Additional file 1 of Complement C3 deficiency alleviates alkylation-induced retinal degeneration in mice

Additional file 1: Figure S1. DNA damage in sodium iodate (NaIO3)-treated mouse retinas. a. Representative images of DNA damage staining in retinal cross sections from each group before and 7 days after NaIO3 injection. b. Semi-quantification of DNA damage using ImageJ. There are DNA damage-positive cells in the retina after NaIO3 injection (n = 6, ***P < 0.001 compared with control)

Loss of p300/CBP leads to chromatin decondensation and changes in distribution of histone marks in <i>R-DCKO</i> nuclei.

<p><b>A.</b> Electron micrographs of nuclei in the ONL of P22 retinas. Compared to <i>Cre neg</i> control littermates, compound heterozygotes (<i>p300 CH</i> and <i>Cbp CH</i>) show slight increases in euchromatin (light areas within nuclei). In <i>R-DCKO</i> nuclei areas of euchromatin are greatly increased, and electron-dense heterochromatin appears reduced. <b>B.</b> Heterochromatin was quantified as a percentage of the total nuclear area in 50 nuclei from 10 micrographs for each genotype. Error bars  = 1 SD. Differences from <i>Cre neg</i> values were significant at p<0.0001. <b>C & D.</b> Comparison of immunoreactivity patterns for repressive histone marks H3K9me3 (green in panel C, white in insets) and H3K27me3 (green in panel D, white in insets) in control (left image) and <i>R-DCKO</i> (right image) retinas confirm loss of the characteristic rod chromatin condensation pattern in <i>R-DCKO</i> outer retina cells. Anti-PKC-alpha (red) marks bipolar cells. <b>E & F.</b> Comparison of immunoreactivity patterns for acetylated histone H3 (AcH3, green in panel D) and H4 (AcH4, green in panel E) reveals the redistribution of these activation marks in <i>R-DCKO</i> cells, corresponding to loss of the characteristic peripheral rod euchromatin distribution pattern. DNA is counterstained with Draq-5 (red). Scale bars: cross-sections  = 20 µm, insets  = 10 µm. <b>G.</b> Western blots of acid-extracted retinal histones from 15-week-old <i>Cre-negative</i> (1) or <i>R-DCKO</i> (2) retinas. CB, Coomassie blue stained gel. AcH3, blot stained for acetylated histone H3; AcH4, blot stained for acetylated histone H4. <b>H.</b> Quantification of band fluorescence intensities for AcH3 levels relative to total H2B levels, and AcH4 levels relative to total H3 levels at P20 did not show significant differences between <i>Cre neg</i> and <i>R-DCKO</i> samples.</p

Knockout of both <i>Ep300</i> and <i>Cbp</i> in rods disrupts photoreceptor architecture and function.

<p><b>A</b>–<b>G.</b> Cross-sections of 4-week-old retinas of the indicated genotypes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069721#pone-0069721-t001" target="_blank">Table 1</a>), stained with hematoxylin and eosin (H&E). <b>H.</b> Section from the same <i>R-DCKO</i> eye as in panel G, fluorescently labeled with anti-PKCα (green, for bipolar cells) and DAPI (red), to show the boundary between the outer and inner nuclear layers. Scale bar  = 50 μm for all 8 panels. <b>I</b>–<b>K.</b> Immunofluorescent staining for p300 protein verified expression in all nuclei in <i>Cre neg</i> controls (<b>I</b>). <i>Ep300</i> expression is lost in the outer nuclear layer (<b>ONL</b>) of <i>p300KO</i> (<b>J</b>) and <i>R-DCKO</i> (<b>K</b>) retinas. <b>L.</b> <i>R-DCKO</i> section stained for p300 (green) and cone arrestin (CARR, red), showing that the few remaining p300-positive cells in the outer retina are cones. Scale bar  = 20 μm for all 4 panels. <b>OS,</b> outer segments; <b>ONL,</b> outer nuclear layer; <b>OPL,</b> outer plexiform layer; <b>INL,</b> inner nuclear layer; <b>IPL,</b> inner plexiform layer; <b>GC,</b> ganglion cell layer. <b>M</b>–<b>O.</b> Amplitudes of dark-adapted (“Dark”) and light-adapted (“Light”) flash electroretinograms (ERG) at 4 weeks of age. Flash intensities (log [CdSec/M²]) are indicated on the X-axis. Error bars indicate +/− 1SD of the mean amplitude for 6 animals of each genotype tested. Two-way repeated measures ANOVA showed significant interactions between genotype and log light level at p<0.0001 for dark-adapted a-waves (Panel M), b-waves (Panel N), and light-adapted b-waves (Panel O). Asterisks (*) indicate values significantly different (p<0.001) from <i>Cre negative</i> controls in post-hoc tests.</p

Genotypes of mice used in this study.

