AAV-Mediated Expression of miR-17 Enhances Neurite and Axon Regeneration In Vitro
Abstract
:1. Introduction
2. Results
2.1. AAV.miR-17 Efficiently Transduce Primary Cortical Neurons
2.2. AAV.miR-17 Does Not Affect Neurite Outgrowth and Arborization
2.3. AAV.miR-17 Enhances Neurite and Axon Regeneration
2.4. miR-17 Is Predicted to Regulate the Expression of Genes Involved in Autophagy and Cell Metabolism
3. Discussion
4. Materials and Methods
4.1. Cloning and Production of AAV Vectors
4.2. Primary Culture of Cortical Neurons and AAV Vector Transduction
4.3. Neurite Outgrowth Experiment and Quantification
4.4. Evaluation of Neurite Arborization by Sholl Analysis
4.5. Mechanical Scratch Lesion and Quantification of Neurite Regeneration
4.6. Culture of Cortical Neurons in Microfluidic Chambers
4.7. Axotomy, Live-Imaging, and Quantification of Axonal Regeneration of Primary Cortical Neurons Cultured in Microfluidic Chambers
4.8. Immunofluorescence
4.9. MiRNA-Target Prediction and Gene Ontology Analyses
4.10. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Neukomm, L.J.; Freeman, M.R. Diverse cellular and molecular modes of axon degeneration. Cell Press 2014, 24, 515–523. [Google Scholar] [CrossRef] [PubMed]
- Goldberg, J.L.; Klassen, M.P.; Hua, Y.; Barres, B.A. Amacrine-Signaled Loss of Intrinsic Axon Growth Ability by Retinal Ganglion Cells. Science 2002, 296, 1860–1864. [Google Scholar] [CrossRef] [PubMed]
- Yiu, G.; He, Z. Glial inhibition of CNS axon regeneration. Nature 2006, 7, 617–627. [Google Scholar] [CrossRef]
- Bouslama-Oueghlani, L.; Wehrle, R.; Sotelo, C.; Dusart, I. The Developmental Loss of the Ability of Purkinje Cells to Regenerate Their Axons Occurs in the Absence of Myelin: Na In Vitro Model to Prevent Myelination. J. Neurosci. 2003, 23, 8318–8329. [Google Scholar] [CrossRef] [PubMed]
- Blackmore, M.; Letourneau, P.C. Changes within Maturing Neurons Limit Axonal Regeneration in the Developing Spinal Cord. J. Neurobiol. 2006, 66, 348–360. [Google Scholar] [CrossRef] [PubMed]
- Ribas, T.V.; Costa, M.R. Gene Manipulation Strategies to Identify Molecular Regulators of Axon Regeneration in the Central Nervous System. Front. Cell. Neurosci. 2017, 11, 231. [Google Scholar] [CrossRef] [PubMed]
- Moore, D.L.; Blackmore, M.G.; Hu, Y.; Kaestner, K.H.; Bixby, J.L.; Lemmon, V.P.; Goldberg, J.L. KLF Family Members Regulate Intrinsic Axon Regeneration Ability. Science 2009, 326, 298–301. [Google Scholar] [CrossRef] [PubMed]
- Bushati, N.; Cohen, S.M. microRNA Functions. Annu. Rev. Cell Dev. Biol. 2007, 23, 175–205. [Google Scholar] [CrossRef] [PubMed]
- Juźwika, C.A.; Drakea, S.S.; Zhanga, Y.; Paradis-Islera, N.; Sylvestera, A.; Amar-Zifkinb, A.; Douglasc, C.; Morquettea, B.; Moore, C.S.; Fourniera, A.E. microRNA dysregulation in neurodegenerative diseases: A systematic review. Prog. Neurobiol. 2019, 182, 1–12. [Google Scholar] [CrossRef] [PubMed]
- Mendell, J.T. miRiad Roles for the miR-17-92 Cluster in Development and Disease. Cell 2008, 133, 217–222. [Google Scholar] [CrossRef]
- Mao, S.; Li, H.; Sun, Q.; Zen, K.; Zhang, C.Y.; Li, L. miR-17 regulates the proliferation and differentiation of the neural precursor cells during mouse corticogenesis. FEBS J. 2014, 281, 1144–1158. [Google Scholar] [CrossRef]
