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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳俊宏(Chun-Hong Chen) | |
| dc.contributor.author | Hui-Ying Tsai | en |
| dc.contributor.author | 蔡惠穎 | zh_TW |
| dc.date.accessioned | 2021-07-11T15:10:40Z | - |
| dc.date.available | 2021-08-14 | |
| dc.date.copyright | 2019-08-14 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-08-08 | |
| dc.identifier.citation | 1. MartinLS. Maple Syrup Urine Disease. In: Encyclopedia of the Neurological Sciences. ; 2014. doi:10.1016/B978-0-12-385157-4.00076-2
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JingK, LimK. Why is autophagy important in human diseases? Exp Mol Med. 2012. doi:10.3858/emm.2012.44.2.028 37. SheP, VanHornC, ReidT, HutsonSM, CooneyRN, LynchCJ. Obesity-related elevations in plasma leucine are associated with alterations in enzymes involved in branched-chain amino acid metabolism. Am J Physiol Metab. 2007. doi:10.1152/ajpendo.00134.2007 38. ZabetianCP. Review of Rosenberg’s Molecular and Genetic Basis of Neurological and Psychiatric Disease, 5th ed . JAMA Neurol. 2015. doi:10.1001/jamaneurol.2015.2634 39. ZinnantiWJ, LazovicJ, GriffinK, et al. Dual mechanism of brain injury and novel treatment strategy in maple syrup urine disease. Brain. 2009. doi:10.1093/brain/awp024 40. DuranM, WadmanSK. Thiamine-responsive inborn errors of metabolism. J Inherit Metab Dis. 1985. doi:10.1007/BF01800663 41. KhannaA, HartM, NyhanWL, HassaneinT, Panyard-DavisJ, BarshopBA. Domino liver transplantation in maple syrup urine disease. Liver Transplant. 2006. doi:10.1002/lt.20744 42. HarperPAW, HealyPJ, DennisJA. Ultrastructural findings in maple syrup urine disease in Poll Hereford calves. Acta Neuropathol. 1986. doi:10.1007/BF00688055 43. HomanicsGE, SkvorakK, FergusonC, WatkinsS, PaulHS. Production and characterization of murine models of classic and intermediate maple syrup urine disease. BMC Med Genet. 2006. doi:10.1186/1471-2350-7-33 44. RobertsNB. Maple syrup urine disease: new insights from a zebrafish model. Dis Model Mech. 2012. doi:10.1242/dmm.010272 45. FriedrichT, LambertAM, MasinoMA, DownesGB. Mutation of zebrafish dihydrolipoamide branched-chain transacylase E2 results in motor dysfunction and models maple syrup urine disease. Dis Model Mech. 2012. doi:10.1242/dmm.008383 46. SonnetDS, O’LearyMN, GutierrezMA, et al. Metformin inhibits Branched Chain Amino Acid (BCAA) derived ketoacidosis and promotes metabolic homeostasis in MSUD. Sci Rep. 2016. doi:10.1038/srep28775 47. HostalekU, GwiltM, HildemannS. Therapeutic Use of Metformin in Prediabetes and Diabetes Prevention. Drugs. 2015. doi:10.1007/s40265-015-0416-8 48. RenaG, PearsonER, SakamotoK. Molecular mechanism of action of metformin: Old or new insights? Diabetologia. 2013. doi:10.1007/s00125-013-2991-0 49. MadirajuAK, ErionDM, RahimiY, et al. Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase. Nature. 2014. doi:10.1038/nature13270 50. KimYD, ParkKG, LeeYS, et al. Metformin inhibits hepatic gluconeogenesis through AMP-activated protein kinase-dependent regulation of the orphan nuclear receptor SHP. Diabetes. 2008. doi:10.2337/db07-0381 51. MengS, CaoJ, HeQ, et al. Metformin activates AMP-activated protein kinase by promoting formation of the αβγheterotrimeric complex. J Biol Chem. 2015. doi:10.1074/jbc.M114.604421 52. CollierCA, BruceCR, SmithAC, LopaschukG, DyckDJ. 