X-Box Binding Protein 1 Is Essential for the Anti-Oxidant Defense and Cell Survival in the Retinal Pigment Epithelium

Damage to the retinal pigment epithelium (RPE) is an early event in the pathogenesis of age-related macular degeneration (AMD). X-box binding protein 1 (XBP1) is a key transcription factor that regulates endoplasmic reticulum (ER) homeostasis and cell survival. This study aimed to delineate the role of endogenous XBP1 in the RPE. Our results show that in a rat model of light-induced retinal degeneration, XBP1 activation was suppressed in the RPE/choroid complex, accompanied by decreased anti-oxidant genes and increased oxidative stress. Knockdown of XBP1 by siRNA resulted in reduced expression of SOD1, SOD2, catalase, and glutathione synthase and sensitized RPE cells to oxidative damage. Using Cre/LoxP system, we generated a mouse line that lacks XBP1 only in RPE cells. Compared to wildtype littermates, RPE-XBP1 KO mice expressed less SOD1, SOD2, and catalase in the RPE, and had increased oxidative stress. At age 3 months and older, these mice exhibited apoptosis of RPE cells, decreased number of cone photoreceptors, shortened photoreceptor outer segment, reduced ONL thickness, and deficit in retinal function. Electron microscopy showed abnormal ultrastructure, Bruch's membrane thickening, and disrupted basal membrane infolding in XBP1-deficient RPE. These results indicate that XBP1 is an important gene involved in regulation of the anti-oxidant defense in the RPE, and that impaired activation of XBP1 may contribute to RPE dysfunction and cell death during retinal degeneration and AMD

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Electron microscopy of RPE and outer retina in old XBP1 KO mice.

<p><b>A</b>). Electron micrograph of a 6-month-old WT mouse retina showing normal RPE and adjacent OS disk membranes. <b>B–C</b>). Highly disorganized OS and disrupted microvilla in a 6-month-old XBP1 KO mouse. Arrows indicate vesiculated OS disc membrane. Asterisks indicate atrophic area of OS. <b>D–E</b>). XBP1 KO mouse eyes display marked vacuolization in RPE and irregular basal infoldings outlined by a red dotted line. Arrows indicate autophagosomes containing undigested OS disc membranes. Arrowheads indicate swollen mitochondria in RPE. Note that the BrM is highly irregularly thickened in XBP1 KO mice. <b>F</b>). Ultrastructure of basal infoldings in a WT mouse. <b>G</b>). Disorganized basal infoldings in a XBP1 KO mouse. Asterisks indicate the accumulation of material between the RPE basal infoldings. <b>H</b>). Discontinuous collagen and elastin layers in the BrM in a XBP1 KO mouse. [Scale bar: 2 µm (A–D, H); scale bar: 500 nm (E–G)]. RPE, retinal pigment epithelium; OS, outer segment; Mv, microvilla; BrM, Bruch's membrane.</p

Decreased expression of antioxidant genes in the RPE in XBP1 KO mice.

<p><b>A</b>). mRNA level of SOD2, SOD1, and catalase in the RPE was determined by real time RT-PCR (mean ± SD, n = 6, ** <i>P</i><0.01). <b>B</b>). Retinal sections were stained with an anti-SOD1 antibody (green) and nuclei were stained with DAPI (blue). <b>C</b>). Immunostaining of SOD2 in the RPE in XBP1 KO and WT mice. Images represent 3 animals in each group. <b>D–E</b>). Western blot analysis revealed significant down-regulated expression of SOD1 and SOD2 in the eyecups of XBP1 KO mice. The protein levels of SOD1 and SOD2 were quantified using densitometry (mean ± SD, n = 6, **<i>P</i><0.01). [scale bar, 50 µm (B); scale bar, 20 µm (C)]. RPE, retinal pigment epithelium; ONL, outer nuclear layer; Ch, choroid.</p

Altered retinal morphology and visual function in XBP1 KO mice.

<p>Histology and retinal function recorded by ERG in 4-month-old XBP1 KO and WT mice. <b>A</b>). Representative retinal sections. <b>B</b>). Mean length of photoreceptor IS/OS (mean ± SD, n = 5). <b>C</b>). Quantification of ONL thickness (mean ± SD, n = 7). <b>D</b>). ERG recordings show significantly decreased amplitude of a and b waves in both photopic and scotopic ERG in 4-month-old, but not in 2-month-old XBP1 KO mice.</p

Sequences of primers used in real-time RT-PCR.