- Benavente, F.; Pinto, C.; Parada, M.; Henríquez, J.P.; Osses, N. Bone morphogenetic protein 2 inhibits neurite outgrowth of motor neuron-like NSC-34 cells and up-regulates its type II receptor. J. Neurochem. 2012, 122, 594–604. [Google Scholar] [CrossRef]
- Rishal, I.; Fainzilber, M. Axon–soma communication in neuronal injury. Nature 2014, 15, 32–42. [Google Scholar] [CrossRef] [PubMed]
- Vahsen, B.F.; Ribas, V.T.; Sundermeyer, J.; Boecker, A.; Dambeck, V.; Lenz, C.; Shomroni, O.; Gomes, L.C.; Tatenhorst, L.; Barski, E.; et al. Inhibition of the autophagic protein ULK1 attenuates axonal degeneration in vitro and in vivo, enhances translation, and modulates splicing. Cell Death Differ. 2020, 27, 2810–2827. [Google Scholar] [CrossRef] [PubMed]
- Ribas, V.T.; Vahsen, B.F.; Tatenhorst, L.; Estrada, V.; Dambeck, V.; Almeida, R.A.; Bähr, M.; Michel, U.; Koch, J.C.; Müller, H.W.; et al. AAV-mediated inhibition of ULK1 promotes axonal regeneration in the central nervous system in vitro and in vivo. Cell Death Dis. 2021, 12, 213. [Google Scholar] [CrossRef] [PubMed]
- Sticht, C.; Torre, C.D.L.; Parveen, A.; Gretz, N. miRWalk: An online resource for prediction of microRNA binding sites. PLoS ONE 2018, 13, e0206239. [Google Scholar] [CrossRef]
- Naka-Kaneda, H.; Nakamura, S.; Igarashi, M.; Aoi, H.; Kanki, H.; Tsuyama, J.; Tsutsumi, S.; Aburatani, H.; Shimazaki, T.; Okano, H. The miR-17/106-p38 axis is a key regulator of the neurogenic-to-gliogenic transition in developing neural stem/progenitor cells. Proc. Natl. Acad. Sci. USA 2014, 111, 1604–1609. [Google Scholar] [CrossRef] [PubMed]
- Chen, J.A.; Huang, Y.P.; Mazzoni, E.O.; Tan, G.C.; Zavadil, J.; Wichterle, H. Mir-17-3p controls spinal neural progenitor patterning by regulating Olig2/Irx3 cross-repressive loop. Neuron 2011, 69, 721–735. [Google Scholar] [CrossRef]
- Altounian, M.; Bellon, A.; Mann, F. Neuronal miR-17-5p contributes to interhemispheric cortical connectivity defects induced by prenatal alcohol exposure. Cell Rep. 2023, 42, 113020. [Google Scholar] [CrossRef] [PubMed]
- Zhang, Y.; Ueno, Y.; Liu, X.S.; Buller, B.; Wang, X.; Chopp, M.; Zhang, Z.G. The MicroRNA-17-92 cluster enhances axonal outgrowth in embryonic cortical neurons. J. Neurosci. 2013, 33, 6885–6894. [Google Scholar] [CrossRef]
- Park, J.W.; Vahidi, B.; Taylor, A.M.; Rhee, S.W.; Jeon, N.L. Microfluidic culture platform for neuroscience research. Nat. Protoc. 2006, 1, 2128–2136. [Google Scholar] [CrossRef] [PubMed]
- Tran, A.P.; Warren, P.M.; Silver, J. Regulation of autophagy by inhibitory CSPG interactions with receptor PTPσ and its impact on plasticity and regeneration after spinal cord injury. Exp. Neurol. 2020, 328, 113276. [Google Scholar] [CrossRef] [PubMed]
- Beckers, A.; Vanhunsel, S.; Van Dyck, A.; Bergmans, S.; Masin, L.; Moons, L. Injury-induced Autophagy Delays Axonal Regeneration after Optic Nerve Damage in Adult Zebrafish. Neuroscience 2021, 470, 52–69. [Google Scholar] [CrossRef]
- Oropesa, M.; de la Mata, M.; Maraver, J.G.; Cordero, M.D.; Cotán, D.; Rodríguez-Hernández, A.; Domínguez-Moñino, I.; de Miguel, M.; Navas, P.; Sánchez-Alcázar, J.A. Apoptotic microtubule network organization and maintenance depend on high cellular ATP levels and energized mitochondria. Apoptosis 2011, 16, 404–424. [Google Scholar] [CrossRef]