<p>Genotypes of mice used in this study.</p

<i>Ep300/Cbp</i> conditional knockout in cones also disrupts cone structure, gene expression and function.

<p><i>Cone opsin</i>-driven <i>Cre (CCre)</i> was used to knock out <i>Ep300/Cbp</i> in cone photoreceptors; morphology, cone gene expression/distribution, and ERG function were assessed at 6–7 weeks of age. <b>A</b>–<b>D.</b> Compared to <i>Cre negative</i> controls (Panel A; inset shows two presumptive cones), H&E staining of <i>CCre</i> conditional knockout retinas reveals no major abnormalities (panels B–D), but cells with large nuclei can be seen scattered throughout the outer retina in <i>C-DCKO</i> mice (Panel D arrowheads and high-magnification inset). <b>E.</b> Cone arrestin (red) and p300 (green) expression are decreased in these cells (arrowheads). <b>F.</b> S-opsin expression (red) is also decreased in these cells (arrowheads), which lack outer segments. <b>G.</b> Peanut agglutinin labelling (red) identifies the displaced, abnormal cells in the outer retina (arrowheads) as cones. Blue in Panels E–G is DAPI labelling of nuclei. <b>H.</b> Cone α-transducin (green) is decreased and mislocalized to the cell bodies. Draq5 (red) marks nuclei. <b>I.</b> ERGs performed on 6 week old <i>CCre</i> mice confirmed decreases in cone-driven responses in <i>C-DCKO</i> retinas (red lines). Two-way repeated measures ANOVA indicated significance at p<0.0001 for dark-adapted and light-adapted b-waves. Asterisks (*) indicate significance differences (p<0.001) from <i>Cre negative</i> controls in post-hoc tests.</p

Expression of photoreceptor genes is decreased in <i>R-DCKO</i> retinas.

<p><b>A.</b> Summarized microarray findings for <i>R-DCKO</i> vs. <i>Cre neg</i> retinas. Each gene was categorized by the cell process in which it functioned, and results for each category are represented as a percentage of all the down- or up-regulated genes (see Supplemental Tables 2 & 3 for details). <b>B.</b> Schematic distribution of the 62 down-regulated photoreceptor or phototransduction-related genes in <i>R-DCKO</i> microarrays (red), compared with the 247 retinal disease loci listed in RetNet (<a href="https://sph.uth.edu/RetNet/home.htm" target="_blank">https://sph.uth.edu/RetNet/home.htm</a>; green) and a list of 230 genes down-regulated in <i>Crx^−/−</i> retinas compiled from published sources <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069721#pone.0069721-Blackshaw1" target="_blank">[51]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069721#pone.0069721-Livesey1" target="_blank">[53]</a>. Numbers in overlapping areas indicate the numbers of genes affected in both/all three conditions. All overlapping genes are listed in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069721#pone.0069721.s009" target="_blank">Tables S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069721#pone.0069721.s010" target="_blank">S5</a>. <b>C</b>–<b>F.</b> Expression of the indicated rod gene (<b>C. </b><b><i>Nrl</i></b><b>; D. </b><b><i>Crx;</i></b><b> E.</b> <b><i>Rhodopsin (Rho);</i></b><b> F. </b><b><i>Rod Transducin (Gnat1)</i></b>) was assessed by quantitative RT-PCR (<b>qRT-PCR</b>) at P14, and is expressed as percent of the level of <i>Cre-negative</i> littermate controls (<b><i>% Cre neg</i></b>). Protein localization was verified by immunohistochemistry (<b>IHC</b>) at P30. Scale bar  = 20 µm for all images. Levels of acetylated histone H3 <b>(AcH3)</b> or H4 <b>(AcH4)</b> on the respective promoter was determined by quantitative chromatin immunoprecipitation (<b>qChIP</b>) at P14, and is expressed as the value from the immunoprecipitated sample divided by the value from the untreated “input” sample, multiplied by 100 (“IP/input”).</p

Image_2_Complement C3a receptor inactivation attenuates retinal degeneration induced by oxidative damage.TIF

Retinal degeneration causes vision loss and threatens the health of elderly individuals worldwide. Evidence indicates that the activation of the complement system is associated with retinal degeneration. However, the mechanism of complement signaling in retinal degeneration needs to be further studied. In this study, we show that the expression of C3 and C3a receptor (C3ar1) is positively associated with the inflammatory response and retinal degeneration. Genetic deletion of C3 and pharmacological inhibition of C3ar1 resulted in the alleviation of neuroinflammation, prevention of photoreceptor cell apoptosis and restoration of visual function. RNA sequencing (RNA-seq) identified a C3ar1-dependent network shown to regulate microglial activation and astrocyte gliosis formation. Mechanistically, we found that STAT3 functioned downstream of the C3-C3ar1 pathway and that the C3ar1-STAT3 pathway functionally mediated the immune response and photoreceptor cell degeneration in response to oxidative stress. These findings reveal an important role of C3ar1 in oxidative-induced retinal degeneration and suggest that intervention of the C3ar1 pathway may alleviate retinal degeneration.</p

Development of conditional knockout phenotypes.

