Molecular and Cellular Biochemistry

, Volume 384, Issue 1–2, pp 7–19 | Cite as

Nogo/RTN4 isoforms and RTN3 expression protect SH-SY5Y cells against multiple death insults

  • Felicia Yu Hsuan Teng
  • Bor Luen TangEmail author


Among the members of the reticulon (RTN) family, Nogo-A/RTN4A, a prominent myelin-associated neurite growth inhibitory protein, and RTN3 are highly expressed in neurons. However, neuronal cell-autonomous functions of Nogo-A, as well as other members of the RTN family, are unclear. We show here that SH-SY5Y neuroblastoma cells stably over-expressing either two of the three major isoforms of Nogo/RTN4 (Nogo-A and Nogo-B) or a major isoform of RTN3 were protected against cell death induced by a battery of apoptosis-inducing agents (including serum deprivation, staurosporine, etoposide, and H2O2) compared to vector-transfected control cells. Nogo-A, -B, and RTN3 are particularly effective in terms of protection against H2O2-induced increase in intracellular reactive oxygen species levels and ensuing apoptotic and autophagic cell death. Expression of these RTNs upregulated basal levels of Bax, activated Bax, and activated caspase 3, but did not exhibit an enhanced ER stress response. The protective effect of RTNs is also not dependent on classical survival-promoting signaling pathways such as Akt and Erk kinase pathways. Neuron-enriched Nogo-A/Rtn4A and RTN3 may, therefore, exert a protective effect on neuronal cells against death stimuli, and elevation of their levels during injury may have a cell-autonomous survival-promoting function.


CNS injury Neuroprotection Reticulon 3 (RTN3) Nogo 



Work was supported by research Grant number 06/1/21/19/438 from the Agency for Science, Technology and Research (A*STAR)’s Biomedical Research council (BMRC). We are grateful to Dr Luc Dupuis (Inserm U692, Strasbourg, France) for the pan-Nogo isoform antibody. We thank Dr Marie Clement (Department of Biochemistry, National University of Singapore) and her lab members for discussions and sharing of reagents.

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

11010_2013_1776_MOESM1_ESM.tif (122 kb)
Expression of Nogo isoforms and RTN3 do not significantly elevate ER stress. A Western immunoblot analysis of SH-SY5Y vector-transfected control (v.c.) and SH-SY5Y cells stably expressing Nogo-A, -B, -C or RTN3 for markers of ER stress response. Lysates were resolved by SDS-PAGE, blotted and probed with antibodies against phosphorylated and total eIF2α, GRP78, GRP94 and γ-tubulin (for loading normalization). B Western immunoblot analysis of SH-SY5Y vector-transfected control (v.c.) and SH-SHSY5Y cells stably expressing Nogo-A for changes in the levels of ER stress response markers at various timepoints from 0 to 48 hr after treatment with 100 μM H2O2 for 30 min. Lysates were resolved by SDS-PAGE, blotted and probed with antibodies against phosphorylated and total eIF2α, GRP78 and GRP94. Nogo-A was probed to show Nogo-A over-expression in the Nogo-A stably expressing cells while γ-tubulin was probed for loading normalization (TIFF 122 kb)


