博碩士論文 111821011 詳細資訊



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姓名 龍庭翔(Ting-Hsiang Lung)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 探討以RNA修飾為基礎的基因治療對於肌萎縮側索硬化症的應用
(Exploring the Application of RNA Modification-Based Gene Therapy for Amyotrophic Lateral Sclerosis)
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摘要(中) 肌萎縮側索硬化症（Amyotrophic lateral sclerosis, ALS），又稱漸凍症，是一種好發於成年人的運動神經元退化性疾病，控制肌肉動作的大腦以及脊髓神經細胞會在發病後會逐漸死亡，目前ALS的致病機制以及治療方式還有待研究。近期，我們實驗室發現N6-甲基腺嘌呤（N6-methyladenosine, m6A）的RNA化學修飾在細胞以及動物的ALS疾病模型中有下降的趨勢。先前實驗中，我們發現利用藥物提升m6A的修飾量，能夠延緩由ALS病人誘導型幹細胞所分化的運動神經元的退化速度。基於上述的在細胞實驗的發現，我們在此研究中想要驗證上述的理論是否也能應用在動物的模型當中，並藉此尋找新的ALS治療方式。首先，我們利用化學化合物和RNA干擾技術並測試在分化的神經母細胞瘤細胞，驗證m6A在神經性退化疾病的甲基化程度。之後，為了建立應用在動物模型的治療方法，我們使用腺相關病毒使其表達針對m6A的去甲基化酶基因「Fto」的short hairpin RNA(scAAV9-shFto)，並利用脊髓鞘內注射的方式將scAAV9-shFto打入ALS的疾病小鼠模型（SOD1G93A 小鼠）。我們的研究結果顯示，在接受scAAV9-shFto注射的SOD1G93A小鼠在運動行為有較好的表現；通過電生理的測試，我們也發現SOD1G93A 小鼠在肌肉收縮能力方面也有改善的效果。更重要的是，scAAV9-shFto的注射能夠延緩了SOD1G93A小鼠的發病時間並且延長其壽命。此外，實驗室先前利用Nanopore定序找出了一些帶有m6A修飾的ALS致病基因，為了更進一步的驗證這些數據，我們利用了m6A的抗體進行RNA的免疫沈澱反應並結合定量反轉錄聚合酶連鎖反應實驗。結果顯示，利用此方式發現大部分的ALS致病基因接受到m6A的甲基修飾，提供了m6A修飾在ALS疾病中的重要性，並結合先前實驗結果，也為ALS找到一個新的治療策略的方向。
摘要(英) Amyotrophic lateral sclerosis (ALS) is an adult-onset, irreversible motor neuron (MN) degenerative disease affecting brain and spinal cord nerve cells that control voluntary muscle movement. Our recent finding observed decreased N6-methyladenosine (m6A) RNA modification in both the in vitro and in vivo ALS disease models. Surprisingly, restoring the m6A level in ALS iPSC-derived MNs will delay the MN degeneration process. Based on those findings and rationales, we aim to investigate whether this phenomenon can be proven in vivo and identify a new therapeutic approach for ALS. Before that, we investigated the role of m6A and its writer, which can deposit the m6A on mRNA in the differentiated neuroblastoma cell line using chemical compounds and RNA interference. To establish the therapy tool, we utilized scAAV9 expressing short hairpin RNA to target one of the m6A eraser enzymes, Fto, which can demethylate the methyl group on m6A. We perform the intrathecal injection to deliver scAAV9-shFto into the SOD1G93A mice. Our result found that SOD1G93A mice with scAAV9-shFto intrathecal delivery demonstrated improved motor activity performance and better neuromuscular function measured by motor behavior tests and electrophysiology tests, respectively. Furthermore, the administration of scAAV9-shFto resulted in a notable delay in the disease onset and prolongation in overall survival in SOD1G93A mice. In addition, we have discovered some ALS-related genes with m6A modification by using m6A nanopore sequencing. To further confirm these data, we performed m6A immunoprecipitation combined with RT-qPCR (m6A-RIP-qPCR) and found that several ALS risk genes are m6A-modified. These findings provide a new therapy perspective on ALS disease and the importance of m6A RNA modification in ALS disease.
關鍵字(中) ★ RNA修飾
★ 基因治療
★ 肌萎縮側索硬化症		 關鍵字(英) ★ RNA modification
★ Gene Therapy
★ Amyotrophic Lateral Sclerosis
論文目次 Table of Contents
摘要 v
Abstract vi
誌謝 vii
Table of Contents viii
Abbreviation xi
Chapter I Introduction 1
1-1 N6‐methyladenosine (m6A) 1
1-2 m6A detection methods 3
1-3 Amyotrophic lateral sclerosis (ALS) 5
1-4 ALS disease model 8
1-5 Therapy for ALS 10
1-6 The relationship between m6A and neural development/degeneration 11
1-7 Overview of this project 12
Chapter II Research Context and Methods 14
2-1 Generation of an inducible METTL14-knockdown SH-SY5Y cell line 14
2-2 Inducible METTL14-knockdown SH-SY5Y cell line differentiation 14
2-3 RNA extraction 14
2-4 Western blot 15
2-5 m6A ELISA assay and quantification 15
2-6 Reverse transcription and real-time quantitative PCR (RT-qPCR) 16
2-7 m6A dot blot assay 17
2-8 Immunofluorescence staining 17
2-9 scAAV9 plasmid construction 18
2-10 scAAV9 virus preparation and injection 18
2-11 Spinal motor neuron quantification 18
2-12 GFAP-positive cell quantification 19
2-13 Mouse breeding 19
2-14 Survival analysis 19
2-15 Behavioral and Electrophysiologic Assay 19
2-15-1 Rotarod 20
2-15-2 Grip strength 20
2-15-3 Compound muscle action potential (CMAP) 20
2-16 Mouse motor neuron differentiation from mouse embryonic stem cell (mESC) 21
2-17 m6A immunoprecipitation and quantitative real-time PCR 21
2-17-1 RNA preparation 21
2-17-2 RNA immunoprecipitation 22
2-17-3 m6A enrichment calculation by RT-qPCR 22
Chapter III Results 23
3-1 The chemical compound-induced m6A hypomethylation caused neuron degeneration in differentiated SH-SY5Y 23
3-2 Establish the METTL14 knockdown-inducible system in SH-SY5Y cell line 23
