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Endothelin-2-mediated protection of mutant photoreceptors in inherited photoreceptor degeneration.

Bramall AN, Szego MJ, Pacione LR, Chang I, Diez E, D'Orleans-Juste P, Stewart DJ, Hauswirth WW, Yanagisawa M, McInnes RR - PLoS ONE (2013)

Bottom Line: Together, these findings suggest that increased Edn2 expression is protective to mutant PRs.Notably, increased expression of the FGF2 protein in Tg(RHO P347S) PRs was ablated in Tg(RHO P347S); Edn2(-/-) retinas.Our findings indicate that the increased expression of PR Edn2 increases PR survival, and suggest that the Edn2-dependent increase in PR expression of FGF2 may contribute to the augmented survival.

View Article: PubMed Central - PubMed

Affiliation: Program in Developmental Biology, The Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada.

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

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Expression of GFP and Edn2 from subretinally injected scAAV5.(A) The temporal and spatial expression of GFP in WT retinas injected subretinally with 1X PBS or scAAV5-smCBA-Gfp 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-Gfp 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 [68]). The regions of the Edn2 mRNA corresponding to the cDNAs cloned into the scAAV5-smCBA-preproEdn2 and scAAV5-smCBA-matEdn2 vectors are shown. (C) Expression of scAAV5-derived Edn2 mRNA in Pde6brd1/rd1 retinas at PN12 after injection of the scAAV5-smCBA-preproEdn2 and scAAV5-smCBA-matEdn2 constructs at PN8. scAAV5-derived Edn2 mRNA expression values are shown relative to the levels of endogenous Edn2 mRNA (from the same retina) and all values were normalized to Gapdh. scAAV5-preproEdn2 transcripts were increased between 1.7 and 7.2-fold (n = 4; average 4.3-fold) over endogenous Edn2 mRNA, while scAAV5-matEdn2 transcripts increased between 2.5 to 11.3-fold over the endogenous Edn2 mRNA (n = 4; average 6.9-fold). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Error bars indicate SEM.
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pone-0058023-g004: Expression of GFP and Edn2 from subretinally injected scAAV5.(A) The temporal and spatial expression of GFP in WT retinas injected subretinally with 1X PBS or scAAV5-smCBA-Gfp 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-Gfp 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 [68]). The regions of the Edn2 mRNA corresponding to the cDNAs cloned into the scAAV5-smCBA-preproEdn2 and scAAV5-smCBA-matEdn2 vectors are shown. (C) Expression of scAAV5-derived Edn2 mRNA in Pde6brd1/rd1 retinas at PN12 after injection of the scAAV5-smCBA-preproEdn2 and scAAV5-smCBA-matEdn2 constructs at PN8. scAAV5-derived Edn2 mRNA expression values are shown relative to the levels of endogenous Edn2 mRNA (from the same retina) and all values were normalized to Gapdh. scAAV5-preproEdn2 transcripts were increased between 1.7 and 7.2-fold (n = 4; average 4.3-fold) over endogenous Edn2 mRNA, while scAAV5-matEdn2 transcripts increased between 2.5 to 11.3-fold over the endogenous Edn2 mRNA (n = 4; average 6.9-fold). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Error bars indicate SEM.

Mentions: EDN2 is processed from a prepro peptide that undergoes sequential cleavage events to generate mature EDN2 (mat EDN2) (Fig. 4B). To determine where endothelin-converting enzyme 1 (ECE-1), an enzyme implicated in the processing of big EDN2 to mature EDN2 [37], is expressed in retina, we used immunofluorescence staining. We found that ECE-1 was expressed in Müller cell bodies and radial fibres (Fig. S2A), as shown by comparison with the Müller cell-specific marker glutamine synthetase [38].


Endothelin-2-mediated protection of mutant photoreceptors in inherited photoreceptor degeneration.

Bramall AN, Szego MJ, Pacione LR, Chang I, Diez E, D'Orleans-Juste P, Stewart DJ, Hauswirth WW, Yanagisawa M, McInnes RR - PLoS ONE (2013)

Expression of GFP and Edn2 from subretinally injected scAAV5.(A) The temporal and spatial expression of GFP in WT retinas injected subretinally with 1X PBS or scAAV5-smCBA-Gfp 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-Gfp 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 [68]). The regions of the Edn2 mRNA corresponding to the cDNAs cloned into the scAAV5-smCBA-preproEdn2 and scAAV5-smCBA-matEdn2 vectors are shown. (C) Expression of scAAV5-derived Edn2 mRNA in Pde6brd1/rd1 retinas at PN12 after injection of the scAAV5-smCBA-preproEdn2 and scAAV5-smCBA-matEdn2 constructs at PN8. scAAV5-derived Edn2 mRNA expression values are shown relative to the levels of endogenous Edn2 mRNA (from the same retina) and all values were normalized to Gapdh. scAAV5-preproEdn2 transcripts were increased between 1.7 and 7.2-fold (n = 4; average 4.3-fold) over endogenous Edn2 mRNA, while scAAV5-matEdn2 transcripts increased between 2.5 to 11.3-fold over the endogenous Edn2 mRNA (n = 4; average 6.9-fold). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Error bars indicate SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585171&req=5

pone-0058023-g004: Expression of GFP and Edn2 from subretinally injected scAAV5.(A) The temporal and spatial expression of GFP in WT retinas injected subretinally with 1X PBS or scAAV5-smCBA-Gfp 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-Gfp 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 [68]). The regions of the Edn2 mRNA corresponding to the cDNAs cloned into the scAAV5-smCBA-preproEdn2 and scAAV5-smCBA-matEdn2 vectors are shown. (C) Expression of scAAV5-derived Edn2 mRNA in Pde6brd1/rd1 retinas at PN12 after injection of the scAAV5-smCBA-preproEdn2 and scAAV5-smCBA-matEdn2 constructs at PN8. scAAV5-derived Edn2 mRNA expression values are shown relative to the levels of endogenous Edn2 mRNA (from the same retina) and all values were normalized to Gapdh. scAAV5-preproEdn2 transcripts were increased between 1.7 and 7.2-fold (n = 4; average 4.3-fold) over endogenous Edn2 mRNA, while scAAV5-matEdn2 transcripts increased between 2.5 to 11.3-fold over the endogenous Edn2 mRNA (n = 4; average 6.9-fold). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Error bars indicate SEM.
Mentions: EDN2 is processed from a prepro peptide that undergoes sequential cleavage events to generate mature EDN2 (mat EDN2) (Fig. 4B). To determine where endothelin-converting enzyme 1 (ECE-1), an enzyme implicated in the processing of big EDN2 to mature EDN2 [37], is expressed in retina, we used immunofluorescence staining. We found that ECE-1 was expressed in Müller cell bodies and radial fibres (Fig. S2A), as shown by comparison with the Müller cell-specific marker glutamine synthetase [38].

Bottom Line: Together, these findings suggest that increased Edn2 expression is protective to mutant PRs.Notably, increased expression of the FGF2 protein in Tg(RHO P347S) PRs was ablated in Tg(RHO P347S); Edn2(-/-) retinas.Our findings indicate that the increased expression of PR Edn2 increases PR survival, and suggest that the Edn2-dependent increase in PR expression of FGF2 may contribute to the augmented survival.

View Article: PubMed Central - PubMed

Affiliation: Program in Developmental Biology, The Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada.

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

Show MeSH
Related in: MedlinePlus