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Distal Axonal Proteins and Their Related MiRNAs in Cultured Cortical Neurons

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Abstract

Proteins and microRNAs (miRNAs) within the axon locally regulate axonal development. However, protein profiles of distal axons of cortical neurons have not been fully investigated. In particular, networks of genes encoding axonal proteins and their related miRNAs in sub compartments of neurons such as axons remain unknown. Using embryonic cortical neurons cultured in a microfluidic device and proteomic approaches, we found that distal axons contain 883 proteins. Bioinformatics analysis revealed that 94 out of these 883 proteins are related to regulating axonal growth. Of the 94 genes encoding these proteins, there were 56 candidate genes that can be putatively targeted by axon-enriched 62 miRNAs with 8mer sites that exactly match these target genes. Among them, we validated 11 proteins and 11 miRNAs, by means of western blot and RT-PCR, respectively. Treatment of distal axons with chondroitin sulfate proteoglycans (CSPGs) that inhibit axonal growth elevated miR-133b, -203a, -29a, and -92a, which were associated with reduced protein level of AKT, MTOR, PI3K, DPYSL2, MAP1B, and PPP2CA. In contrast, reduction of miR-128, -15b, -195, -26b, -34b, -376b, and -381 by CSPGs was accompanied by increased EZR, KIF5A, DCX, GSK3B, and ROCK2 proteins. In silico pathway analysis revealed an interconnected network of these miRNAs and protein coding genes that is highly related to regulating axonal growth. Our data provide new insights into networks of miRNAs and their related proteins in distal axons in mediating axonal growth.

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References

  1. Jung H, Yoon BC, Holt CE (2012) Axonal mRNA localization and local protein synthesis in nervous system assembly, maintenance and repair. Nat Rev Neurosci 13(5):308–324. https://doi.org/10.1038/nrn3210

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Gomes C, Merianda TT, Lee SJ, Yoo S, Twiss JL (2014) Molecular determinants of the axonal mRNA transcriptome. Dev Neurobiol 74(3):218–232. https://doi.org/10.1002/dneu.22123

    Article  CAS  PubMed  Google Scholar 

  3. Estrada-Bernal ASS, Sosa LJ, Simon GC, Hansen KC et al (2012) Functional complexity of the axonal growth cone: a proteomic analysis. PLoS One 7(2):e31858

    Article  CAS  Google Scholar 

  4. Igarashi M (2014) Proteomic identification of the molecular basis of mammalian CNS growth cones. Neurosci Res 88:1–15. https://doi.org/10.1016/j.neures.2014.07.005

    Article  CAS  PubMed  Google Scholar 

  5. van Niekerk EA, Tuszynski MH, Lu P, Dulin JN (2016) Molecular and cellular mechanisms of axonal regeneration after spinal cord injury. Mol Cell Proteomics 15(2):394–408. https://doi.org/10.1074/mcp.R115.053751

    Article  CAS  PubMed  Google Scholar 

  6. Natera-Naranjo O, Aschrafi A, Gioio AE, Kaplan BB (2010) Identification and quantitative analyses of microRNAs located in the distal axons of sympathetic neurons. RNA 16(8):1516–1529. https://doi.org/10.1261/rna.1833310

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Aschrafi A, Schwechter AD, Mameza MG, Natera-Naranjo O, Gioio AE, Kaplan BB (2008) MicroRNA-338 regulates local cytochrome c oxidase IV mRNA levels and oxidative phosphorylation in the axons of sympathetic neurons. J Neurosci 28(47):12581–12590. https://doi.org/10.1523/JNEUROSCI.3338-08.2008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zhang Y, Ueno Y, Liu XS, Buller B, Wang X, Chopp M, Zhang ZG (2013) The microRNA-17-92 cluster enhances axonal outgrowth in embryonic cortical neurons. J Neurosci 33(16):6885–6894. https://doi.org/10.1523/JNEUROSCI.5180-12.2013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kaplan BB, Kar AN, Gioio AE, Aschrafi A (2013) MicroRNAs in the axon and presynaptic nerve terminal. Front Cell Neurosci 7:126. https://doi.org/10.3389/fncel.2013.00126

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Liu B, Li J, Cairns MJ (2014) Identifying miRNAs, targets and functions. Brief Bioinform 15(1):1–19. https://doi.org/10.1093/bib/bbs075