<p><b>A.</b> H&E-stained sagittal sections of retinas from mice representing each indicated genotype (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069721#pone-0069721-t001" target="_blank">Table 1</a>), at the indicated ages. Irregularities appear in the ONL of <i>R-DCKO</i> mice at P10, and in <i>Cbp CH</i> retinas at P14. Scale bar  = 50 μm. <b>B.</b> Immunofluorescence staining for p300 (green) shows progressive loss of p300 from ONL cells of <i>R-DCKO</i> retinas between P7 and P14. Scale bar  = 25 μm. <b>C.</b> TUNEL staining for cell death was performed on three retinas of each genotype at P10, P14, and P32. TUNEL positive cells were only increased relative to age-matched <i>Cre negative</i> controls in <i>Cbp CH</i> retinas at P32 (arrow). <b>D.</b> P32 <i>Cbp CH</i> retina showing TUNEL positive cells (black arrowheads). These are frequently seen near ONL irregularities. <b>OS</b>, outer segments. Scale bar  = 50 μm. <b>E.</b> P32 <i>R-DCKO</i> retina containing one TUNEL+ cell (black arrowhead). <i>R-DCKO</i> retinas do not show increased cell death relative to <i>Cre-negative</i> littermates at any age examined.</p

Compound heterozygotes show age-dependent phenotypes.

<p><b>A</b>–<b>D.</b> Cross-sections of 8-week-old retinas stained with H&E show disrupted morphology similar to that seen at 4 weeks in <i>Cbp CH</i> and <i>R-DCKO</i> mice. Scale bar  = 50 μm. <b>E</b>–<b>G.</b> ERG testing shows persistence of the functional impairment in <i>R-DCKO</i> retinas (panels F and G). Dark-adapted b-wave deficits in some <i>p300 CH</i> mice tested at this time are reflected in the slightly decreased average and broad error bars for this genotype (Panel F orange line). Two-way repeated measures ANOVA indicated significance at p<0.0001 for dark-adapted a-waves (Panel E), b-waves (Panel F), and light-adapted b-waves (Panel G). <b>H</b>–<b>K.</b> Cross-sections of 12-week-old retinas stained with H&E. Morphologic abnormalities in <i>Cbp CH</i> retinas (panel J) have resolved, although whorls and rosettes are still seen in <i>R-DCKO</i> retinas (panel K). Scale bar  = 50 μm. <b>L</b>–<b>N.</b> ERG testing at 12 weeks revealed decreases in function in both <i>Cbp CH</i> and <i>P300 CH</i>, and <i>R-DCKO</i> retinas have lost cone responses in addition to rod function. Two-way repeated measures ANOVA indicated significance at p<0.0001 for dark-adapted a-waves (Panel L), b-waves (Panel M), and light-adapted b-waves (Panel N). Asterisks (*) indicate p<0.001 vs. <i>Cre negative</i> controls in post-hoc tests.</p

Image_1_Complement C3a receptor inactivation attenuates retinal degeneration induced by oxidative damage.TIF

Retinal degeneration causes vision loss and threatens the health of elderly individuals worldwide. Evidence indicates that the activation of the complement system is associated with retinal degeneration. However, the mechanism of complement signaling in retinal degeneration needs to be further studied. In this study, we show that the expression of C3 and C3a receptor (C3ar1) is positively associated with the inflammatory response and retinal degeneration. Genetic deletion of C3 and pharmacological inhibition of C3ar1 resulted in the alleviation of neuroinflammation, prevention of photoreceptor cell apoptosis and restoration of visual function. RNA sequencing (RNA-seq) identified a C3ar1-dependent network shown to regulate microglial activation and astrocyte gliosis formation. Mechanistically, we found that STAT3 functioned downstream of the C3-C3ar1 pathway and that the C3ar1-STAT3 pathway functionally mediated the immune response and photoreceptor cell degeneration in response to oxidative stress. These findings reveal an important role of C3ar1 in oxidative-induced retinal degeneration and suggest that intervention of the C3ar1 pathway may alleviate retinal degeneration.</p