3-3 The neurites of SH-SY5Y cells showed no prominent defects upon induction of METTL14 knockdown 24
3-4 Generation of scAAV9-shFto and the functional test 25
3-5 Explore the gene therapy function of scAAV9-shFto in SOD1G93A mice 27
3-6 Verify the m6A-modified sites in ALS-related gene 29
Chapter IV Discussion 31
Chapter V Future Perspective 36
5-1 Investigate the relationship between SNPs of m6A-related genes and MN degeneration 36
5-2 Dissect the effect of FTO knockdown on ALS disease 37
5-3 Identification and verification of potential m6A-modified transcript linked to ALS disease from m6A-RIP -seq and nanopore sequencing 37
Chapter VI Figure and Figure Legends 39
Figure 1. METTL3 inhibitor causes differentiated SH-SY5Y degeneration. 39
Figure 2. The METTL14 knockdown-inducible system can successfully regulate the m6A level. 41
Figure 3. Differentiated SH-SY5Y-Tet-shMETTL14 demonstrate no prominent defect upon doxycycline induction. 43
Figure 4. The shRNA targeting Fto effectively knocked down Fto expression and led to a notable increase in the m6A level. 45
Figure 5. The AAV delivery through intrathecal injection successfully transmits to motor neurons and decreases the Fto expression in the mouse spinal cord. 46
Figure 6. The intrathecal injection of scAAV9-shFto mitigates the pathology of SOD1G93A mice. 48
Figure 7. The intrathecal injection of scAAV9-shFto protects neuromuscular function and delays the disease onset of SOD1G93A mice. 50
Figure 8. To verify the m6A-modified sites in ALS-related genes in differentiated mouse motor neuron 53
Appendix 1: Previous data from Dr. Ya-Ping Yen 54
Appendix 2: Plasmid, Primer, Antibody list, the Recipe of Buffer and Medium 55
Reference 60
參考文獻 Reference
1. Liu, J., Yue, Y., Han, D., Wang, X., Fu, Y., Zhang, L., Jia, G., Yu, M., Lu, Z., Deng, X., et al. (2014). A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat Chem Biol 10, 93-95. 10.1038/nchembio.1432.
2. Meyer, K.D., Saletore, Y., Zumbo, P., Elemento, O., Mason, C.E., and Jaffrey, S.R. (2012). Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell 149, 1635-1646. 10.1016/j.cell.2012.05.003.
3. Ma, S., Chen, C., Ji, X., Liu, J., Zhou, Q., Wang, G., Yuan, W., Kan, Q., and Sun, Z. (2019). The interplay between m6A RNA methylation and noncoding RNA in cancer. J Hematol Oncol 12, 121. 10.1186/s13045-019-0805-7.
4. Coker, H., Wei, G., and Brockdorff, N. (2019). m6A modification of non-coding RNA and the control of mammalian gene expression. Biochim Biophys Acta Gene Regul Mech 1862, 310-318. 10.1016/j.bbagrm.2018.12.002.
5. Fu, Y., Dominissini, D., Rechavi, G., and He, C. (2014). Gene expression regulation mediated through reversible m(6)A RNA methylation. Nat Rev Genet 15, 293-306. 10.1038/nrg3724.
6. Patil, D.P., Chen, C.K., Pickering, B.F., Chow, A., Jackson, C., Guttman, M., and Jaffrey, S.R. (2016). m(6)A RNA methylation promotes XIST-mediated transcriptional repression. Nature 537, 369-373. 10.1038/nature19342.
7. Wen, J., Lv, R., Ma, H., Shen, H., He, C., Wang, J., Jiao, F., Liu, H., Yang, P., Tan, L., et al. (2018). Zc3h13 Regulates Nuclear RNA m(6)A Methylation and Mouse Embryonic Stem Cell Self-Renewal. Mol Cell 69, 1028-1038 e1026. 10.1016/j.molcel.2018.02.015.
8. Bawankar, P., Lence, T., Paolantoni, C., Haussmann, I.U., Kazlauskiene, M., Jacob, D., Heidelberger, J.B., Richter, F.M., Nallasivan, M.P., Morin, V., et al. (2021). Hakai is required for stabilization of core components of the m(6)A mRNA methylation machinery. Nat Commun 12, 3778. 10.1038/s41467-021-23892-5.
9. Jia, G., Fu, Y., Zhao, X., Dai, Q., Zheng, G., Yang, Y., Yi, C., Lindahl, T., Pan, T., Yang, Y.G., and He, C. (2011). N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol 7, 885-887. 10.1038/nchembio.687.
10. Zheng, G., Dahl, J.A., Niu, Y., Fedorcsak, P., Huang, C.M., Li, C.J., Vagbo, C.B., Shi, Y., Wang, W.L., Song, S.H., et al. (2013). ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol Cell 49, 18-29. 10.1016/j.molcel.2012.10.015.
11. Yen, Y.P., and Chen, J.A. (2021). The m(6)A epitranscriptome on neural development and degeneration. J Biomed Sci 28, 40. 10.1186/s12929-021-00734-6.
12. Alarcon, C.R., Goodarzi, H., Lee, H., Liu, X., Tavazoie, S., and Tavazoie, S.F. (2015). HNRNPA2B1 Is a Mediator of m(6)A-Dependent Nuclear RNA Processing Events. Cell 162, 1299-1308. 10.1016/j.cell.2015.08.011.
13. Xiao, W., Adhikari, S., Dahal, U., Chen, Y.S., Hao, Y.J., Sun, B.F., Sun, H.Y., Li, A., Ping, X.L., Lai, W.Y., et al. (2016). Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing. Mol Cell 61, 507-519. 10.1016/j.molcel.2016.01.012.
