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ER stress protects from retinal degeneration

2009; Springer Nature; Volume: 28; Issue: 9 Linguagem: Inglês

10.1038/emboj.2009.76

1460-2075

César S. Mendes, Clémence Levet, Gilles Chatelain, Pierre Dourlen, Antoine Fouillet, Marie-Laure Dichtel-Danjoy, Alexis Gambis, Hyung Don Ryoo, Hermann Steller, Bertrand Mollereau,

Genetics, Aging, and Longevity in Model Organisms

Article2 April 2009free access ER stress protects from retinal degeneration César S Mendes César S Mendes Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USAPresent address: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA Search for more papers by this author Clémence Levet Clémence Levet LBMC, UMR5239 CNRS/Ecole Normale Supérieure de Lyon, IFR 128 Biosciences Lyon Gerland, Université de Lyon, Lyon, France Search for more papers by this author Gilles Chatelain Gilles Chatelain LBMC, UMR5239 CNRS/Ecole Normale Supérieure de Lyon, IFR 128 Biosciences Lyon Gerland, Université de Lyon, Lyon, France Search for more papers by this author Pierre Dourlen Pierre Dourlen LBMC, UMR5239 CNRS/Ecole Normale Supérieure de Lyon, IFR 128 Biosciences Lyon Gerland, Université de Lyon, Lyon, France Search for more papers by this author Antoine Fouillet Antoine Fouillet LBMC, UMR5239 CNRS/Ecole Normale Supérieure de Lyon, IFR 128 Biosciences Lyon Gerland, Université de Lyon, Lyon, France Search for more papers by this author Marie-Laure Dichtel-Danjoy Marie-Laure Dichtel-Danjoy LBMC, UMR5239 CNRS/Ecole Normale Supérieure de Lyon, IFR 128 Biosciences Lyon Gerland, Université de Lyon, Lyon, France Search for more papers by this author Alexis Gambis Alexis Gambis Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Hyung Don Ryoo Hyung Don Ryoo Department of Cell Biology, New York University School of Medicine, New York, NY, USA Search for more papers by this author Hermann Steller Hermann Steller Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Bertrand Mollereau Corresponding Author Bertrand Mollereau LBMC, UMR5239 CNRS/Ecole Normale Supérieure de Lyon, IFR 128 Biosciences Lyon Gerland, Université de Lyon, Lyon, France Search for more papers by this author César S Mendes César S Mendes Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USAPresent address: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA Search for more papers by this author Clémence Levet Clémence Levet LBMC, UMR5239 CNRS/Ecole Normale Supérieure de Lyon, IFR 128 Biosciences Lyon Gerland, Université de Lyon, Lyon, France Search for more papers by this author Gilles Chatelain Gilles Chatelain LBMC, UMR5239 CNRS/Ecole Normale Supérieure de Lyon, IFR 128 Biosciences Lyon Gerland, Université de Lyon, Lyon, France Search for more papers by this author Pierre Dourlen Pierre Dourlen LBMC, UMR5239 CNRS/Ecole Normale Supérieure de Lyon, IFR 128 Biosciences Lyon Gerland, Université de Lyon, Lyon, France Search for more papers by this author Antoine Fouillet Antoine Fouillet LBMC, UMR5239 CNRS/Ecole Normale Supérieure de Lyon, IFR 128 Biosciences Lyon Gerland, Université de Lyon, Lyon, France Search for more papers by this author Marie-Laure Dichtel-Danjoy Marie-Laure Dichtel-Danjoy LBMC, UMR5239 CNRS/Ecole Normale Supérieure de Lyon, IFR 128 Biosciences Lyon Gerland, Université de Lyon, Lyon, France Search for more papers by this author Alexis Gambis Alexis Gambis Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Hyung Don Ryoo Hyung Don Ryoo Department of Cell Biology, New York University School of Medicine, New York, NY, USA Search for more papers by this author Hermann Steller Hermann Steller Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Bertrand Mollereau Corresponding Author Bertrand Mollereau LBMC, UMR5239 CNRS/Ecole Normale Supérieure de Lyon, IFR 128 Biosciences Lyon Gerland, Université de Lyon, Lyon, France Search for more papers by this author Author Information César S Mendes1, Clémence Levet2, Gilles Chatelain2, Pierre Dourlen2, Antoine Fouillet2, Marie-Laure Dichtel-Danjoy2, Alexis Gambis1, Hyung Don Ryoo3, Hermann Steller1 and Bertrand Mollereau 2 1Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA 2LBMC, UMR5239 CNRS/Ecole Normale Supérieure de