figure 1. ...................................................................... 77
Supplementary Figure 2. ...................................................................... 78
Supplementary Figure 3. ...................................................................... 79
Supplementary Figure 4. ...................................................................... 80
Supplementary Figure 5. ...................................................................... 81
Supplementary Data 1. ......................................................................... 82
References .................................................................................. 87
                                

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