Endothelin-2-Mediated Protection of Mutant Photoreceptors in Inherited Photoreceptor Degeneration

<div><p>Expression of the <i>Endothelin-2 (Edn2)</i> mRNA is greatly increased in the photoreceptors (PRs) of mouse models of inherited PR degeneration (IPD). To examine the role of <i>Edn2</i> in mutant PR survival, we generated <i>Edn2^−/−</i> mice carrying homozygous <i>Pde6b^rd1</i> alleles or the <i>Tg(RHO P347S)</i> transgene. In the <i>Edn2^−/−</i> background, PR survival increased 110% in <i>Pde6b^rd1/rd1</i> mice at post-natal (PN) day 15, and 60% in <i>Tg(RHO P347S)</i> mice at PN40. In contrast, PR survival was not increased in retinal explants of <i>Pde6b^rd1/rd1</i>; <i>Edn2^−/−</i> mice. This finding, together with systemic abnormalities in <i>Edn2^−/−</i> mice, suggested that the increased survival of mutant PRs in the <i>Edn2^−/−</i> background resulted at least partly from the systemic EDN2 loss of function. To examine directly the role of EDN2 in mutant PRs, we used a scAAV5-<i>Edn2</i> cDNA vector to restore <i>Edn2</i> expression in <i>Pde6b^rd1/rd1</i>; <i>Edn2^−/−</i> PRs and observed an 18% increase in PR survival at PN14. Importantly, PR survival was also increased after injection of scAAV5-<i>Edn2</i> into <i>Pde6b^rd1/rd1</i> retinas, by 31% at PN15. Together, these findings suggest that increased <i>Edn2</i> expression is protective to mutant PRs. To begin to elucidate <i>Edn2</i>-mediated mechanisms that contribute to PR survival, we used microarray analysis and identified a cohort of 20 genes with >4-fold increased expression in <i>Tg(RHO P347S)</i> retinas, including <i>Fgf2</i>. Notably, increased expression of the FGF2 protein in <i>Tg(RHO P347S)</i> PRs was ablated in <i>Tg(RHO P347S)</i>; <i>Edn2^−/−</i> retinas. Our findings indicate that the increased expression of PR <i>Edn2</i> increases PR survival, and suggest that the <i>Edn2</i>-dependent increase in PR expression of FGF2 may contribute to the augmented survival.</p> </div

Expression of GFP and <i>Edn2</i> from subretinally injected scAAV5.

<p>(A) The temporal and spatial expression of GFP in WT retinas injected subretinally with 1X PBS or scAAV5-smCBA-<i>Gfp</i> at PN8 and evaluated at PN10, PN12, and PN14 by GFP immunofluorescence (A, Panels 1–4). Significant GFP staining was not observed until PN12, with stronger staining at PN14, especially in the vicinity of the subretinal injection site (arrow). The spatial expression of GFP in WT retinas injected with scAAV5-smCBA-<i>Gfp</i> was also evaluated in paraffin sections at PN12 (A, Panel 5). GFP expression was observed predominantly in the ONL and RPE; sporadic expression of GFP in Müller cells was also observed in paraffin sections. (Bar = 25 µm.) (B) Schematic of the EDN2 cleavage events required to produce the mature EDN2 peptide. EDN2 is first produced as prepro EDN2 (175 aa) which is rapidly processed by furin-like endopeptidases to yield big EDN2 (38 aa). Big EDN2 must then be cleaved by an endothelin-specific converting enzyme (ECE) to produce the 21 a.a. mature EDN2 peptide that can bind to endothelin receptors (figure adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058023#pone.0058023-Saida1" target="_blank">[68]</a>). The regions of the <i>Edn2</i> mRNA corresponding to the cDNAs cloned into the scAAV5-<i>smCBA</i>-prepro<i>Edn2</i> and scAAV5-<i>smCBA</i>-mat<i>Edn2</i> vectors are shown. (C) Expression of scAAV5-derived <i>Edn2</i> mRNA in <i>Pde6b^rd1/rd1</i> retinas at PN12 after injection of the scAAV5-<i>smCBA</i>-prepro<i>Edn2</i> and scAAV5-<i>smCBA</i>-mat<i>Edn2</i> constructs at PN8. scAAV5-derived <i>Edn2</i> mRNA expression values are shown relative to the levels of endogenous <i>Edn2</i> mRNA (from the same retina) and all values were normalized to <i>Gapdh</i>. scAAV5-prepro<i>Edn2</i> transcripts were increased between 1.7 and 7.2-fold (n = 4; average 4.3-fold) over endogenous <i>Edn2</i> mRNA, while scAAV5-mat<i>Edn2</i> transcripts increased between 2.5 to 11.3-fold over the endogenous <i>Edn2</i> mRNA (n = 4; average 6.9-fold). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Error bars indicate SEM.</p

Systemic erythropoietin (EPO) and retinal vascular endothelial growth factor (VEGF) is increased in <i>Edn2^−/−</i> mice.

