Advertisement

Molecular Neurobiology

, Volume 56, Issue 4, pp 2703–2713 | Cite as

Distal Axonal Proteins and Their Related MiRNAs in Cultured Cortical Neurons

  • Chao Li
  • Yi Zhang
  • Albert M. Levin
  • Bao Yan Fan
  • Hua Teng
  • Moleca M. Ghannam
  • Michael Chopp
  • Zheng Gang ZhangEmail author
Article

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.

Keywords

Axonal growth Axonal proteins and MiRNAs Bioinformatics 

Notes

Author Contribution

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.

Funding information

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

Compliance with Ethical Standards

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.

Supplementary material

12035_2018_1266_MOESM1_ESM.xlsx (154 kb)
ESM 1 (XLSX 154 kb)
12035_2018_1266_MOESM2_ESM.xlsx (31 kb)
ESM 2 (XLSX 31 kb)
12035_2018_1266_MOESM3_ESM.xlsx (12 kb)
ESM 3 (XLSX 11 kb)
12035_2018_1266_Fig5_ESM.png (425 kb)
ESM 4

(PNG 424 kb)

12035_2018_1266_MOESM4_ESM.tif (741 kb)
High Resolution Image (TIF 740 kb)

References

  1. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 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 CrossRefPubMedGoogle Scholar
  3. 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):e31858CrossRefGoogle Scholar
  4. 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 CrossRefPubMedGoogle Scholar
  5. 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 CrossRefPubMedGoogle Scholar
  6. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 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 CrossRefPubMedGoogle Scholar
  11. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 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 CrossRefGoogle Scholar
  13. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 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 CrossRefPubMedGoogle Scholar
  15. 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):3395–3407CrossRefGoogle Scholar
  16. 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):722–724CrossRefGoogle Scholar
  17. 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):96–103CrossRefGoogle Scholar
  18. 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 CrossRefPubMedGoogle Scholar
  19. 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 5:635–651CrossRefGoogle Scholar
  20. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 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 CrossRefPubMedGoogle Scholar
  23. 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 CrossRefPubMedGoogle Scholar
  24. 24.
    Tohda C, Kuboyama T, Komatsu K (2005) Search for natural products related to regeneration of the neuronal network. Neurosignals 14(1–2):34–45CrossRefGoogle Scholar
  25. 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):6728–6753CrossRefGoogle Scholar
  26. 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 CrossRefPubMedGoogle Scholar
  27. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  31. 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 CrossRefPubMedGoogle Scholar
  32. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 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 CrossRefPubMedGoogle Scholar
  39. 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 CrossRefPubMedGoogle Scholar
  40. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Silver J, Miller JH (2004) Regeneration beyond the glial scar. Nat Rev Neurosci 5(2):146–156CrossRefGoogle Scholar
  42. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Irena Ivanovska MAC (2008) Combinatorial microRNAs: working together to make a difference. Cell Cycle 7(20):3137–3142CrossRefGoogle Scholar
  44. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  45. 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 CrossRefGoogle Scholar
  46. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  47. 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 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of NeurologyHenry Ford HospitalDetroitUSA
  2. 2.Department of Public Health SciencesHenry Ford HospitalDetroitUSA
  3. 3.Center of BioinformaticsHenry Ford HospitalDetroitUSA
  4. 4.Oakland University William Beaumont School of MedicineRoyal OakUSA
  5. 5.Department of PhysicsOakland UniversityRochesterUSA

Personalised recommendations