14. Wang, X., Lu, Z., Gomez, A., Hon, G.C., Yue, Y., Han, D., Fu, Y., Parisien, M., Dai, Q., Jia, G., et al. (2014). N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117-120. 10.1038/nature12730.
15. Roundtree, I.A., Luo, G.Z., Zhang, Z., Wang, X., Zhou, T., Cui, Y., Sha, J., Huang, X., Guerrero, L., Xie, P., et al. (2017). YTHDC1 mediates nuclear export of N(6)-methyladenosine methylated mRNAs. Elife 6. 10.7554/eLife.31311.
16. Wang, X., Zhao, B.S., Roundtree, I.A., Lu, Z., Han, D., Ma, H., Weng, X., Chen, K., Shi, H., and He, C. (2015). N(6)-methyladenosine Modulates Messenger RNA Translation Efficiency. Cell 161, 1388-1399. 10.1016/j.cell.2015.05.014.
17. Shi, H., Wang, X., Lu, Z., Zhao, B.S., Ma, H., Hsu, P.J., Liu, C., and He, C. (2017). YTHDF3 facilitates translation and decay of N(6)-methyladenosine-modified RNA. Cell Res 27, 315-328. 10.1038/cr.2017.15.
18. Hsu, P.J., Zhu, Y., Ma, H., Guo, Y., Shi, X., Liu, Y., Qi, M., Lu, Z., Shi, H., Wang, J., et al. (2017). Ythdc2 is an N(6)-methyladenosine binding protein that regulates mammalian spermatogenesis. Cell Res 27, 1115-1127. 10.1038/cr.2017.99.
19. Huang, H., Weng, H., Sun, W., Qin, X., Shi, H., Wu, H., Zhao, B.S., Mesquita, A., Liu, C., Yuan, C.L., et al. (2018). Recognition of RNA N(6)-methyladenosine by IGF2BP proteins enhances mRNA stability and translation. Nat Cell Biol 20, 285-295. 10.1038/s41556-018-0045-z.
20. Jiang, X., Liu, B., Nie, Z., Duan, L., Xiong, Q., Jin, Z., Yang, C., and Chen, Y. (2021). The role of m6A modification in the biological functions and diseases. Signal Transduct Target Ther 6, 74. 10.1038/s41392-020-00450-x.
21. Zhuang, H., Yu, B., Tao, D., Xu, X., Xu, Y., Wang, J., Jiao, Y., and Wang, L. (2023). The role of m6A methylation in therapy resistance in cancer. Mol Cancer 22, 91. 10.1186/s12943-023-01782-2.
22. Zhang, Y., Lu, L., and Li, X. (2022). Detection technologies for RNA modifications. Exp Mol Med 54, 1601-1616. 10.1038/s12276-022-00821-0.
23. Nagarajan, A., Janostiak, R., and Wajapeyee, N. (2019). Dot Blot Analysis for Measuring Global N(6)-Methyladenosine Modification of RNA. Methods Mol Biol 1870, 263-271. 10.1007/978-1-4939-8808-2_20.
24. Mathur, L., Jung, S., Jang, C., and Lee, G. (2021). Quantitative analysis of m(6)A RNA modification by LC-MS. STAR Protoc 2, 100724. 10.1016/j.xpro.2021.100724.
25. Castellanos-Rubio, A., Santin, I., Olazagoitia-Garmendia, A., Romero-Garmendia, I., Jauregi-Miguel, A., Legarda, M., and Bilbao, J.R. (2019). A novel RT-QPCR-based assay for the relative quantification of residue specific m6A RNA methylation. Sci Rep 9, 4220. 10.1038/s41598-019-40018-6.
26. Dominissini, D., Moshitch-Moshkovitz, S., Schwartz, S., Salmon-Divon, M., Ungar, L., Osenberg, S., Cesarkas, K., Jacob-Hirsch, J., Amariglio, N., Kupiec, M., et al. (2012). Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485, 201-206. 10.1038/nature11112.
27. Meyer, K.D. (2019). DART-seq: an antibody-free method for global m(6)A detection. Nat Methods 16, 1275-1280. 10.1038/s41592-019-0570-0.
28. Wang, Y., Xiao, Y., Dong, S., Yu, Q., and Jia, G. (2020). Antibody-free enzyme-assisted chemical approach for detection of N(6)-methyladenosine. Nat Chem Biol 16, 896-903. 10.1038/s41589-020-0525-x.
29. Shu, X., Cao, J., Cheng, M., Xiang, S., Gao, M., Li, T., Ying, X., Wang, F., Yue, Y., Lu, Z., et al. (2020). A metabolic labeling method detects m(6)A transcriptome-wide at single base resolution. Nat Chem Biol 16, 887-895. 10.1038/s41589-020-0526-9.
30. Wang, Y., Zhao, Y., Bollas, A., Wang, Y., and Au, K.F. (2021). Nanopore sequencing technology, bioinformatics and applications. Nat Biotechnol 39, 1348-1365. 10.1038/s41587-021-01108-x.
31. Liu, H., Begik, O., and Novoa, E.M. (2021). EpiNano: Detection of m(6)A RNA Modifications Using Oxford Nanopore Direct RNA Sequencing. Methods Mol Biol 2298, 31-52. 10.1007/978-1-0716-1374-0_3.
32. Hendra, C., Pratanwanich, P.N., Wan, Y.K., Goh, W.S.S., Thiery, A., and Goke, J. (2022). Detection of m6A from direct RNA sequencing using a multiple instance learning framework. Nat Methods 19, 1590-1598. 10.1038/s41592-022-01666-1.
33. Owens, B. (2017). Amyotrophic lateral sclerosis. Nature 550, S105. 10.1038/550S105a.
34. Ranganathan, R., Haque, S., Coley, K., Shepheard, S., Cooper-Knock, J., and Kirby, J. (2020). Multifaceted Genes in Amyotrophic Lateral Sclerosis-Frontotemporal Dementia. Front Neurosci 14, 684. 10.3389/fnins.2020.00684.