Lyon, IFR 128 Biosciences Lyon Gerland, Université de Lyon, Lyon, France 3Department of Cell Biology, New York University School of Medicine, New York, NY, USA *Corresponding author. Laboratory of Molecular Biology of the Cell, Ecole Normale Supérieure de Lyon, 9 rue du Vercors, Lyon 69007, France. Tel.: +33 472 728 163; Fax: +33 472 728 674; E-mail: [email protected] The EMBO Journal (2009)28:1296-1307https://doi.org/10.1038/emboj.2009.76 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The unfolded protein response (UPR) is a specific cellular process that allows the cell to cope with the overload of unfolded/misfolded proteins in the endoplasmic reticulum (ER). ER stress is commonly associated with degenerative pathologies, but its role in disease progression is still a matter for debate. Here, we found that mutations in the ER-resident chaperone, neither inactivation nor afterpotential A (NinaA), lead to mild ER stress, protecting photoreceptor neurons from various death stimuli in adult Drosophila. In addition, Drosophila S2 cultured cells, when pre-exposed to mild ER stress, are protected from H2O2, cycloheximide- or ultraviolet-induced cell death. We show that a specific ER-mediated signal promotes antioxidant defences and inhibits caspase-dependent cell death. We propose that an immediate consequence of the UPR not only limits the accumulation of misfolded proteins but also protects tissues from harmful exogenous stresses. Introduction The unfolded protein response (UPR) is an evolutionary conserved adaptive response to perturbations of normal endoplasmic reticulum (ER) physiology. Strikingly, the UPR is implicated in several human pathologies, such as neurodegeneration, diabetes and cancer (Marciniak and Ron, 2006; Lin et al, 2008). The UPR engages several responses, including transcriptional upregulation of ER-resident chaperones, selective inhibition of translation and activation of ER-associated degradation (ERAD) (reviewed in Ron and Walter, 2007). Despite a large body of work in this field, little is known about the cellular consequences of the UPR upon physiological levels of ER stress in vivo. Genetic and cell culture studies have unveiled the core of the UPR pathway, linking the detection of ER stress to the effector responses. In unstressed cells, the ER stress sensor and chaperone, Bip/Hsc3, binds and restricts the molecular components of the UPR. In stressed cells, Bip/Hsc3 is titrated away by unfolded proteins, relieving its inhibition on the UPR. This leads to trans-autophosphorylation of the PKR-like ER kinase (Perk) (Harding et al, 1999). Active Perk phosphorylates eIF2α, inhibiting protein synthesis and limiting the flux of misfolded proteins to the ER. In addition, the UPR promotes the phosphorylation of Inositol-requiring enzyme 1 (Ire1). Activated Ire1 splices an intron from the mRNA of x-box binding protein-1 (xbp1), creating a translational frameshift (Yoshida et al, 2001; Calfon et al, 2002). The spliced form of xbp1 acts as a transcriptional activator, resulting in the expression of ER-stress target genes, including protein chaperones and components of the ERAD pathway (Travers et al, 2000). In parallel, the membrane-bound protein Atf6 translocates to the golgi, where it is cleaved by SP1 and SP2. The cleaved peptide translocates to the nucleus, where it acts as a transcription factor (Ye et al, 2000). If UPR activation cannot overcome ER stress, then its target pathways induce the activation of the apoptotic program (reviewed by Szegezdi et al, 2006). Apoptosis is executed by specific caspases and regulated both by pro- and anti-apoptotic Bcl-2 proteins. However, the UPR has also been shown to induce an antioxidant response, limiting the deleterious effect of prolonged ER stress (Cullinan and Diehl, 2004). How the UPR switches between inducing two such opposite programs is not clear. Drosophila melanogaster has recently emerged as a useful in vivo model system to study the UPR pathway (Pomar et al, 2003; Hollien and Weissman, 2006; Ryoo and Steller, 2007; Souid et al, 2007). Most UPR components found in yeast and mammalian cells, such as Atf4 (Crc), Perk and eIF2α, have Drosophila homologs (Pomar et al, 2003 and reviewed