<p>PrimerBank: <a href="http://pga.mgh.harvard.edu/primerbank/" target="_blank">http://pga.mgh.harvard.edu/primerbank/</a>.</p

Increased oxidative stress in the RPE and photoreceptors in RPE-specific XBP1 KO mice.

<p><b>A</b>). XBP1 expression in the RPE in XBP1 KO and littermate WT mice. The single layer of RPE cells was isolated as a sheet from 2-month-old XBP1 KO and littermate WT mice using dispase as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038616#s4" target="_blank">Methods</a> section. Two RPE sheets from the same mouse were pooled and used for RNA isolation. XBP1 expression was measured by real-time RT-PCR. Results are expressed as mean ± SD (n = 6). <b>B–C</b>). Activation of XBP1 induced by ER stress in XBP1 KO and WT mice. Eyecups containing RPE, choroid, and sclera were incubated with 10 µg/ml tunicamycin for 6 h. Proteins were extract from the RPE by incubation of lysis buffer with the inner surface of the eyecups and subjected to Western blot analysis. Results show that spliced XBP1 (XBP1S) expression was undetectable in unstimulated eyecups (<b>B</b>, upper panel), but was markedly increased in WT mice compared to XBP1 KO mice (<b>B</b>, lower panel). XBP1S expression was quantified by densitometry (<b>C</b>) (mean ± SD, n = 6, **<i>P</i><0.01). <b>D</b>). Immunostaining of XBP1 (green) in retinal cryosections from 2-month-old XBP1 KO and WT mice. Blue: nuclear staining with DAPI. <b>E</b>). mRNA expression of ERdj4 and P58IPK in the RPE was measured by real-time RT-PCR (mean ± SD, n = 6). <b>F</b>). Immunostaining of 3-NT (green) in retinal cryosections from XBP1 KO and WT mice. Blue: nuclear staining with DAPI. <b>G–J</b>). <i>In situ</i> dihydroethidium (DHE) and 2,7-CM-H₂DCFDA (DCF) staining of fresh retinal cryosections from XBP1 KO and WT mice. Representative images from 4 animals in each group are shown in <b>G</b>. Note intensive staining of DHE (indicative of O₂⁻) and DCF (indicative of ROS) in RPE and photoreceptor cells in XBP1 KO mice. <b>H–I</b>). Quantification of fluorescence intensity in the RPE layer shows a significant increase in O₂⁻ and ROS levels in XBP1 KO mice (mean ± SD, n = 4). * <i>P</i><0.05, ** <i>P</i><0.01. Scale bar: 50 µm in D and F; 20 µm in G. RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; Ch, choroid.</p

Knockdown of XBP1 down-regulates anti-oxidant genes in human RPE cells.

<p>Human RPE (ARPE-19) cells were transfected with XBP1 siRNA or control siRNA for 48 h. <b>A</b>). mRNA expression of antioxidant gene catalase, SOD2, SOD1, Nrf2, and GSH synthase was measured by real-time RT-PCR. Data were expressed as mean ± SD (n = 3 independent experiments). <b>B</b>). Protein levels of spliced XBP1and SOD2 were determined by Western blot analysis and semi-quantified by densitometry. <b>C</b>). Intracellular superoxide production was detected by Dihydroethidium (DHE) (upper panel). Mitochondrial superoxide level was analyzed by MitoSOX™ Red assay (lower panel). Representative images from 3 independent experiments are shown. <b>D</b>). Intracellular ROS generation was determined by DCF. The fluorescence density was quantified by using a fluorescence plate reader with wavelength of 485/535 nm (mean ± SD, n = 3). <b>E</b>). Cell viability was determined by MTT assay. The numbers of viable cells are expressed as % of control, averaged from 3 independent experiments (mean ± SD). <b>F</b>). Apoptosis was detected by Annexin V staining in ARPE-19 cells transfected with XBP1 siRNA or control siRNA. * <i>P</i><0.05, ** <i>P</i><0.01 vs. control siRNA. <b>G</b>). Transfected cells were exposed to 4-HNE (50 µM) for 24 h, apoptosis was detected by TUNEL assay. Left panels show representative images of TUNEL staining (red). Nuclei were stained with DAPI (blue). Right panel shows the quantitative results of apoptotic cells. * <i>P</i><0.05 vs. control siRNA; ‡ <i>P</i><0.01 vs. control siRNA+4-HNE.</p