35. Mead, R.J., Shan, N., Reiser, H.J., Marshall, F., and Shaw, P.J. (2023). Amyotrophic lateral sclerosis: a neurodegenerative disorder poised for successful therapeutic translation. Nat Rev Drug Discov 22, 185-212. 10.1038/s41573-022-00612-2.
36. Akcimen, F., Lopez, E.R., Landers, J.E., Nath, A., Chio, A., Chia, R., and Traynor, B.J. (2023). Amyotrophic lateral sclerosis: translating genetic discoveries into therapies. Nat Rev Genet 24, 642-658. 10.1038/s41576-023-00592-y.
37. Cleveland, D.W., and Rothstein, J.D. (2001). From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS. Nat Rev Neurosci 2, 806-819. 10.1038/35097565.
38. Peggion, C., Scalcon, V., Massimino, M.L., Nies, K., Lopreiato, R., Rigobello, M.P., and Bertoli, A. (2022). SOD1 in ALS: Taking Stock in Pathogenic Mechanisms and the Role of Glial and Muscle Cells. Antioxidants (Basel) 11. 10.3390/antiox11040614.
39. Chen, L. (2021). The important functional role of TDP-43 plays in amyotrophic lateral sclerosis-frontotemporal dementia. Neural Regen Res 16, 682-683. 10.4103/1673-5374.293142.
40. Renton, A.E., Chio, A., and Traynor, B.J. (2014). State of play in amyotrophic lateral sclerosis genetics. Nat Neurosci 17, 17-23. 10.1038/nn.3584.
41. Turner, M.R., Hardiman, O., Benatar, M., Brooks, B.R., Chio, A., de Carvalho, M., Ince, P.G., Lin, C., Miller, R.G., Mitsumoto, H., et al. (2013). Controversies and priorities in amyotrophic lateral sclerosis. Lancet Neurol 12, 310-322. 10.1016/S1474-4422(13)70036-X.
42. Brown, A.L., Wilkins, O.G., Keuss, M.J., Hill, S.E., Zanovello, M., Lee, W.C., Bampton, A., Lee, F.C.Y., Masino, L., Qi, Y.A., et al. (2022). TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A. Nature 603, 131-137. 10.1038/s41586-022-04436-3.
43. Ma, X.R., Prudencio, M., Koike, Y., Vatsavayai, S.C., Kim, G., Harbinski, F., Briner, A., Rodriguez, C.M., Guo, C., Akiyama, T., et al. (2022). TDP-43 represses cryptic exon inclusion in the FTD-ALS gene UNC13A. Nature 603, 124-130. 10.1038/s41586-022-04424-7.
44. Baughn, M.W., Melamed, Z., Lopez-Erauskin, J., Beccari, M.S., Ling, K., Zuberi, A., Presa, M., Gonzalo-Gil, E., Maimon, R., Vazquez-Sanchez, S., et al. (2023). Mechanism of STMN2 cryptic splice-polyadenylation and its correction for TDP-43 proteinopathies. Science 379, 1140-1149. 10.1126/science.abq5622.
45. Suk, T.R., and Rousseaux, M.W.C. (2020). The role of TDP-43 mislocalization in amyotrophic lateral sclerosis. Mol Neurodegener 15, 45. 10.1186/s13024-020-00397-1.
46. Tan, A.Y., Riley, T.R., Coady, T., Bussemaker, H.J., and Manley, J.L. (2012). TLS/FUS (translocated in liposarcoma/fused in sarcoma) regulates target gene transcription via single-stranded DNA response elements. Proc Natl Acad Sci U S A 109, 6030-6035. 10.1073/pnas.1203028109.
47. Bertolotti, A., Lutz, Y., Heard, D.J., Chambon, P., and Tora, L. (1996). hTAF(II)68, a novel RNA/ssDNA-binding protein with homology to the pro-oncoproteins TLS/FUS and EWS is associated with both TFIID and RNA polymerase II. EMBO J 15, 5022-5031.
48. Ishigaki, S., and Sobue, G. (2018). Importance of Functional Loss of FUS in FTLD/ALS. Front Mol Biosci 5, 44. 10.3389/fmolb.2018.00044.
49. Sun, S., Ling, S.C., Qiu, J., Albuquerque, C.P., Zhou, Y., Tokunaga, S., Li, H., Qiu, H., Bui, A., Yeo, G.W., et al. (2015). ALS-causative mutations in FUS/TLS confer gain and loss of function by altered association with SMN and U1-snRNP. Nat Commun 6, 6171. 10.1038/ncomms7171.
50. Renton, A.E., Majounie, E., Waite, A., Simon-Sanchez, J., Rollinson, S., Gibbs, J.R., Schymick, J.C., Laaksovirta, H., van Swieten, J.C., Myllykangas, L., et al. (2011). A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257-268. 10.1016/j.neuron.2011.09.010.
51. Kobayashi, H., Abe, K., Matsuura, T., Ikeda, Y., Hitomi, T., Akechi, Y., Habu, T., Liu, W., Okuda, H., and Koizumi, A. (2011). Expansion of intronic GGCCTG hexanucleotide repeat in NOP56 causes SCA36, a type of spinocerebellar ataxia accompanied by motor neuron involvement. Am J Hum Genet 89, 121-130. 10.1016/j.ajhg.2011.05.015.
52. Majounie, E., Renton, A.E., Mok, K., Dopper, E.G., Waite, A., Rollinson, S., Chio, A., Restagno, G., Nicolaou, N., Simon-Sanchez, J., et al. (2012). Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study. Lancet Neurol 11, 323-330. 10.1016/S1474-4422(12)70043-1.
53. Rutherford, N.J., Heckman, M.G., Dejesus-Hernandez, M., Baker, M.C., Soto-Ortolaza, A.I., Rayaprolu, S., Stewart, H., Finger, E., Volkening, K., Seeley, W.W., et al. (2012). Length of normal alleles of C9ORF72 GGGGCC repeat do not influence disease phenotype. Neurobiol Aging 33, 2950 e2955-2957. 10.1016/j.neurobiolaging.2012.07.005.