    Article  CAS  PubMed  Google Scholar 

  11. Zhang Y, Chopp M, Liu XS, Kassis H, Wang X, Li C, An G, Zhang ZG (2015) MicroRNAs in the axon locally mediate the effects of chondroitin sulfate proteoglycans and cGMP on axonal growth. Dev Neurobiol 75:1402–1419. https://doi.org/10.1002/dneu.22292

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lee D-C, Hassan SS, Romero R, Tarca AL, Bhatti G, Gervasi MT, Caruso JA, Stemmer PM et al (2011) Protein profiling underscores immunological functions of uterine cervical mucus plug in human pregnancy. J Proteome 74(6):817–828. https://doi.org/10.1016/j.jprot.2011.02.025

    Article  CAS  Google Scholar 

  13. Taylor AM, Blurton-Jones M, Rhee SW, Cribbs DH, Cotman CW, Jeon NL (2005) A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nat Methods 2(8):599–605. https://doi.org/10.1038/nmeth777

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Teunissen CE, Dijkstra C, Polman C (2005) Biological markers in CSF and blood for axonal degeneration in multiple sclerosis. Lancet Neurol 4(1):32–41. https://doi.org/10.1016/S1474-4422(04)00964-0

    Article  PubMed  Google Scholar 

  15. López-Bendito G, Flames N, Ma L, Fouquet C, Di Meglio T, Chedotal A, Tessier-Lavigne M, Marín O (2007) Robo1 and Robo2 cooperate to control the guidance of major axonal tracts in the mammalian forebrain. J Neurosci 27(13):3395–3407

    Article  Google Scholar 

  16. Martínez–Yélamos A, Saiz A, Sanchez-Valle R, Casado V, Ramón JM, Graus F, Arbizu T (2001) 14-3-3 protein in the CSF as prognostic marker in early multiple sclerosis. Neurology 57(4):722–724

    Article  Google Scholar 

  17. Skene JHP, Willard M (1981) Axonally transported proteins associated with axon growth in rabbit central and peripheral nervous systems. J Cell Biol 89(1):96–103

    Article  CAS  Google Scholar 

  18. De Vos KJ, Grierson AJ, Ackerley S, Miller CCJ (2008) Role of axonal transport in neurodegenerative diseases. Annu Rev Neurosci 31(1):151–173. https://doi.org/10.1146/annurev.neuro.31.061307.090711

    Article  CAS  PubMed  Google Scholar 

  19. Elvira G, Wasiak S, Blandford V, Tong X-K, Serrano A, Fan X, del Rayo Sánchez-Carbente M, Servant F et al (2006) Characterization of an RNA granule from developing brain. Mol Cell Proteomics 5:635–651

    Article  CAS  Google Scholar 

  20. Hörnberg H, Holt C (2013) RNA-binding proteins and translational regulation in axons and growth cones. Front Neurosci 7:81. https://doi.org/10.3389/fnins.2013.00081

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Tripathi VB, Baskaran P, Shaw CE, Guthrie S (2014) Tar DNA-binding protein-43 (TDP-43) regulates axon growth in vitro and in vivo. Neurobiol Dis 65(100):25–34. https://doi.org/10.1016/j.nbd.2014.01.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sotelo-Silveira JR, Calliari A, Kun A, Koenig E, Sotelo JR (2006) RNA trafficking in axons. Traffic 7(5):508–515. https://doi.org/10.1111/j.1600-0854.2006.00405.x

    Article  CAS  PubMed  Google Scholar 

  23. Read D, Gorman A (2009) Involvement of Akt in neurite outgrowth. Cell Mol Life Sci 66(18):2975–2984. https://doi.org/10.1007/s00018-009-0057-8

    Article  CAS  PubMed  Google Scholar 

  24. Tohda C, Kuboyama T, Komatsu K (2005) Search for natural products related to regeneration of the neuronal network. Neurosignals 14(1–2):34–45

    Article  CAS  Google Scholar 

  25. More SV, Koppula S, Kim I-S, Kumar H, Kim B-W, Choi D-K (2012) The role of bioactive compounds on the promotion of neurite outgrowth. Molecules 17(6):6728–6753

    Article  CAS  Google Scholar 

  26. Miyaguchi K (2004) Localization of selenium-binding protein at the tips of rapidly extending protrusions. Histochem Cell Biol 121(5):371–376. https://doi.org/10.1007/s00418-004-0623-y

