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.
This is a preview of subscription content, log in to check access.
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.
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)
Ng CEL, Tang BL (2002) Nogos and the Nogo-66 receptor: factors inhibiting CNS neuron regeneration. J Neurosci Res 67:559–565PubMedCrossRefGoogle Scholar
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:10.1038/35000219PubMedCrossRefGoogle Scholar
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
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
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:20026323PubMedGoogle Scholar
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:20026582PubMedGoogle Scholar
Liu H, Ng CEL, Tang BL (2002) Nogo-A expression in mouse central nervous system neurons. Neurosci Lett 328:257–260PubMedCrossRefGoogle Scholar
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.02-0453fjePubMedCrossRefGoogle Scholar
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.2005.08.029PubMedCrossRefGoogle Scholar
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.1203948PubMedCrossRefGoogle Scholar
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
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
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.4402233PubMedCrossRefGoogle Scholar
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/nm1088PubMedCrossRefGoogle Scholar
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.20340PubMedCrossRefGoogle Scholar
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.2011.206PubMedCrossRefGoogle Scholar
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.2005.07.012PubMedCrossRefGoogle Scholar
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