54. van Blitterswijk, M., DeJesus-Hernandez, M., Niemantsverdriet, E., Murray, M.E., Heckman, M.G., Diehl, N.N., Brown, P.H., Baker, M.C., Finch, N.A., Bauer, P.O., et al. (2013). Association between repeat sizes and clinical and pathological characteristics in carriers of C9ORF72 repeat expansions (Xpansize-72): a cross-sectional cohort study. Lancet Neurol 12, 978-988. 10.1016/S1474-4422(13)70210-2.
55. Gijselinck, I., Van Mossevelde, S., van der Zee, J., Sieben, A., Engelborghs, S., De Bleecker, J., Ivanoiu, A., Deryck, O., Edbauer, D., Zhang, M., et al. (2016). The C9orf72 repeat size correlates with onset age of disease, DNA methylation and transcriptional downregulation of the promoter. Mol Psychiatry 21, 1112-1124. 10.1038/mp.2015.159.
56. Nordin, A., Akimoto, C., Wuolikainen, A., Alstermark, H., Jonsson, P., Birve, A., Marklund, S.L., Graffmo, K.S., Forsberg, K., Brannstrom, T., and Andersen, P.M. (2015). Extensive size variability of the GGGGCC expansion in C9orf72 in both neuronal and non-neuronal tissues in 18 patients with ALS or FTD. Hum Mol Genet 24, 3133-3142. 10.1093/hmg/ddv064.
57. Beck, J., Poulter, M., Hensman, D., Rohrer, J.D., Mahoney, C.J., Adamson, G., Campbell, T., Uphill, J., Borg, A., Fratta, P., et al. (2013). Large C9orf72 hexanucleotide repeat expansions are seen in multiple neurodegenerative syndromes and are more frequent than expected in the UK population. Am J Hum Genet 92, 345-353. 10.1016/j.ajhg.2013.01.011.
58. Balendra, R., and Isaacs, A.M. (2018). C9orf72-mediated ALS and FTD: multiple pathways to disease. Nat Rev Neurol 14, 544-558. 10.1038/s41582-018-0047-2.
59. Otey, C.A., Boukhelifa, M., and Maness, P. (2003). B35 neuroblastoma cells: an easily transfected, cultured cell model of central nervous system neurons. Methods Cell Biol 71, 287-304. 10.1016/s0091-679x(03)01013-6.
60. LePage, K.T., Dickey, R.W., Gerwick, W.H., Jester, E.L., and Murray, T.F. (2005). On the use of neuro-2a neuroblastoma cells versus intact neurons in primary culture for neurotoxicity studies. Crit Rev Neurobiol 17, 27-50. 10.1615/critrevneurobiol.v17.i1.20.
61. Shafer, T.J., and Atchison, W.D. (1991). Transmitter, ion channel and receptor properties of pheochromocytoma (PC12) cells: a model for neurotoxicological studies. Neurotoxicology 12, 473-492.
62. Biedler, J.L., Helson, L., and Spengler, B.A. (1973). Morphology and growth, tumorigenicity, and cytogenetics of human neuroblastoma cells in continuous culture. Cancer Res 33, 2643-2652.
63. Shipley, M.M., Mangold, C.A., and Szpara, M.L. (2016). Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line. J Vis Exp, 53193. 10.3791/53193.
64. Agholme, L., Lindstrom, T., Kagedal, K., Marcusson, J., and Hallbeck, M. (2010). An in vitro model for neuroscience: differentiation of SH-SY5Y cells into cells with morphological and biochemical characteristics of mature neurons. J Alzheimers Dis 20, 1069-1082. 10.3233/JAD-2010-091363.
65. Liu, Y., Eaton, E.D., Wills, T.E., McCann, S.K., Antonic, A., and Howells, D.W. (2018). Human Ischaemic Cascade Studies Using SH-SY5Y Cells: a Systematic Review and Meta-Analysis. Transl Stroke Res 9, 564-574. 10.1007/s12975-018-0620-4.
66. Xicoy, H., Wieringa, B., and Martens, G.J. (2017). The SH-SY5Y cell line in Parkinson′s disease research: a systematic review. Mol Neurodegener 12, 10. 10.1186/s13024-017-0149-0.
67. Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676. 10.1016/j.cell.2006.07.024.
68. Fujimori, K., Ishikawa, M., Otomo, A., Atsuta, N., Nakamura, R., Akiyama, T., Hadano, S., Aoki, M., Saya, H., Sobue, G., and Okano, H. (2018). Modeling sporadic ALS in iPSC-derived motor neurons identifies a potential therapeutic agent. Nat Med 24, 1579-1589. 10.1038/s41591-018-0140-5.
69. Roybon, L., Lamas, N.J., Garcia, A.D., Yang, E.J., Sattler, R., Lewis, V.J., Kim, Y.A., Kachel, C.A., Rothstein, J.D., Przedborski, S., et al. (2013). Human stem cell-derived spinal cord astrocytes with defined mature or reactive phenotypes. Cell Rep 4, 1035-1048. 10.1016/j.celrep.2013.06.021.
70. Amoroso, M.W., Croft, G.F., Williams, D.J., O′Keeffe, S., Carrasco, M.A., Davis, A.R., Roybon, L., Oakley, D.H., Maniatis, T., Henderson, C.E., and Wichterle, H. (2013). Accelerated high-yield generation of limb-innervating motor neurons from human stem cells. J Neurosci 33, 574-586. 10.1523/JNEUROSCI.0906-12.2013.
71. Ogawa, S., Tokumoto, Y., Miyake, J., and Nagamune, T. (2011). Induction of oligodendrocyte differentiation from adult human fibroblast-derived induced pluripotent stem cells. In Vitro Cell Dev Biol Anim 47, 464-469. 10.1007/s11626-011-9435-2.
72. Todd, T.W., and Petrucelli, L. (2022). Modelling amyotrophic lateral sclerosis in rodents. Nat Rev Neurosci 23, 231-251. 10.1038/s41583-022-00564-x.
73. Knippenberg, S., Thau, N., Dengler, R., and Petri, S. (2010). Significance of behavioural tests in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). Behav Brain Res 213, 82-87. 10.1016/j.bbr.2010.04.042.