in Ryoo and Steller, 2007). In the Drosophila model of autosomal dominant retinitis pigmentosa (ADRP), the misfolding rhodopsin-1 (Rh1) mutation (ninaEG69D, termed rh1G69D for clarity) induces UPR activation by increasing xbp1 splicing and Ire1-dependent expression of the heat shock cognate protein 3 (Hsc3, the Drosophila Bip homolog; Ryoo et al, 2007). The reduction of xbp1 expression leads to increased retinal degeneration, indicating that xbp1 and its transcriptional targets are required to reduce the accumulation of misfolded Rh1 in the ER. Although the UPR pathway is conserved in Drosophila, the relationship between ER stress and apoptosis remains to be explored. Searching for genes that protect adult photoreceptor cells (PRCs) against apoptosis, we identified mutations that disrupt the maturation and folding of the Rh1 protein as strong suppressors of neurodegeneration. These mutations lead to the activation of the UPR, which promotes neuroprotection against cellular insults by inducing an antioxidant response while inhibiting caspase activation. These results are not restricted to photoreceptor neurons, as cultured S2 cells become more resistant to cell death stimuli if pre-treated with ER stress-inducing chemical agents. Results PRCs exposed to ER stress exhibit death resistance In a dominant modifier screen to identify cell death regulators in differentiated Drosophila PRCs (CSM, HS and BM, unpublished data), we have found that mutations in the Drosophila cyclophilin B homolog ninaA suppress dp53-induced cell death in PRCs (Figure 1; Table I). NinaA is a membrane-bound chaperone localized in the ER devoted exclusively to the biogenesis of the light sensing Rh1 protein. It has been shown that the peptidyl-prolyl cis-trans isomerase activity of NinaA allows proper Rh1 folding. In addition, NinaA functions as a chaperone, escorting Rh1 through the secretory pathway from the ER to the rhabdomeres (microvillar membrane in which rhodopsins accumulate) of outer PRCs (the rods-like PRCs of Drosophila) (Colley et al, 1991; Baker et al, 1994). NinaA amorphs lead to the accumulation of misfolded Rh1 protein in the ER, likely resulting in ER stress (Colley et al, 1991). Figure 1.Loss of ninaA function protects PRCs from cell death. PRC integrity is visualized in outer PRCs submitted to the ectopic expression of rpr, dp53 and dcp-1, in a wild-type or ninaA mutant background. (A–J) PRC rhabdomeres are visualized in semi-thin tangential retinal sections. Scale bar, 10 μm. Outer PRC rhabdomeres of a representative ommatidium are circled in yellow. (A–C) PRC rhabdomeres are organized in a trapezoidal pattern in control retinas (5-day-old flies). (A) Wild type. (B) ninaAE110V/+. (C) ninaAE110V. (D–F) Loss of ninaA protects from rpr-induced cell death (1-day-old flies). (D) Ectopic expression of rpr in outer PRCs with rh1-Gal4;uas-rpr (rh1>rpr) induces a massive loss of PRC integrity. (E) Reduced PRC killing in rh1>rpr;ninaAE110V/+. (F) Reduced PRC killing in rh1>rpr;ninaAE110V. (G, H) Loss of ninaA protects from dp53-induced cell death (8-day-old flies). (G) Ectopic expression of dp53 in outer PRCs with rh1-Gal4;uas-dp53 (rh1>dp53) induces a massive PRC killing. (H) Reduced PRC killing in rh1>dp53/ninaAE110V/+. (I, J) Loss of ninaA protects from dcp-1-induced cell death (1-day-old flies). (I) Loss of PRCs in rh1-Gal4;;uas-dcp-1 (rh1>dcp-1). (J) Reduced killing in rh1-Gal4; ninaAE110V/+;uas-dcp-1. (K) Quantitative analysis of photoreceptor survival seen in (A, D–J). The results are expressed as mean percentage of wild-type control ±s.e.m. of three animals (*P dp53). Viable PRCs expressing GFP (rh1-GFP) were visualized by the immersion technique (Pichaud and Desplan, 2001) in wild-type or ninaA mutant retinas. NinaA protein levels obtained from western blot analysis (from Ondek et al, 1992). ninaAE110V/+, ninaAQ137L/+, ninaAG98D/+ and ninaA[email protected]/+ mutant retinas are protected from dp53-induced PRC death. The results are expressed as amount of outer PRC/ommatidium ±s.e.m. of three animals (*P dp53) induces PRC killing. (C) PRC killing is reduced in rh1>dp53; ninaAE110V/+. (D) Reduced rh1 dosage does not protect from dp53-induced PRC killing (rh1>dp53; rh1I17/+). (E) Reduced rh1 dosage suppresses ninaAE110V/+ protective effect in PRCs submitted to dp53-induced killing (rh1>dp53; ninaAE110V/+; rh1I17/+). (F) A missense mutation in rh1 protects from dp53-induced PRC killing (rh1>dp53; rh1G69D/+). (G) Quantitative analysis of photoreceptor survival seen in (A–F). Results are expressed as mean percentage of survival ±s.e.m. of three animals (*P<0.05; ***P xbp1:GFP). Scale bar, 100 μm. (A) Wild-type PRCs expressing xbp1:GFP show weak GFP staining. (C) rh1G69D/+, (E) ninaAE110V/+or (G) ninaAE110V retina expressing xbp1:GFP display an increased GFP staining in PRC nuclei (yellow arrowheads). (B) Wild-type retinas do not exhibit Hsc3 staining. (D) rh1G69D/+ retinas show a marked Hsc3 staining. (F) ninaAE110V/+ and (H) ninaAE110V retinas show weak Hsc3 staining. (I) Western blot analysis performed on fly eye tissue. ninaAE110V/+ or ninaA1/+ exhibit a clear Hsc3 increase compared with wild-type retina. Hsc3 protein level is further increased in ninaA1 compared with ninaA1/+. The Hsc3 level in rh1G69D/+ is increased to a level similar to that in ninaA amorphs. Representative blot of four independent experiments. (J) Quantification of Hsc3 immunoreactivity. The results are expressed as mean percentage of wild-type control ±s.e.m. of four independent experiments (*P<0.05; **P<0.01; ***P<0.001 in Student's t-test). Download figure Download PowerPoint To further characterize the extent of UPR activation, we evaluated the levels of Hsc3 expression in ninaA and rh1 mutant retinas (Figure 3). Similarly to Xbp1:GFP, rh1G69D mutant retinas show a robust increase of Hsc3 expression as seen in horizontal cryosections and western blots (Figure 3, D, I, J; Ryoo et al, 2007). The loss of ninaA also led to an increase in the levels of Hsc3 (Figure 3F and H). Interestingly, western blot quantification shows that the increase in Hsc3 protein is dependent on the dosage of ninaA gene expression, as flies heterozygous for ninaA exhibit less of an increase when compared with ninaA amorphs (Figure 3I and J). This is supported by the fact that the amount of unfolded Rh1 is dependent on the genomic dosage of ninaA (Baker et al, 1994). For the rest of the study, we will consider that heterozygous ninaA mutants induce moderate ER stress, while homozygous ninaA or rh1G69D mutants trigger strong ER stress. Taken together, the intensity of UPR activation, visualized by xbp1 splicing and Hsc3 expression, depends on the amount of misfolded Rh1 proteins that accumulate in the ER. Prolonged and moderate ER stress does not lead to neurodegeneration Accumulation of misfolded proteins and UPR activation protect PRCs from death in young adult retina (Figures 1 and 2). We next asked whether prolonged ER stress and long-term UPR activation under normal physiological conditions can affect long-term PRC survival. PRC integrity was determined in aged flies (60 days old) carrying ninaAE110V or rh1G69D mutations, reared under normal temperature (25°C) and light:dark cycle (12:12 h) conditions. In 60-day-old adults, we found that both homozygous ninaAE110V and rh1G69D mutant retinas (strong ER stress) display defects in the ommatidia trapezoidal arrangement and occasional PRC loss (Figure 4C and D, and data not shown). These results correspond with an earlier study showing that long-term PRC degeneration occurs in homozygous ninaA mutant retinas (Rosenbaum et al, 2006). Conversely, heterozygous ninaAE110V retinas (moderate ER stress) remained intact, showing normal morphology (Figure 4B) despite displaying hallmarks of ER stress and UPR activation (Figure 3E and F, and data not shown). This result indicates that a prolonged moderate ER stress does not cause PRC death. Figure 4.Mild and long-term accumulation of unfolded Rh1 protein does not promote age-related retinal degeneration. (A–D) Semi-thin tangential retina sections in 60-day-old flies raised at 25°C in a 12:12 light–dark cycle. Outer PRC rhabdomeres of representative ommatidium are circled in yellow. Scale bar, 10 μm. (A) Wild type and (B) ninaAE110V/+ retinas exhibit a preserved retina structure with intact PRC rhabdomeres (100% ±0.0 PRC survival). (C) ninaAE110V (98.37% ±0.74 PRC