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V. Pernet, S. Joly, F. Christ, J. L. Martin, M. E. Schwab; The Up-Regulation of the Reticulon Protein Nogo-A/RTN4-A Does Not Change the ER Stress Response in Axotomized Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2246.
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In the present study, we investigated the role of neuronal Nogo-A in axotomized retinal ganglion cells (RGCs) and its possible influence on the ER stress response in vivo.
Nogo-A was observed by immunohistochemistry on retinal cross sections and on retinal flat mounts from C57Bl6 male mice. RGCs and Mueller cells were identified using β3-tubulin and glutamine synthetase antibodies respectively. Adult mice were axotomized and retinal flat-mounts were double labeled for Nogo-A and β3-tubulin between 3 and 14 days after injury. To visualize apoptotic RGCs annexin V was injected in the vitreous body at 7 or 10 days following axotomy. The ER stress markers CHOP and Bip, as well as ATF3 and c-jun, were followed by quantitative RT-PCR in injured retinal lysates. RGC survival was evaluated after intraocular injections of different adeno-associated viruses 2 (AAV2). AAV2 treatments were performed 4 weeks before axotomy to block the intracellular up-regulation of Nogo-A (AAV2.ShRNA-Nogo-A) or to enhance Nogo-A gene expression (AAV2.Nogo-A) relative to a control virus containing the green fluorescent protein (GFP) gene.
Nogo-A was increased in 55% of RGCs at 7 and 14 days after optic nerve transection. The protein and mRNA levels of CHOP were strongly and selectively enhanced in RGCs, 3 and 5 days post axotomy, suggesting a fast ER stress induction prior to the RGC loss. Surprisingly, big cells up-regulating Nogo-A protein presented a low level of the CHOP protein, suggesting a negative correlation between the ER stress-associated apoptosis and the elevation of Nogo-A post-lesion. Moreover, Nogo-A immunopositive cells were not labeled by annexin V at 7 days, implying that the increase of Nogo-A may be protective against apoptosis. However, the blockade of Nogo-A up-regulation with an AAV2.shRNA-Nogo-A did not alter the increase of CHOP mRNA compared with the AAV.GFP treatment 5 days after injury. The density of surviving RGCs was not significantly changed by inhibiting Nogo-A. In contrast, the exogenous increase of Nogo-A after AAV2.Nogo-A treatment exacerbated the RGC death but did not affect CHOP mRNA levels in intact or injured retinas.
Our data show that Nogo-A and the ER stress are quickly up-regulated in RGCs after optic nerve lesion. We show that Nogo-A does not seem to influence the ER stress induction and apoptosis triggered by the optic nerve injury.
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