74. Arnold, W.D., Sheth, K.A., Wier, C.G., Kissel, J.T., Burghes, A.H., and Kolb, S.J. (2015). Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles. J Vis Exp. 10.3791/52899.
75. Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P., Hentati, A., Donaldson, D., Goto, J., O′Regan, J.P., Deng, H.X., and et al. (1993). Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362, 59-62. 10.1038/362059a0.
76. Gurney, M.E., Pu, H., Chiu, A.Y., Dal Canto, M.C., Polchow, C.Y., Alexander, D.D., Caliendo, J., Hentati, A., Kwon, Y.W., Deng, H.X., and et al. (1994). Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science 264, 1772-1775. 10.1126/science.8209258.
77. Vinsant, S., Mansfield, C., Jimenez-Moreno, R., Del Gaizo Moore, V., Yoshikawa, M., Hampton, T.G., Prevette, D., Caress, J., Oppenheim, R.W., and Milligan, C. (2013). Characterization of early pathogenesis in the SOD1(G93A) mouse model of ALS: part II, results and discussion. Brain Behav 3, 431-457. 10.1002/brb3.142.
78. Vinsant, S., Mansfield, C., Jimenez-Moreno, R., Del Gaizo Moore, V., Yoshikawa, M., Hampton, T.G., Prevette, D., Caress, J., Oppenheim, R.W., and Milligan, C. (2013). Characterization of early pathogenesis in the SOD1(G93A) mouse model of ALS: part I, background and methods. Brain Behav 3, 335-350. 10.1002/brb3.143.
79. Gurney, M.E., Fleck, T.J., Himes, C.S., and Hall, E.D. (1998). Riluzole preserves motor function in a transgenic model of familial amyotrophic lateral sclerosis. Neurology 50, 62-66. 10.1212/wnl.50.1.62.
80. Ito, H., Wate, R., Zhang, J., Ohnishi, S., Kaneko, S., Ito, H., Nakano, S., and Kusaka, H. (2008). Treatment with edaravone, initiated at symptom onset, slows motor decline and decreases SOD1 deposition in ALS mice. Exp Neurol 213, 448-455. 10.1016/j.expneurol.2008.07.017.
81. Bensimon, G., Lacomblez, L., and Meininger, V. (1994). A controlled trial of riluzole in amyotrophic lateral sclerosis. ALS/Riluzole Study Group. N Engl J Med 330, 585-591. 10.1056/NEJM199403033300901.
82. Writing, G., and Edaravone, A.L.S.S.G. (2017). Safety and efficacy of edaravone in well defined patients with amyotrophic lateral sclerosis: a randomised, double-blind, placebo-controlled trial. Lancet Neurol 16, 505-512. 10.1016/S1474-4422(17)30115-1.
83. Paganoni, S., Macklin, E.A., Hendrix, S., Berry, J.D., Elliott, M.A., Maiser, S., Karam, C., Caress, J.B., Owegi, M.A., Quick, A., et al. (2020). Trial of Sodium Phenylbutyrate-Taurursodiol for Amyotrophic Lateral Sclerosis. N Engl J Med 383, 919-930. 10.1056/NEJMoa1916945.
84. Miller, T., Cudkowicz, M., Shaw, P.J., Andersen, P.M., Atassi, N., Bucelli, R.C., Genge, A., Glass, J., Ladha, S., Ludolph, A.L., et al. (2020). Phase 1-2 Trial of Antisense Oligonucleotide Tofersen for SOD1 ALS. N Engl J Med 383, 109-119. 10.1056/NEJMoa2003715.
85. Amado, D.A., and Davidson, B.L. (2021). Gene therapy for ALS: A review. Mol Ther 29, 3345-3358. 10.1016/j.ymthe.2021.04.008.
86. Naso, M.F., Tomkowicz, B., Perry, W.L., 3rd, and Strohl, W.R. (2017). Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs 31, 317-334. 10.1007/s40259-017-0234-5.
87. Colella, P., Ronzitti, G., and Mingozzi, F. (2018). Emerging Issues in AAV-Mediated In Vivo Gene Therapy. Mol Ther Methods Clin Dev 8, 87-104. 10.1016/j.omtm.2017.11.007.
88. Cappella, M., Ciotti, C., Cohen-Tannoudji, M., and Biferi, M.G. (2019). Gene Therapy for ALS-A Perspective. Int J Mol Sci 20. 10.3390/ijms20184388.
89. Foust, K.D., Salazar, D.L., Likhite, S., Ferraiuolo, L., Ditsworth, D., Ilieva, H., Meyer, K., Schmelzer, L., Braun, L., Cleveland, D.W., and Kaspar, B.K. (2013). Therapeutic AAV9-mediated suppression of mutant SOD1 slows disease progression and extends survival in models of inherited ALS. Mol Ther 21, 2148-2159. 10.1038/mt.2013.211.
90. Jiang, J., Zhu, Q., Gendron, T.F., Saberi, S., McAlonis-Downes, M., Seelman, A., Stauffer, J.E., Jafar-Nejad, P., Drenner, K., Schulte, D., et al. (2016). Gain of Toxicity from ALS/FTD-Linked Repeat Expansions in C9ORF72 Is Alleviated by Antisense Oligonucleotides Targeting GGGGCC-Containing RNAs. Neuron 90, 535-550. 10.1016/j.neuron.2016.04.006.
91. Becker, L.A., Huang, B., Bieri, G., Ma, R., Knowles, D.A., Jafar-Nejad, P., Messing, J., Kim, H.J., Soriano, A., Auburger, G., et al. (2017). Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice. Nature 544, 367-371. 10.1038/nature22038.
92. Yoon, K.J., Ringeling, F.R., Vissers, C., Jacob, F., Pokrass, M., Jimenez-Cyrus, D., Su, Y., Kim, N.S., Zhu, Y., Zheng, L., et al. (2017). Temporal Control of Mammalian Cortical Neurogenesis by m(6)A Methylation. Cell 171, 877-889 e817. 10.1016/j.cell.2017.09.003.