  1. 1.
    Ng CEL, Tang BL (2002) Nogos and the Nogo-66 receptor: factors inhibiting CNS neuron regeneration. J Neurosci Res 67:559–565PubMedCrossRefGoogle Scholar
  2. 2.
    Schwab ME (2004) Nogo and axon regeneration. Curr Opin Neurobiol 14:118–124. doi: 10.1016/j.conb.2004.01.004 PubMedCrossRefGoogle Scholar
  3. 3.
    Schweigreiter R, Bandtlow CE (2006) Nogo in the injured spinal cord. J Neurotrauma 23:384–396. doi: 10.1089/neu.2006.23.384 PubMedCrossRefGoogle Scholar
  4. 4.
    Fournier AE, GrandPre T, Strittmatter SM (2001) Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration. Nature 409:341–346. doi: 10.1038/35053072 PubMedCrossRefGoogle Scholar
  5. 5.
    He Z, Koprivica V (2004) The Nogo signaling pathway for regeneration block. Annu Rev Neurosci 27:341–368. doi: 10.1146/annurev.neuro.27.070203.144340 PubMedCrossRefGoogle Scholar
  6. 6.
    Park JB, Yiu G, Kaneko S, Wang J, Chang J, He XL et al (2005) A TNF receptor family member, TROY, is a coreceptor with Nogo receptor in mediating the inhibitory activity of myelin inhibitors. Neuron 45:345–351. doi: 10.1016/j.neuron.2004.12.040 PubMedCrossRefGoogle Scholar
  7. 7.
    Shao Z, Browning JL, Lee X, Scott ML, Shulga-Morskaya S, Allaire N et al (2005) TAJ/TROY, an orphan TNF receptor family member, binds Nogo-66 receptor 1 and regulates axonal regeneration. Neuron 45:353–359. doi: 10.1016/j.neuron.2004.12.050 PubMedCrossRefGoogle Scholar
  8. 8.
    Mi S, Sandrock A, Miller RH (2008) LINGO-1 and its role in CNS repair. Int J Biochem Cell Biol 40:1971–1978. doi: 10.1016/j.biocel.2008.03.018 PubMedCrossRefGoogle Scholar
  9. 9.
    Yiu G, He Z (2006) Glial inhibition of CNS axon regeneration. Nat Rev Neurosci 7:617–627. doi: 10.1038/nrn1956 PubMedCrossRefGoogle Scholar
  10. 10.
    Chen MS, Huber AB, van der Haar ME, Frank M, Schnell L, Spillmann AA et al (2000) Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature 403:434–439. doi: 10.1038/35000219 PubMedCrossRefGoogle Scholar
  11. 11.
    GrandPré T, Nakamura F, Vartanian T, Strittmatter SM (2000) Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein. Nature 403:439–444. doi: 10.1038/35000226 PubMedCrossRefGoogle Scholar
  12. 12.
    Hunt D, Mason MRJ, Campbell G, Coffin R, Anderson PN (2002) Nogo receptor mRNA expression in intact and regenerating CNS neurons. Mol Cell Neurosci 20:537–552PubMedCrossRefGoogle Scholar
  13. 13.
    Hunt D, Coffin RS, Prinjha RK, Campbell G, Anderson PN (2003) Nogo-A expression in the intact and injured nervous system. Mol Cell Neurosci 24:1083–1102PubMedCrossRefGoogle Scholar
  14. 14.
    Huber AB, Weinmann O, Brösamle C, Oertle T, Schwab ME (2002) Patterns of Nogo mRNA and protein expression in the developing and adult rat and after CNS lesions. J Neurosci 22:3553–3567. doi: 20026323 PubMedGoogle Scholar
  15. 15.
    Wang X, Chun SJ, Treloar H, Vartanian T, Greer CA, Strittmatter SM et al (2002) Localization of Nogo-A and Nogo-66 receptor proteins at sites of axon-myelin and synaptic contact. J Neurosci 22:5505–5515. doi: 20026582 PubMedGoogle Scholar
  16. 16.
    Liu H, Ng CEL, Tang BL (2002) Nogo-A expression in mouse central nervous system neurons. Neurosci Lett 328:257–260PubMedCrossRefGoogle Scholar
  17. 17.
    Jin WL, Liu YY, Liu HL, Yang H, Wang Y, Jiao XY et al (2003) Intraneuronal localization of Nogo-A in the rat. J Comp Neurol 458:1–10. doi: 10.1002/cne.10547 PubMedCrossRefGoogle Scholar
  18. 18.
    Josephson A, Widenfalk J, Widmer HW, Olson L, Spenger C (2001) NOGO mRNA expression in adult and fetal human and rat nervous tissue and in weight drop injury. Exp Neurol 169:319–328. doi: 10.1006/exnr.2001.7659 PubMedCrossRefGoogle Scholar
  19. 19.
    Meier S, Bräuer AU, Heimrich B, Schwab ME, Nitsch R, Savaskan NE et al (2003) Molecular analysis of Nogo expression in the hippocampus during development and following lesion and seizure. FASEB J 17:1153–1155. doi: 10.1096/fj.02-0453fje PubMedCrossRefGoogle Scholar