survival) and (D) rh1G69D/+ (95.94% ±1.88 PRC survival) retinas show occasional signs of degeneration. Download figure Download PowerPoint An ER-mediated signal inhibits caspase activation Mutations leading to the accumulation of unfolded Rh1 and activation of the UPR can protect PRCs from cell death induced by dp53, rpr or the caspase dcp-1 (Figures 1, 2 and 3). We first tested whether the UPR activation could lead to a reduction of the rh1-GAL4/UAS system and a subsequent decrease of the ectopic expression of the apoptotic proteins. To address this question, we compared GFP expression levels (ectopically expressed using GAL4/UAS system), in ninaA, rh1 mutant and wild-type retinas (Figure 5). Although GFP levels were decreased in homozygous ninaAE110V or rh1G69D mutant retinas (strong ER stress), no reduction of GFP was observed in heterozygous ninaAE110V mutants (moderate ER stress) compared with the β-tubulin loading control (Figure 5A and B). These results suggest that while attenuation of the rh1-GAL/UAS system can contribute to cell death inhibition under strong ER stress conditions, it does not play a role in moderate ER stress situations. In addition, we tested whether the UPR could regulate rh1 promoter activity. We measured rh1 promoter activity (using a rh1-LacZ transgenic line) by quantifying X-Gal staining of retinal sections and measuring β-galactosidase activity with an ONPG assay in ninaAE110V and rh1G69D mutants (Supplementary Figure 3). No difference was observed between the different genotypes, indicating that rh1 promoter activity functions normally in PRCs under ER stress. This result corroborates an earlier study showing that rh1 transcription is unaffected in ninaA mutants (Zuker et al, 1988). Although we cannot exclude that subtle translation attenuation can push the balance in favour of anti-apoptotic genes in ninaAE110V heterozygous retina, it suggests that another ER-mediated mechanism inhibits cell death. Figure 5.Effect of ER stress on the GAL4/UAS ectopic expression system. (A) Western blot analysis against GFP on head extracts expressing GFP under the control of the rh1-Gal4 driver in wild-type, ninaAE110V/+, ninaAE110V or rh1G69D/+ backgrounds. Representative blot from eight independent experiments. (B) Quantification of the amount of GFP detected in (A), normalized to the loading control and to the amount of GFP expressed in a wild-type background. There is no reduction of GFP protein levels in ninaAE110V/+ compared with wild type. ninaAE110V or rh1G69D/+ exhibit reproducible reductions in GFP levels compared with wild-type tissues. Reprobing with β-tubulin antibody serves as a loading control. The results are expressed as a mean percentage of wild-type control ±s.e.m. of eight independent experiments (**P<0.01; ***P dp53) induces outer PRC, but not inner PRC death that do not express dp53 (orange arrowhead). (B) dp53-induced PRC death is reduced by p35 concomitant expression (rh1>dp53; gmr-p35). (C) dp53-induced PRC death is reduced in ninaA mutants (rh1>dp53; ninaAE110V/+). (D) dp53-induced PRC death is reduced by p35 expression in the ninaA mutant (rh1>dp53; ninaAE110V/+; gmr-p35). (E) Quantitative analysis of photoreceptor survival seen in (A–D). The results are expressed as mean percentage of survival ±s.e.m. of three animals. (F) Caspase activity measured by the appearance of a cleaved PARP (cPARP) epitope by western blot using an anti-cPARP antibody. cPARP is present in tissue expressing dp53 in wild type but strongly reduced in ninaAE110V/+ background. Reprobing with β-tubulin antibody serves as loading control. Representative blot of three independent experiments. (G) Quantification of cPARP immunoreactivity. The results are expressed as mean percentage of total ±s.e.m. of three independent experiments. (H) Caspase activity measured by luminescence activity. The results are expressed as luminescence signal relative to the amount of protein±s.e.m. of three independent experiments (**P<0.01; ***P<0.001 in Student's t-test). Download figure Download PowerPoint We then examined the effector caspase activation levels in PRCs submitted to ER stress by measuring PARP cleavage and using a DEVD-based caspase activity assay