93. Zhao, F., Xu, Y., Gao, S., Qin, L., Austria, Q., Siedlak, S.L., Pajdzik, K., Dai, Q., He, C., Wang, W., et al. (2021). METTL3-dependent RNA m(6)A dysregulation contributes to neurodegeneration in Alzheimer′s disease through aberrant cell cycle events. Mol Neurodegener 16, 70. 10.1186/s13024-021-00484-x.
94. Castro-Hernandez, R., Berulava, T., Metelova, M., Epple, R., Pena Centeno, T., Richter, J., Kaurani, L., Pradhan, R., Sakib, M.S., Burkhardt, S., et al. (2023). Conserved reduction of m(6)A RNA modifications during aging and neurodegeneration is linked to changes in synaptic transcripts. Proc Natl Acad Sci U S A 120, e2204933120. 10.1073/pnas.2204933120.
95. Mandrioli, J., Mediani, L., Alberti, S., and Carra, S. (2020). ALS and FTD: Where RNA metabolism meets protein quality control. Semin Cell Dev Biol 99, 183-192. 10.1016/j.semcdb.2019.06.003.
96. McMillan, M., Gomez, N., Hsieh, C., Bekier, M., Li, X., Miguez, R., Tank, E.M.H., and Barmada, S.J. (2023). RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia. Mol Cell 83, 219-236 e217. 10.1016/j.molcel.2022.12.019.
97. Li, Y., Dou, X., Liu, J., Xiao, Y., Zhang, Z., Hayes, L., Wu, R., Fu, X., Ye, Y., Yang, B., et al. (2023). Globally reduced N(6)-methyladenosine (m(6)A) in C9ORF72-ALS/FTD dysregulates RNA metabolism and contributes to neurodegeneration. Nat Neurosci 26, 1328-1338. 10.1038/s41593-023-01374-9.
98. Yen, Y.P., Hsieh, W.F., Tsai, Y.Y., Lu, Y.L., Liau, E.S., Hsu, H.C., Chen, Y.C., Liu, T.C., Chang, M., Li, J., et al. (2018). Dlk1-Dio3 locus-derived lncRNAs perpetuate postmitotic motor neuron cell fate and subtype identity. Elife 7. 10.7554/eLife.38080.
99. Scott, S., Kranz, J.E., Cole, J., Lincecum, J.M., Thompson, K., Kelly, N., Bostrom, A., Theodoss, J., Al-Nakhala, B.M., Vieira, F.G., et al. (2008). Design, power, and interpretation of studies in the standard murine model of ALS. Amyotroph Lateral Scler 9, 4-15. 10.1080/17482960701856300.
100. Choi, S.H., Flamand, M.N., Liu, B., Zhu, H., Hu, M., Wang, M., Sewell, J., Holley, C.L., Al-Hashimi, H.M., and Meyer, K.D. (2022). RBM45 is an m(6)A-binding protein that affects neuronal differentiation and the splicing of a subset of mRNAs. Cell Rep 40, 111293. 10.1016/j.celrep.2022.111293.
101. Lim, C.K.W., Gapinske, M., Brooks, A.K., Woods, W.S., Powell, J.E., Zeballos, C.M., Winter, J., Perez-Pinera, P., and Gaj, T. (2020). Treatment of a Mouse Model of ALS by In Vivo Base Editing. Mol Ther 28, 1177-1189. 10.1016/j.ymthe.2020.01.005.
102. McCampbell, A., Cole, T., Wegener, A.J., Tomassy, G.S., Setnicka, A., Farley, B.J., Schoch, K.M., Hoye, M.L., Shabsovich, M., Sun, L., et al. (2018). Antisense oligonucleotides extend survival and reverse decrement in muscle response in ALS models. J Clin Invest 128, 3558-3567. 10.1172/JCI99081.
103. Gao, X., Shin, Y.H., Li, M., Wang, F., Tong, Q., and Zhang, P. (2010). The fat mass and obesity associated gene FTO functions in the brain to regulate postnatal growth in mice. PLoS One 5, e14005. 10.1371/journal.pone.0014005.
104. Ragagnin, A.M.G., Shadfar, S., Vidal, M., Jamali, M.S., and Atkin, J.D. (2019). Motor Neuron Susceptibility in ALS/FTD. Front Neurosci 13, 532. 10.3389/fnins.2019.00532.
105. Wu, Z., Lu, M., Liu, D., Shi, Y., Ren, J., Wang, S., Jing, Y., Zhang, S., Zhao, Q., Li, H., et al. (2023). m(6)A epitranscriptomic regulation of tissue homeostasis during primate aging. Nat Aging 3, 705-721. 10.1038/s43587-023-00393-2.
106. Van Hoecke, A., Schoonaert, L., Lemmens, R., Timmers, M., Staats, K.A., Laird, A.S., Peeters, E., Philips, T., Goris, A., Dubois, B., et al. (2012). EPHA4 is a disease modifier of amyotrophic lateral sclerosis in animal models and in humans. Nat Med 18, 1418-1422. 10.1038/nm.2901.
107. Mitsui, S., Otomo, A., Sato, K., Ishiyama, M., Shimakura, K., Okada-Yamaguchi, C., Warabi, E., Yanagawa, T., Aoki, M., Shang, H.F., and Hadano, S. (2022). SQSTM1, a protective factor of SOD1-linked motor neuron disease, regulates the accumulation and distribution of ubiquitinated protein aggregates in neuron. Neurochem Int 158, 105364. 10.1016/j.neuint.2022.105364.
108. Mitsui, S., Otomo, A., Nozaki, M., Ono, S., Sato, K., Shirakawa, R., Adachi, H., Aoki, M., Sobue, G., Shang, H.F., and Hadano, S. (2018). Systemic overexpression of SQSTM1/p62 accelerates disease onset in a SOD1(H46R)-expressing ALS mouse model. Mol Brain 11, 30. 10.1186/s13041-018-0373-8.
109. Liang, D., Lin, W.J., Ren, M., Qiu, J., Yang, C., Wang, X., Li, N., Zeng, T., Sun, K., You, L., et al. (2022). m(6)A reader YTHDC1 modulates autophagy by targeting SQSTM1 in diabetic skin. Autophagy 18, 1318-1337. 10.1080/15548627.2021.1974175.