- Cassimeris, L.; Silva, V.C.; Miller, E.; Ton, Q.; Molnar, C.; Fong, J. Fueled by microtubules: Does tubulin dimer/polymer partitioning regulate intracellular metabolism? Cytoskeleton 2012, 69, 133–143. [Google Scholar] [CrossRef]
- Duan, Q.; Zheng, H.; Qin, Y.; Yan, J.; Wang, J.; Burgess, S.M.; Fan, C. Stat3 Has a Different Role in Axon Growth During Development Than It Does in Axon Regeneration After Injury. Mol. Neurobiol. 2024, 61, 1753–1768. [Google Scholar] [CrossRef] [PubMed]
- Kim, S.H.; Trinh, A.T.; Larsen, M.C.; Mastrocola, A.S.; Jefcoate, C.R.; Bushel, P.R.; Tibbetts, R.S. Tunable regulation of CREB DNA binding activity couples genotoxic stress response and metabolism. Nucleic Acids Res. 2016, 44, 9667–9680. [Google Scholar] [CrossRef]
- Sancho, M.; Leiva, D.; Lucendo, E.; Orzáez, M. Understanding MCL1: From cellular function and regulation to pharmacological inhibition. FEBS J. 2022, 289, 6209–6234. [Google Scholar] [CrossRef]
- You, L.; Wang, Z.; Li, H.; Shou, J.; Jing, Z.; Xie, J.; Sui, X.; Pan, H.; Han, W. The role of STAT3 in autophagy. Autophagy 2015, 11, 729–739. [Google Scholar] [CrossRef] [PubMed]
- Bian, H.; Li, F.; Wang, W.; Zhao, Q.; Gao, S.; Ma, J.; Li, X.; Ren, W.; Qin, C.; Qi, J. MAPK/p38 regulation of cytoskeleton rearrangement accelerates induction of macrophage activation by TLR4, but not TLR3. Int. J. Mol. Med. 2017, 40, 1495–1503. [Google Scholar] [CrossRef]
- Wu, S.Y.; Lan, S.H.; Liu, H.S. Degradative autophagy selectively regulates CCND1 (cyclin D1) and MIR224, two oncogenic factors involved in hepatocellular carcinoma tumorigenesis. Autophagy 2019, 15, 729–730. [Google Scholar] [CrossRef]
- Jonchère, B.; Bélanger, A.; Guette, C.; Barré, B.; Coqueret, O. STAT3 as a new autophagy regulator. Jak-Stat 2013, 2, e24353. [Google Scholar] [CrossRef]
- Shen, S.; Niso-Santano, M.; Adjemian, S.; Takehara, T.; Malik, S.A.; Minoux, H.; Souquere, S.; Mariño, G.; Lachkar, S.; Senovilla, L.; et al. Cytoplasmic STAT3 represses autophagy by inhibiting PKR activity. Mol. Cell 2012, 48, 667–680. [Google Scholar] [CrossRef] [PubMed]
- Yang, J.; Liao, X.; Agarwal, M.K.; Barnes, L.; Auron, P.E.; Stark, G.R. Unphosphorylated STAT3 accumulates in response to IL-6 and activates transcription by binding to NFkappaB. Genes Dev. 2007, 21, 1396–1408. [Google Scholar] [CrossRef]
- Zhang, H. CCND1 silencing suppresses liver cancer stem cell differentiation through inhibiting autophagy. Hum. Cell 2020, 33, 140–147. [Google Scholar] [CrossRef]
- Su, Y.; Zhou, H.; Ma, Z.; Liu, J.; Li, C. CCND1-Induced Autophagy Contributes to Lymph Node Metastasis in Endometrial Cancer. Horm. Metab. Res. 2023, 55, 413–419. [Google Scholar] [CrossRef]
- Man, G.; Yang, H.; Shen, K.; Zhang, D.; Zhang, J.; Wu, H.; Zhang, H.; Wang, J. Circular RNA RHOT1 Regulates miR-142-5p/CCND1 to Participate in Chondrocyte Autophagy and Proliferation in Osteoarthritis. J. Immunol. Res. 2022, 2022, 4370873. [Google Scholar] [CrossRef]
- Nardone, V.; Barbarino, M.; Angrisani, A.; Correale, P.; Pastina, P.; Cappabianca, S.; Reginelli, A.; Mutti, L.; Miracco, C.; Giannicola, R.; et al. CDK4, CDK6/cyclin-D1 Complex Inhibition and Radiotherapy for Cancer Control: A Role for Autophagy. Int. J. Mol. Sci. 2021, 22, 8391. [Google Scholar] [CrossRef]
