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

Abstract

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.

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Acknowledgments

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.

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The authors declare no conflict of interest.

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Correspondence to Bor Luen Tang.

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A

Expression of Nogo isoforms and RTN3 do not significantly elevate ER stress. 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)

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Teng, F.Y.H., Tang, B.L. Nogo/RTN4 isoforms and RTN3 expression protect SH-SY5Y cells against multiple death insults. Mol Cell Biochem 384, 7–19 (2013). https://doi.org/10.1007/s11010-013-1776-6

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Keywords

  • CNS injury
  • Neuroprotection
  • Reticulon 3 (RTN3)
  • Nogo