<p>At PN21, serum EPO was increased 11-fold in Edn2^−/− vs. Edn2^+/+ mice (n = 7; p<0.005) (A) and retinal VEGF was increased 4-fold (n = 4; p<0.005) (B). EPO and VEGF were both measured using ELISA assays. Error bars indicate SEM.</p

FGF2 expression in <i>Tg(RHO P347S)</i> PRs returns to WT levels in the absence of EDN2.

<p>(A) qRT-PCR quantification of <i>Edn2</i>, <i>Gm12541</i>, <i>Fgf2</i> and <i>Gfap</i> expression in <i>Tg(RHO P347S)</i> retinas in the presence or absence of EDN2 function. Bar graphs show the expression of mRNAs in retinas of the indicated genotypes relative to the expression levels seen in <i>Edn2^+/+</i> (WT) retinas. Fold down-regulation in <i>Tg(RHO P347S)</i>; <i>Edn2^−/−</i> retinas vs. <i>Tg(RHO P347S); Edn2^+/+</i> retinas is shown to the right of the vertical bars (n = 3;p<0.05 for all mRNAs). All qRT-PCR values were normalized to <i>Gapdh</i> mRNA. ND (not detected). (B) Immunostaining for FGF2 showed low levels of expression in all three retinal nuclear layers in <i>Edn2^+/+</i> and <i>Edn2^−/−</i> retinas, but FGF2 expression increased significantly in the PRs of <i>Tg(RHO P347S); Edn2^+/+</i> retinas at PN21 (third top panel). In contrast, FGF2 staining was similar to WT retinas in <i>Tg(RHO P347S)</i>; <i>Edn2^−/−</i> retinas and, most notably, from PRs (fourth upper panel). GFAP expression in Müller cells was increased in <i>Tg(RHO P347S); Edn2^+/+</i> retinas (third bottom panel), but reduced in <i>Tg(RHO P347S)</i>; <i>Edn2^−/−</i> retinas (fourth bottom panel). GFAP expression in <i>Tg(RHO P347S)</i>; <i>Edn2^−/−</i> retinas was higher than in <i>Edn2^+/+</i> retinas. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Bar = 25 µm). Error bars indicate SEM.</p

Retinal <i>Edn2</i> mRNA is increased in several models of IPD.

<p>(A) qRT-PCR assays of the <i>Edn2</i> mRNA. Edn2 was increased 32-fold, 70-fold, and 72-fold in the <i>Prph2^rds/+</i> (7 weeks), <i>Tg(RHO P347S)</i> (3 weeks) and <i>Pde6b^rd1/rd1</i> (PN12) models of IPD, respectively (^**n = 3; Student's t-test p<0.005). At the three time points chosen, >40% of the PR population remained. <i>Edn2</i> mRNA expression was assigned a value of 1 in WT retinas, to calculate its relative expression in the IPD models. qRT-PCR values were normalized to the mRNA expression of <i>Gapdh</i>. (B) <i>In situ</i> hybridization of <i>Edn2</i> mRNA in <i>Prph2^rds/+</i> and <i>Tg(RHO P347S)</i> retinas. <i>Edn2</i> transcripts were undetectable in WT retina, but were detected exclusively in the ONL of the mutant PRs (arrowheads) in <i>Prph2^rds/+</i> and <i>Tg(RHO P347S)</i> retinas. (C,D) Temporal expression of <i>Edn2</i> transcripts during PR degeneration in <i>Pde6b^rd1/rd1</i> retinas, relative to WT <i>Edn2</i> mRNA expression at PN12 and normalized to <i>Gapdh</i> mRNA (C), or rhodopsin mRNA (D). <i>Edn2</i> mRNA was not significantly increased until PN10 (^*n = 3; p<0.05 vs. WT). RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Error bars indicate SEM; scale bar = 10 µm.</p

scAAV5-mediated transfer of the mature <i>Edn2</i> cDNA at PN8 increases mutant PR survival.