  20. 20.
    Marklund N, Fulp CT, Shimizu S, Puri R, McMillan A, Strittmatter SM et al (2006) Selective temporal and regional alterations of Nogo-A and small proline-rich repeat protein 1A (SPRR1A) but not Nogo-66 receptor (NgR) occur following traumatic brain injury in the rat. Exp Neurol 197:70–83. doi: 10.1016/j.expneurol.2005.08.029 PubMedCrossRefGoogle Scholar
  21. 21.
    Wang H, Yao Y, Jiang X, Chen D, Xiong Y, Mu D et al (2006) Expression of Nogo-A and NgR in the developing rat brain after hypoxia-ischemia. Brain Res 1114:212–220. doi: 10.1016/j.brainres.2006.07.056 PubMedCrossRefGoogle Scholar
  22. 22.
    Cheatwood JL, Emerick AJ, Schwab ME, Kartje GL (2008) Nogo-A expression after focal ischemic stroke in the adult rat. Stroke 39:2091–2098. doi: 10.1161/STROKEAHA.107.507426 PubMedCrossRefGoogle Scholar
  23. 23.
    Eslamboli A, Grundy RI, Irving EA (2006) Time-dependent increase in Nogo-A expression after focal cerebral ischemia in marmoset monkeys. Neurosci Lett 408:89–93. doi: 10.1016/j.neulet.2006.08.056 PubMedCrossRefGoogle Scholar
  24. 24.
    Tagami S, Eguchi Y, Kinoshita M, Takeda M, Tsujimoto Y (2000) A novel protein, RTN-XS, interacts with both Bcl-XL and Bcl-2 on endoplasmic reticulum and reduces their anti-apoptotic activity. Oncogene 19:5736–5746. doi: 10.1038/sj.onc.1203948 PubMedCrossRefGoogle Scholar
  25. 25.
    Oertle T, Merkler D, Schwab ME (2003) Do cancer cells die because of Nogo-B? Oncogene 22:1390–1399. doi: 10.1038/sj.onc.1206278 PubMedCrossRefGoogle Scholar
  26. 26.
    Chen Y, Tang X, Cao X, Chen H, Zhang X (2006) Human Nogo-C overexpression induces HEK293 cell apoptosis via a mechanism that involves JNK-c-Jun pathway. Biochem Biophys Res Commun 348:923–928. doi: 10.1016/j.bbrc.2006.07.166 PubMedCrossRefGoogle Scholar
  27. 27.
    Wan Q, Kuang E, Dong W, Zhou S, Xu H, Qi Y et al (2007) Reticulon 3 mediates Bcl-2 accumulation in mitochondria in response to endoplasmic reticulum stress. Apoptosis 12:319–328. doi: 10.1007/s10495-006-0574-y PubMedCrossRefGoogle Scholar
  28. 28.
    Zhu L, Xiang R, Dong W, Liu Y, Qi Y (2007) Anti-apoptotic activity of Bcl-2 is enhanced by its interaction with RTN3. Cell Biol Int 31:825–830. doi: 10.1016/j.cellbi.2007.01.032 PubMedCrossRefGoogle Scholar
  29. 29.
    Di Sano F, Fazi B, Tufi R, Nardacci R, Piacentini M (2007) Reticulon-1C acts as a molecular switch between endoplasmic reticulum stress and genotoxic cell death pathway in human neuroblastoma cells. J Neurochem 102:345–353. doi: 10.1111/j.1471-4159.2007.04479.x PubMedCrossRefGoogle Scholar
  30. 30.
    Ruch W, Cooper PH, Baggiolini M (1983) Assay of H2O2 production by macrophages and neutrophils with homovanillic acid and horse-radish peroxidase. J Immunol Methods 63:347–357PubMedCrossRefGoogle Scholar
  31. 31.
    Whittemore ER, Loo DT, Watt JA, Cotman CW (1995) A detailed analysis of hydrogen peroxide-induced cell death in primary neuronal culture. Neuroscience 67:921–932PubMedCrossRefGoogle Scholar
  32. 32.
    Xiong Y, Ding H, Xu M, Gao J (2009) Protective effects of asiatic acid on rotenone- or H2O2-induced injury in SH-SY5Y cells. Neurochem Res 34:746–754. doi: 10.1007/s11064-008-9844-0 PubMedCrossRefGoogle Scholar
  33. 33.
    Park SE, Kim S, Sapkota K, Kim SJ (2010) Neuroprotective effect of Rosmarinus officinalis extract on human dopaminergic cell line, SH-SY5Y. Cell Mol Neurobiol 30:759–767. doi: 10.1007/s10571-010-9502-3 PubMedCrossRefGoogle Scholar
  34. 34.
    Scherz-Shouval R, Elazar Z (2007) ROS, mitochondria and the regulation of autophagy. Trends Cell Biol 17:422–427. doi: 10.1016/j.tcb.2007.07.009 PubMedCrossRefGoogle Scholar
  35. 35.
