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

Complement factor H: spatial and temporal expression and localization in the eye

PURPOSE. Complement factor H (CFH) is a component of the mammalian complement system, which regulates the alternative pathway of complement activation and protects the host cell from inappropriate complement activation. CFH is a key regulator of innate immunity, and CFH deficiency leads to membranoproliferative glomerulonephritis type II. A variation in human CFH, Y402H, has been shown to be associated with an increased risk for age-related macular degeneration. The authors describe studies on the spatial and temporal expression of the CFH gene and localization of this protein in ocular tissues to gain insight into its role in the eye. METHODS. CFH expression in human and mouse tissues was studied by quantitative RT-PCR and Western blot analysis, and localization of CFH was studied by immunohistochemical analysis followed by fluorescence microscopy. RESULTS. In human and mouse, CFH expression was found to be similar to the highest level of expression in the liver. In ocular tissue, CFH was detected in the distalmost optic nerve (3 mm) cut from the scleral surface of the eyeball, sclera, RPE-choroid, retina, lens, and ciliary body. In mouse, Cfh expression was observed from early embryonic stages, and in the eye its expression increased with age. CONCLUSIONS. A significant level of CFH expression is maintained in different ocular tissues during development and aging. Sustained high levels of CFH expression in eye tissues suggest that this protein may play a role in protecting these tissues from indiscriminate complement activation and inflammatory insult. (Invest Ophthalmol Vis Sci. 2006;47: 4091-4097

Retinal Sphingolipids and Their Very-Long-Chain Fatty Acid–Containing Species

Sphingolipids and ceramides are involved in photoreceptor cell death, inflammation, and angiogenesis at the photoreceptor–RPE interface. This study was a comprehensive characterization of the abundance and fatty acid composition of retinal sphingolipids, with special emphasis on very-long-chain fatty acid–containing species

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

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

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

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

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