<p>(A) Micrographs showing ONL thickness in individual retinas at PN14–15. (a) Injection of the scAAV5-<i>smCBA</i>-mat<i>Edn2</i> vector into WT retinas at PN8 did not alter retinal morphology at PN15. (b) Injection of the scAAV5-<i>smCBA</i>-mat<i>Edn2</i> vector at PN8 increased PR ONL thickness of <i>Pde6b^rd1/rd1</i> retinas by 31% (n = 9; ^*p<0.05) at PN15, and (c) of <i>Pde6b^rd1/rd1</i>; <i>Edn2^−/−</i> retinas at PN14 by 18% (n = 5; ^*p<0.05). (d) In contrast, injection of the scAAV5-<i>smCBA</i>-prepro<i>Edn2</i> vector at PN8 had no effect on PR ONL thickness of <i>Pde6b^rd1/rd1</i> retinas (n = 6;p>0.05) at PN15, although (e) this vector improved PR survival in <i>Pde6b^rd1/rd1</i>; <i>Edn2^−/−</i> retinas by 14% (n = 6; ^*p<0.05). (B) A bar graph summarizing the effects of AAV vectors expressing <i>Edn2</i> cDNAs, injected at PN8, on mutant PR survival in <i>Pde6b^rd1/rd1</i> and <i>Pde6b^rd1/rd1</i>; <i>Edn2^−/−</i> retinas at PN14–15. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Black bar = 25 µm). Error bars indicate SEM.</p

Identification of possible EDN2 regulated genes using microarrays.

<p>Gene expression was examined using the Mouse 430_2.0 Affymetrix array (4 arrays/genotype) and the 20 most differentially expressed mRNAs in <i>Tg(RHO P347S); Edn2^+/+</i> vs. <i>Edn2^+/+</i> retinas, and in <i>Tg(RHO P347S)</i>; <i>Edn2^−/−</i> vs. <i>Edn2^−/−</i> retinas, at PN21 are shown. All differentially expressed genes in <i>Tg(RHO P347S)</i>; <i>Edn2^+/+</i> retinas showed a significant reduction in expression in <i>Tg(RHO P347S)</i>; <i>Edn2^−/−</i> retinas, with the exception of <i>Nuclear protein 1</i>. The fold reduction is shown to the right of each pair of bars. The detection of <i>Edn2</i> transcripts in <i>Tg(RHO P347S)</i>; <i>Edn2^−/−</i> retinas is due to the expression of partial <i>Edn2</i> mRNAs from the <i>Edn2^−/−</i> locus, in which part of exon 1 and all of exon 2 were replaced with the <i>Neo</i> cassette. n.s. =  no statistically significant increase in expression.</p

The effect of EDN2 loss on mutant PR survival <i>in vivo</i> and in retinal explants.

<p>(A) At PN40, the histology and the thickness of the ONL (n = 5;p>0.05) was normal in toluidine-blue stained <i>Edn2^+/+</i> and <i>Edn2^−/−</i> retinas. (B,C) The loss of EDN2 in <i>Tg(RHO P347S)</i> retinas resulted in a mean 63% increase in ONL thickness at PN40 (n = 6; p<0.005) (C) and a mean 110% increase in ONL thickness in <i>Pde6b^rd1/rd1</i> retinas at PN15 (n = 6; p<0.005) (B). (D) ONL thickness in WT, <i>Pde6b^rd1/rd1</i> and <i>Tg(RHO P347S)</i> retinas in mice expressing or lacking EDN2 (^**p<0.005). (E) qRT−PCR assays of the <i>Edn2</i> mRNA, normalized to <i>Gapdh</i> mRNA, in <i>in vivo</i> WT, WT explants and <i>Pde6b^rd1/rd1</i> explants (n = 3;^*p<0.05). Values were compared to the mean <i>Edn2</i> mRNA levels in WT <i>in vivo</i> samples (arbitrarily given a value of 1). <i>Edn2</i> transcripts were significantly increased in WT as well as <i>Pde6b^rd1/rd1</i> explants at PN12 following retinal dissection at PN7, likely as a result of dissection-induced mechanical stress. (F) WT retinal explants cultured <i>ex vivo</i> from PN7-PN17 had an average of 7–8 rows of PR nuclei at PN17 (n = 10 retinas, one representative shown). Owing to artifacts in frozen sections, the number of nuclei, instead of ONL thickness, was assessed in retinal explants. The absence of EDN2 in <i>Pde6b^rd1/rd1</i>; <i>Edn2^−/−</i> retinal explants did not increase PR survival. Both <i>Pde6b^rd1/rd1</i> and <i>Pde6b^rd1/rd1</i>; <i>Edn2^−/−</i> explants cultured from PN7-PN17 had an average of 3 rows of PR nuclei at PN17 (n = 4;p>0.05, one representative shown) (H&E staining). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Black bar = 25 µm in A–C, and F). Error bars indicate SEM.</p