    Article  CAS  PubMed  Google Scholar 

  27. Lu W-c, Y-x Z, Qiao P, Zheng J, Wu Q, Shen Q (2018) The protocadherin alpha cluster is required for axon extension and myelination in the developing central nervous system. Neural Regen Res 13(3):427–433. https://doi.org/10.4103/1673-5374.228724

    Article  PubMed  PubMed Central  Google Scholar 

  28. Mosca TJ, Luginbuhl DJ, Wang IE, Luo L (2017) Presynaptic LRP4 promotes synapse number and function of excitatory CNS neurons. eLife 6:e27347. https://doi.org/10.7554/eLife.27347

    Article  PubMed  PubMed Central  Google Scholar 

  29. Terada K, Kojima Y, Watanabe T, Izumo N, Chiba K, Karube Y (2014) Inhibition of nerve growth factor-induced neurite outgrowth from PC12 cells by dexamethasone: signaling pathways through the glucocorticoid receptor and phosphorylated Akt and ERK1/2. PLoS One 9(3):e93223. https://doi.org/10.1371/journal.pone.0093223

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ketschek A, Jones S, Spillane M, Korobova F, Svitkina T, Gallo G (2015) Nerve growth factor promotes reorganization of the axonal microtubule array at sites of axon collateral branching. Dev Neurobiol 75(12):1441–1461. https://doi.org/10.1002/dneu.22294

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Higuero AM, Sánchez-Ruiloba L, Doglio LE, Portillo F, Abad-Rodríguez J, Dotti CG, Iglesias T (2010) Kidins220/ARMS modulates the activity of microtubule-regulating proteins and controls neuronal polarity and development. J Biol Chem 285(2):1343–1357. https://doi.org/10.1074/jbc.M109.024703

    Article  CAS  PubMed  Google Scholar 

  32. Liz MA, Mar FM, Santos TE, Pimentel HI, Marques AM, Morgado MM, Vieira S, Sousa VF et al (2014) Neuronal deletion of GSK3β increases microtubule speed in the growth cone and enhances axon regeneration via CRMP-2 and independently of MAP1B and CLASP2. BMC Biol 12:47–47. https://doi.org/10.1186/1741-7007-12-47

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Williams RR, Venkatesh I, Pearse DD, Udvadia AJ, Bunge MB (2015) MASH1/Ascl1a leads to GAP43 expression and axon regeneration in the adult CNS. PLoS One 10(3):e0118918. https://doi.org/10.1371/journal.pone.0118918

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Sosa LJ, Bergman J, Estrada-Bernal A, Glorioso TJ, Kittelson JM, Pfenninger KH (2013) Amyloid precursor protein is an autonomous growth cone adhesion molecule engaged in contact guidance. PLoS One 8(5):e64521. https://doi.org/10.1371/journal.pone.0064521

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Thelen K, Jaehrling S, Spatz JP, Pollerberg GE (2012) Depending on its nano-spacing, ALCAM promotes cell attachment and axon growth. PLoS One 7(12):e40493. https://doi.org/10.1371/journal.pone.0040493

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Hur E-M, Zhou F-Q (2010) GSK3 signaling in neural development. Nat Rev Neurosci 11(8):539–551. https://doi.org/10.1038/nrn2870

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Hur E-M, Saijilafu LBD, Kim S-J, Xu W-L, Zhou F-Q (2011) GSK3 controls axon growth via CLASP-mediated regulation of growth cone microtubules. Genes Dev 25(18):1968–1981. https://doi.org/10.1101/gad.17015911

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Goldie BJ, Cairns MJ (2012) Post-transcriptional trafficking and regulation of neuronal gene expression. Mol Neurobiol 45(1):99–108. https://doi.org/10.1007/s12035-011-8222-0

    Article  CAS  PubMed  Google Scholar 

  39. Sasaki Y, Gross C, Xing L, Goshima Y, Bassell GJ (2014) Identification of axon-enriched microRNAs localized to growth cones of cortical neurons. Dev Neurobiol 74(3):397–406. https://doi.org/10.1002/dneu.22113