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BMC Dev Biol. 2015;15(1):1-12. doi:10.1186/s12861-015-0062-0 58. BrooksDS, VishalK, KawakamiJ, BouyainS, GeisbrechtER. Optimization of wrMTrck to monitor Drosophila larval locomotor activity. J Insect Physiol. 2016. doi:10.1016/j.jinsphys.2016.07.007 59. DavisJW. Bioinformatics and Computational Biology Solutions Using R and Bioconductor. J Am Stat Assoc. 2009. doi:10.1198/jasa.2007.s179 60. JouvetP, RustinP, TaylorDL, et al. Branched Chain Amino Acids Induce Apoptosis in Neural Cells without Mitochondrial Membrane Depolarization or Cytochrome c Release: Implications for Neurological Impairment Associated with Maple Syrup Urine Disease. Mol Biol Cell. 2000;11(5):1919-1932. doi:10.1091/mbc.11.5.1919 61. SteinlinM, BlaserS, BoltshauserE. Cerebellar involvement in metabolic disorders: A pattern-recognition approach. Neuroradiology. 1998. doi:10.1007/s002340050597 62. ShellmerDA, Devito DabbsA, DewMA, et al. Cognitive and adaptive functioning after liver transplantation for maple syrup urine disease: A case series. Pediatr Transplant. 2011. doi:10.1111/j.1399-3046.2010.01411.x 63. LynchCJ, AdamsSH. Branched-chain amino acids in metabolic signalling and insulin resistance. Nat Rev Endocrinol. 2014. doi:10.1038/nrendo.2014.171 64. HouJW. Maple Syrup Urine Disease Complicated with Kyphoscoliosis and Myelopathy. Pediatr Neonatol. 2016. doi:10.1016/j.pedneo.2013.10.013 65. ArcherSL. Mitochondrial Dynamics — Mitochondrial Fission and Fusion in Human Diseases. N Engl J Med. 2013. doi:10.1056/NEJMra1215233 66. WangZH, ClarkC, GeisbrechtER. Analysis of mitochondrial structure and function in the Drosophila larval musculature. Mitochondrion. 2016. doi:10.1016/j.mito.2015.11.005 67. Fernández‐HernándezI, ScheenaardE, PollaroloG, GonzalezC. The translational relevance of Drosophila in drug discovery . EMBO Rep. 2016. doi:10.15252/embr.201642080 68. FujitaS, DreyerHC, DrummondMJ, et al. Nutrient signalling in the regulation of human muscle protein synthesis. J Physiol. 2007. doi:10.1113/jphysiol.2007.134593 69. TsaprasP, JacominAC, NezisIP. Assays to monitor mitophagy in drosophila. In: Methods in Molecular Biology. ; 2019. doi:10.1007/978-1-4939-8873-0_42 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78663 | - |
| dc.description.abstract | 楓糖尿症為罕見的代謝性胺基酸異常疾病，由於患者缺乏支鏈胺基酸代謝酵素-支鏈甲型酮酸脫氫複合物(BCKDH)，導致過量的支鏈胺基酸(BCAAs)累積在血液中，進而對神經系統造成傷害。此病在台灣的發病率為十萬分之一，在嬰兒時期就會發病，如果未立即處理將會有死亡的風險。目前治療方法有兩種，一為飲食控制，避免攝入過多的支鏈胺基酸以減少體內過多的累積，二為嚴重者需要進行肝臟移植手術。本篇研究中，我們以果蠅為模式生物，將參與支鏈胺基酸代謝路徑中的五個酵素基因分別進行剔除。支鏈甲型酮酸脫氫複合物(BCKDH)包含四個亞基E1α、E1β、E2和E3分別對應到果蠅基因的CG8199、CG17691、CG5599和CG7430，此外路徑上游的支鏈胺基酸轉移酶(BCAT)針對基因則是CG1673，也將進行基因剔除。實驗結果顯示，支鏈胺基酸的代謝地點在肌肉，不同於其他胺基酸在肝臟代謝。表現量在肌肉較高的是E1α、E2、E3，表示著這三個是主要代謝支鏈胺基酸的亞基，而其中又以E3表現量最高，被認為是整個代謝路徑中最主要的基因。另外，確認了在五株基因剔除果蠅中支鏈胺基酸會上升之後，發現脂肪細胞的脂滴大小也會改變，顯示支鏈胺基酸的累積也會改變著脂肪的代謝。此外，我們利用視網膜電圖(ERG)，紀錄在果蠅視神經在光刺激下電位的變化，E2和E3的變異株在神經傳導的功能上有明顯的受損，若解開導致神經性退化的機制，將對解決楓糖尿症嬰兒大腦受到支鏈胺基酸堆積是一大里程碑。此外，我們利用一種降血糖藥物-二甲雙胍進行測試，並且看到幼蟲體內支鏈胺基酸含量以及爬行能力有一定程度的恢復，在未來能進行更多的測試，為楓糖尿症帶來藥物治療的契機。 | zh_TW |
| dc.description.abstract | Maple Syrup Urine Disease (MSUD) is a rare autosomal recessive inherited disease caused by dysfunctions of the branched-chain α-keto-acid dehydrogenase (BCKDH) complex; this complex is involved in the branched-chain amino acid (BCAA) degradation pathway. Although it has been reported that patients with MSUD suffer from neuronal injuries, the causes are currently unknown.