    Chen Y, McMillan-Ward E, Kong J, Israels SJ, Gibson SB (2008) Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells. Cell Death Differ 15:171–182. doi: 10.1038/sj.cdd.4402233 PubMedCrossRefGoogle Scholar
  36. 36.
    Kuang E, Wan Q, Li X, Xu H, Zou T, Qi Y et al (2006) ER stress triggers apoptosis induced by Nogo-B/ASY overexpression. Exp Cell Res 312:1983–1988. doi: 10.1016/j.yexcr.2006.02.024 PubMedCrossRefGoogle Scholar
  37. 37.
    Lee DHS, Strittmatter SM, Sah DWY (2003) Targeting the Nogo receptor to treat central nervous system injuries. Nat Rev Drug Discov 2:872–878. doi: 10.1038/nrd1228 PubMedCrossRefGoogle Scholar
  38. 38.
    Raisman G (2004) Myelin inhibitors: does NO mean GO? Nat Rev Neurosci 5:157–161. doi: 10.1038/nrn1328 PubMedCrossRefGoogle Scholar
  39. 39.
    Voeltz GK, Prinz WA, Shibata Y, Rist JM, Rapoport TA (2006) A class of membrane proteins shaping the tubular endoplasmic reticulum. Cell 124:573–586. doi: 10.1016/j.cell.2005.11.047 PubMedCrossRefGoogle Scholar
  40. 40.
    Dawson TR, Lazarus MD, Hetzer MW, Wente SR (2009) ER membrane-bending proteins are necessary for de novo nuclear pore formation. J Cell Biol 184:659–675. doi: 10.1083/jcb.200806174 PubMedCrossRefGoogle Scholar
  41. 41.
    Wang B, Xiao Z, Chen B, Han J, Gao Y, Zhang J et al (2008) Nogo-66 promotes the differentiation of neural progenitors into astroglial lineage cells through mTOR-STAT3 pathway. PLoS ONE 3:e1856. doi: 10.1371/journal.pone.0001856 PubMedCrossRefGoogle Scholar
  42. 42.
    Wang F, Zhu Y (2008) The interaction of Nogo-66 receptor with Nogo-p4 inhibits the neuronal differentiation of neural stem cells. Neuroscience 151:74–81. doi: 10.1016/j.neuroscience.2007.10.034 PubMedCrossRefGoogle Scholar
  43. 43.
    He W, Lu Y, Qahwash I, Hu XY, Chang A, Yan R et al (2004) Reticulon family members modulate BACE1 activity and amyloid-beta peptide generation. Nat Med 10:959–965. doi: 10.1038/nm1088 PubMedCrossRefGoogle Scholar
  44. 44.
    Murayama KS, Kametani F, Saito S, Kume H, Akiyama H, Araki W et al (2006) Reticulons RTN3 and RTN4-B/C interact with BACE1 and inhibit its ability to produce amyloid beta-protein. Eur J Neurosci 24:1237–1244. doi: 10.1111/j.1460-9568.2006.05005.x PubMedCrossRefGoogle Scholar
  45. 45.
    Kuang E, Wan Q, Li X, Xu H, Liu Q, Qi Y et al (2005) ER Ca2+ depletion triggers apoptotic signals for endoplasmic reticulum (ER) overload response induced by overexpressed reticulon 3 (RTN3/HAP). J Cell Physiol 204:549–559. doi: 10.1002/jcp.20340 PubMedCrossRefGoogle Scholar
  46. 46.
    Mi YJ, Hou B, Liao QM, Ma Y, Luo Q, Dai YK et al (2012) Amino-Nogo-A antagonizes reactive oxygen species generation and protects immature primary cortical neurons from oxidative toxicity. Cell Death Differ 19:1175–1186. doi: 10.1038/cdd.2011.206 PubMedCrossRefGoogle Scholar
  47. 47.
    Bell KFS, Hardingham GE (2011) CNS peroxiredoxins and their regulation in health and disease. Antioxid Redox Signal 14:1467–1477. doi: 10.1089/ars.2010.3567 PubMedCrossRefGoogle Scholar
  48. 48.
    De Craene JO, Coleman J, Estrada de Martin P, Pypaert M, Anderson S, Yates JR et al (2006) Rtn1p is involved in structuring the cortical endoplasmic reticulum. Mol Biol Cell 17:3009–3020. doi: 10.1091/mbc.E06-01-0080 PubMedCrossRefGoogle Scholar
  49. 49.
    Wakana Y, Koyama S, Nakajima KI, Hatsuzawa K, Nagahama M, Tani K et al (2005) Reticulon 3 is involved in membrane trafficking between the endoplasmic reticulum and Golgi. Biochem Biophys Res Commun 334:1198–1205. doi: 10.1016/j.bbrc.2005.07.012 PubMedCrossRefGoogle Scholar
  50. 50.
    Kaltschmidt C, Kaltschmidt B, Neumann H, Wekerle H, Baeuerle PA (1994) Constitutive NF-kappa B activity in neurons. Mol Cell Biol 14:3981–3992PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Department of BiochemistryNational University of SingaporeSingaporeRepublic of Singapore

Personalised recommendations