    Article  CAS  PubMed  Google Scholar 

  40. Nam J-W, Rissland OS, Koppstein D, Abreu-Goodger C, Jan CH, Agarwal V, Yildirim MA, Rodriguez A et al (2014) Global analyses of the effect of different cellular contexts on microRNA targeting. Mol Cell 53(6):1031–1043. https://doi.org/10.1016/j.molcel.2014.02.013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Silver J, Miller JH (2004) Regeneration beyond the glial scar. Nat Rev Neurosci 5(2):146–156

    Article  CAS  Google Scholar 

  42. Twiss JL, Kalinski AL, Sachdeva R, Houle JD (2016) Intra-axonal protein synthesis – a new target for neural repair? Neural Regen Res 11(9):1365–1367. https://doi.org/10.4103/1673-5374.191193

    Article  PubMed  PubMed Central  Google Scholar 

  43. Irena Ivanovska MAC (2008) Combinatorial microRNAs: working together to make a difference. Cell Cycle 7(20):3137–3142

    Article  Google Scholar 

  44. Tarang S, Weston MD (2014) Macros in microRNA target identification: a comparative analysis of in silico, in vitro, and in vivo approaches to microRNA target identification. RNA Biol 11(4):324–333. https://doi.org/10.4161/rna.28649

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Dajas-Bailador F, Bonev B, Garcez P, Stanley P, Guillemot F, Papalopulu N (2012) MicroRNA-9 regulates axon extension and branching by targeting Map1b in mouse cortical neurons. Nat Neurosci 15(5):697–699 http://www.nature.com/neuro/journal/v15/n5/abs/nn.3082.html#supplementary-information

    Article  CAS  Google Scholar 

  46. Lai Y-W, Chu S-Y, Wei J-Y, Cheng C-Y, Li J-C, Chen P-L, Chen C-H, Yu H-H (2016) Drosophila microRNA-34 impairs axon pruning of mushroom body γ neurons by downregulating the expression of ecdysone receptor. Sci Rep 6:39141. https://doi.org/10.1038/srep39141

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Sun X, Zhou Z, Fink DJ, Mata M (2013) HspB1 silences translation of PDZ-RhoGEF by enhancing miR-20a and miR-128 expression to promote neurite extension. Mol Cell Neurosci 57:111–119. https://doi.org/10.1016/j.mcn.2013.10.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This work was supported by the National Institutes of Health (RO1 NS088656 and RO1 NS75156) and American Heart Association (16SDG29860003).

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Authors and Affiliations

  1. Department of Neurology, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, MI, 48202, USA

    Chao Li, Yi Zhang, Bao Yan Fan, Hua Teng, Michael Chopp & Zheng Gang Zhang

  2. Department of Public Health Sciences, Henry Ford Hospital, Detroit, MI, 48202, USA

    Albert M. Levin

  3. Center of Bioinformatics, Henry Ford Hospital, Detroit, MI, 48202, USA

    Albert M. Levin

  4. Oakland University William Beaumont School of Medicine, Royal Oak, MI, 48073, USA

    Moleca M. Ghannam

  5. Department of Physics, Oakland University, Rochester, MI, 48309, USA

    Michael Chopp

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  1. Chao Li
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  2. Yi Zhang
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  3. Albert M. Levin
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Contributions

Conceived and designed the experiments: C.L. and Z.Z. Performed the experiments: C.L., Y.Z., M.G., H.T., and B.F. Analyzed the data: C.L., A.L., and Z.Z. Prepared all the figures: C.L., Y.Z., and Z.Z. Wrote the manuscript: C.L., A.L., M.C., and Z.Z.

Corresponding author

Correspondence to Zheng Gang Zhang.

Ethics declarations

The study was carried out in accordance with the NIH Guide for the Care and Use of Laboratory. Animals were approved by the Institutional Animal Care and Use Committee of Henry Ford Hospital.

Competing Interests

The authors declare that they have no competing interests.

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Significance

Axonal proteins locally regulate axonal development. However, the protein profile in the distal axon has not been investigated. Using proteomic and quantitative RT-PCR approaches in combination with bioinformatics analysis, the present study identified a network of miRNAs and their target proteins in distal axons, which mediate axonal growth. This finding provides molecular basis for further investigating the roles of proteins and miRNAs within the distal axon in mediating axonal function.

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Li, C., Zhang, Y., Levin, A.M. et al. Distal Axonal Proteins and Their Related MiRNAs in Cultured Cortical Neurons. Mol Neurobiol 56, 2703–2713 (2019). https://doi.org/10.1007/s12035-018-1266-7

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