In order to investigate the underlying mechanisms of MSUD, we generated Drosophila melanogaster BCKDH enzyme and BCAA degradation pathway mutants using CRISPR-Cas9 techniques. The BCKDH enzyme consists of three components, each of which was targeted for mutation; alpha-keto acid decarboxylase (E1/ composed of the sub-units α (CG8199) and β (CG17691)), dihydrolipoyl transacylase (E2/CG5599) and dihydrolipoamide dehydrogenase (E3/CG7430). Branched-chain amino acid aminotransferase (BCAT), which is upstream of the BCKDH complex, was also targeted (CG1673). We found that BCAAs accumulated and lipid droplet (LD) sizes changed in mutant lines as compared to controls. Furthermore, we used GMR Gal4 to drive RNAi for each of the five genes and found that changes to rhabdomere morphology were only observed in CG5599 and CG7430 RNAi lines, indicating these two genes may be the most important for neuronal development. Using a Drosophila electroretinogram (ERG) to examine mutant physiology, we found that CG5599 and CG7430 RNAi lines lacked on- and off- transients and displayed defective depolarization, indicating that they had a neurodegenerative phenotype. Our results suggest that dihydrolipoyl transacylase and dihydrolipoamide dehydrogenase may be the most promising targets for further research into MSUD associated chronic neuropsychiatric symptoms. Moreover, we treated Metformin in this Drosophila model by detecting BCAAs level and larval locomotor, finding that an extent of rescuing. These results indicated that Metformin probably could be a potential drug for curing MSUD. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:10:40Z (GMT). No. of bitstreams: 1 ntu-108-R06b43019-1.pdf: 3788030 bytes, checksum: b014133fda2b5d2968815c9e123fad96 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | Introduction ...1
Literature review ...4 1. BCAA degradation pathway ...4 2. Lipid metabolism ...6 3. Monomethyl Branched-chain Fatty Acids (mmBCFAs) ...8 4. mTOR and BCAA metabolic pathways ...9 5. Neurodegeneration ...10 6. Autophagy in Drosophila ...11 7. Maple Syrup Urine Disease (MSUD) ...13 8. Animal models of MSUD ...15 9. The biguanide antihyperglycemic agent metformin ...17 Aims of thesis ...19 Materials and Methods ...20 1. Fly/larva stocks and genetics ...20 2. Pupal eclosion rate analysis ...20 3. GC analysis of mmBCFA ...21 4. UPLC-MS-Multiple Reaction Monitoring (MRM) analysis ...22 5. Fat body dissection and imaging ...22 6. Photoreceptor immunofluorescence (IF) and imaging ...23 7. Lysotracker staining and imaging ...24 8. Brain paraffin sectioning ...24 9. Electroretinogram (ERG) ...25 10. Larval locomotion analysis ...25 11. RNA extraction and real-time PCR (qPCR) ...26 1. RNA extraction. ...26 2. Reverse transcription -PCR (RNA→cDNA). ...27 3. SYBR-system. ...27 12. Statistical analysis ...28 Results ...29 1. Generation of BCKDH knock-out mutants via CRISPR-mediated mutagenesis ...29 2. Mutant larvae display elevated levels of BCAAs (valine, leucine and isoleucine). ...29 3. Loss of several BCKDH genes resulted in larval arrested development. ...30 4. Vacuolar lesions increased in number in mutant brains. ...30 5. Knockdown of BCKDH genes causes damage to neurons and results in neurodegeneration. ...31 6. BCKDH complex expression site in WT flies. ...32 7. Lipid droplets accumulate in larval fat bodies. ...32 8. BCKDH mutant found increased lipid droplets size but not CG1673 mutant in high protein diet. ...33 9. Production of mmBCFA increased in MSUD mutants. ...33 10. Starvation-induced autophagy may be downregulated in the larval fat body. ...34 11. Larval movement capability was significantly reduced in BCKDH mutants. ...35 12. Mitochondrial morphology changes in muscle and fat body ...36 13. Metformin treatment reduces the accumulation of leucine and isoleucine in E2 mutant larvae. ...37 14. Metformin treatment rescues locomotor behavior in mutant larvae. ...37 Discussion ...39 References ...45 Figures ...56 Supplementary information ...75 Appendix ...79 | |
| dc.language.iso | en | |
| dc.subject | 神經性退化 | zh_TW |
| dc.subject | 支鏈甲型酮酸脫氫複合物缺乏 | zh_TW |
| dc.subject | 支鏈胺基酸 | zh_TW |
| dc.subject | 楓糖尿症 | zh_TW |
| dc.subject | 二甲雙胍 | zh_TW |
| dc.subject | 果蠅 | zh_TW |
| dc.subject | Drosophila melanogaster | en |
| dc.subject | Maple Syrup Urine Disease | en |
| dc.subject | BCKDH deficiency | en |
| dc.subject | BCAA degradation pathway | en |
| dc.subject | Neurodegeneration | en |
| dc.subject | Metformin | en |
| dc.title | 突變果蠅支鏈甲型酮酸脫氫複合物導致肌肉以及神經性損傷 | zh_TW |
| dc.title | Mutations of the Drosophila branched-chain α-keto acid dehydrogenase complex (BCKDH) result in muscular and neuronal dysfunctions | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 詹智強,王致恬,林靜嫻 | |
| dc.subject.keyword | 楓糖尿症,支鏈胺基酸,支鏈甲型酮酸脫氫複合物缺乏,神經性退化,果蠅,二甲雙胍, | zh_TW |
| dc.subject.keyword | Maple Syrup Urine Disease,BCKDH deficiency,Drosophila melanogaster,BCAA degradation pathway,Neurodegeneration,Metformin, | en |
| dc.relation.page | 83 | |
| dc.identifier.doi | 10.6342/NTU201902658 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2019-08-09 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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