110. Sun, M., Shen, Y., Jia, G., Deng, Z., Shi, F., Jing, Y., and Xia, S. (2023). Activation of the HNRNPA2B1/miR-93-5p/FRMD6 axis facilitates prostate cancer progression in an m6A-dependent manner. J Cancer 14, 1242-1256. 10.7150/jca.83863.
111. Cockova, Z., Honc, O., Telensky, P., Olsen, M.J., and Novotny, J. (2021). Streptozotocin-Induced Astrocyte Mitochondrial Dysfunction Is Ameliorated by FTO Inhibitor MO-I-500. ACS Chem Neurosci 12, 3818-3828. 10.1021/acschemneuro.1c00063.
112. Delaunay, S., Helm, M., and Frye, M. (2024). RNA modifications in physiology and disease: towards clinical applications. Nat Rev Genet 25, 104-122. 10.1038/s41576-023-00645-2.
113. Wei, J., Liu, F., Lu, Z., Fei, Q., Ai, Y., He, P.C., Shi, H., Cui, X., Su, R., Klungland, A., et al. (2018). Differential m(6)A, m(6)A(m), and m(1)A Demethylation Mediated by FTO in the Cell Nucleus and Cytoplasm. Mol Cell 71, 973-985 e975. 10.1016/j.molcel.2018.08.011.
114. Hideyama, T., Yamashita, T., Aizawa, H., Tsuji, S., Kakita, A., Takahashi, H., and Kwak, S. (2012). Profound downregulation of the RNA editing enzyme ADAR2 in ALS spinal motor neurons. Neurobiol Dis 45, 1121-1128. 10.1016/j.nbd.2011.12.033.
115. Li, T., Yu, G., Guo, T., Qi, H., Bing, Y., Xiao, Y., Li, C., Liu, W., Yuan, Y., He, Y., et al. (2015). The plasma S-adenosylmethionine level is associated with the severity of hepatitis B-related liver disease. Medicine (Baltimore) 94, e489. 10.1097/MD.0000000000000489.
116. Ding, W., Smulan, L.J., Hou, N.S., Taubert, S., Watts, J.L., and Walker, A.K. (2015). s-Adenosylmethionine Levels Govern Innate Immunity through Distinct Methylation-Dependent Pathways. Cell Metab 22, 633-645. 10.1016/j.cmet.2015.07.013.
117. Lee, S., and Kim, T.J. (2024). ROS-induced metabolic reprogramming to one-carbon metabolism and S-adenosylmethionine-mediated epigenetic modification in IL-10-producing B cells for the resolution of pneumonia. Cell Mol Immunol 21, 203-204. 10.1038/s41423-023-01117-7.
118. Wang, P., Doxtader, K.A., and Nam, Y. (2016). Structural Basis for Cooperative Function of Mettl3 and Mettl14 Methyltransferases. Mol Cell 63, 306-317. 10.1016/j.molcel.2016.05.041.
119. Wang, X., Feng, J., Xue, Y., Guan, Z., Zhang, D., Liu, Z., Gong, Z., Wang, Q., Huang, J., Tang, C., et al. (2016). Structural basis of N(6)-adenosine methylation by the METTL3-METTL14 complex. Nature 534, 575-578. 10.1038/nature18298.
120. Du, Y., Hou, G., Zhang, H., Dou, J., He, J., Guo, Y., Li, L., Chen, R., Wang, Y., Deng, R., et al. (2018). SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function. Nucleic Acids Res 46, 5195-5208. 10.1093/nar/gky156.
121. Li, S.R., Kang, N.N., Wang, R.R., Li, M.D., Chen, L.H., Zhou, P., Xu, D.X., Zhao, H., and Fu, L. (2024). ALKBH5 SUMOylation-mediated FBXW7 m6A modification regulates alveolar cells senescence during 1-nitropyrene-induced pulmonary fibrosis. J Hazard Mater 468, 133704. 10.1016/j.jhazmat.2024.133704.
122. Sugiokto, F.G., Saiada, F., Zhang, K., and Li, R. (2024). SUMOylation of the m6A reader YTHDF2 by PIAS1 promotes viral RNA decay to restrict EBV replication. mBio 15, e0316823. 10.1128/mbio.03168-23.
123. Porto, E.M., Komor, A.C., Slaymaker, I.M., and Yeo, G.W. (2020). Base editing: advances and therapeutic opportunities. Nat Rev Drug Discov 19, 839-859. 10.1038/s41573-020-0084-6.
124. Papadeas, S.T., Kraig, S.E., O′Banion, C., Lepore, A.C., and Maragakis, N.J. (2011). Astrocytes carrying the superoxide dismutase 1 (SOD1G93A) mutation induce wild-type motor neuron degeneration in vivo. Proc Natl Acad Sci U S A 108, 17803-17808. 10.1073/pnas.1103141108.
125. Liau, E.S., Jin, S., Chen, Y.C., Liu, W.S., Calon, M., Nedelec, S., Nie, Q., and Chen, J.A. (2023). Single-cell transcriptomic analysis reveals diversity within mammalian spinal motor neurons. Nat Commun 14, 46. 10.1038/s41467-022-35574-x.
126. Loedige, I., Baranovskii, A., Mendonsa, S., Dantsuji, S., Popitsch, N., Breimann, L., Zerna, N., Cherepanov, V., Milek, M., Ameres, S., and Chekulaeva, M. (2023). mRNA stability and m(6)A are major determinants of subcellular mRNA localization in neurons. Mol Cell 83, 2709-2725 e2710. 10.1016/j.molcel.2023.06.021.
127. Sheehan, C.J., Marayati, B.F., Bhatia, J., and Meyer, K.D. (2023). In situ visualization of m6A sites in cellular mRNAs. Nucleic Acids Res 51, e101. 10.1093/nar/gkad787.
指導教授 陳俊安 陳盛良(Jun-An Chen Shen-Liang Chen) 審核日期 2024-7-22
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