- Wade, H.; Pan, K.; Su, Q. CREBH: A Complex Array of Regulatory Mechanisms in Nutritional Signaling, Metabolic Inflammation, and Metabolic Disease. Mol. Nutr. Food Res. 2021, 65, e2000771. [Google Scholar] [CrossRef]
- Bruno, N.E.; Nwachukwu, J.C.; Hughes, D.C.; Srinivasan, S.; Hawkins, R.; Sturgill, D.; Hager, G.L.; Hurst, S.; Sheu, S.S.; Bodine, S.C.; et al. Activation of Crtc2/Creb1 in skeletal muscle enhances weight loss during intermittent fasting. FASEB J. 2021, 35, e21999. [Google Scholar] [CrossRef]
- Cui, A.; Ding, D.; Li, Y. Regulation of Hepatic Metabolism and Cell Growth by the ATF/CREB Family of Transcription Factors. Diabetes 2021, 70, 653–664. [Google Scholar] [CrossRef]
- Balke, D.; Tatenhorst, L.; Dambeck, V.; Ribas, V.T.; Vahsen, B.F.; Michel, U.; Bähr, M.; Lingor, P. AAV-Mediated Expression of Dominant-Negative ULK1 Increases Neuronal Survival and Enhances Motor Performance in the MPTP Mouse Model of Pa kinson’s Disease. Mol. Neurobiol. 2019, 57, 685–697. [Google Scholar] [CrossRef] [PubMed]
- Zolotukhin, S.; Byrne, B.J.; Mason, E.; Zolotukhin, I.; Potter, M.; Chesnut, K.; Summerford, C.; Samulski, R.J.; Muzyczka, N. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 1999, 6, 973–985. [Google Scholar] [CrossRef] [PubMed]
- Huang, H.Y.; Lin, Y.C.; Cui, S.; Huang, Y.; Tang, Y.; Xu, J.; Bao, J.; Li, Y.; Wen, J.; Zuo, H.; et al. miRTarBase update 2022: An informative resource for experimentally validated miRNA–target interactions. Nucleic Acids Res. 2022, 50, 222–230. [Google Scholar] [CrossRef] [PubMed]
- Ge, S.X.; Jung, D.; Yao, R. ShinyGO: A graphical gene-set enrichment tool for animals and plants. Bioinformatics 2020, 36, 2628–2629. [Google Scholar] [CrossRef]
- Szklarczyk, D.; Kirsch, R.; Koutrouli, M.; Nastou, K.; Mehryary, F.; Hachilif, R.; Gable, A.L.; Fang, T.; Doncheva, N.T.; Pyysalo, S.; et al. The STRING database in 2023: Protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res. 2023, 51, 638–643. [Google Scholar] [CrossRef]
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Almeida, R.A.; Ferreira, C.G.; Matos, V.U.S.; Nogueira, J.M.; Braga, M.P.; Caldi Gomes, L.; Jorge, E.C.; Soriani, F.M.; Michel, U.; Ribas, V.T. AAV-Mediated Expression of miR-17 Enhances Neurite and Axon Regeneration In Vitro. Int. J. Mol. Sci. 2024, 25, 9057. https://doi.org/10.3390/ijms25169057
Almeida RA, Ferreira CG, Matos VUS, Nogueira JM, Braga MP, Caldi Gomes L, Jorge EC, Soriani FM, Michel U, Ribas VT. AAV-Mediated Expression of miR-17 Enhances Neurite and Axon Regeneration In Vitro. International Journal of Molecular Sciences. 2024; 25(16):9057. https://doi.org/10.3390/ijms25169057
Chicago/Turabian StyleAlmeida, Raquel Alves, Carolina Gomes Ferreira, Victor Ulysses Souza Matos, Julia Meireles Nogueira, Marina Pimenta Braga, Lucas Caldi Gomes, Erika Cristina Jorge, Frederico Marianetti Soriani, Uwe Michel, and Vinicius Toledo Ribas. 2024. "AAV-Mediated Expression of miR-17 Enhances Neurite and Axon Regeneration In Vitro" International Journal of Molecular Sciences 25, no. 16: 9057. https://doi.org/10.3390/ijms25169057