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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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Retinal Edn2 mRNA is increased in several models of IPD.(A) qRT-PCR assays of the Edn2 mRNA. Edn2 was increased 32-fold, 70-fold, and 72-fold in the Prph2rds/+ (7 weeks), Tg(RHO P347S) (3 weeks) and Pde6brd1/rd1 (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. Edn2 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 Gapdh. (B) In situ hybridization of Edn2 mRNA in Prph2rds/+ and Tg(RHO P347S) retinas. Edn2 transcripts were undetectable in WT retina, but were detected exclusively in the ONL of the mutant PRs (arrowheads) in Prph2rds/+ and Tg(RHO P347S) retinas. (C,D) Temporal expression of Edn2 transcripts during PR degeneration in Pde6brd1/rd1 retinas, relative to WT Edn2 mRNA expression at PN12 and normalized to Gapdh mRNA (C), or rhodopsin mRNA (D). Edn2 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.
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pone-0058023-g001: Retinal Edn2 mRNA is increased in several models of IPD.(A) qRT-PCR assays of the Edn2 mRNA. Edn2 was increased 32-fold, 70-fold, and 72-fold in the Prph2rds/+ (7 weeks), Tg(RHO P347S) (3 weeks) and Pde6brd1/rd1 (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. Edn2 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 Gapdh. (B) In situ hybridization of Edn2 mRNA in Prph2rds/+ and Tg(RHO P347S) retinas. Edn2 transcripts were undetectable in WT retina, but were detected exclusively in the ONL of the mutant PRs (arrowheads) in Prph2rds/+ and Tg(RHO P347S) retinas. (C,D) Temporal expression of Edn2 transcripts during PR degeneration in Pde6brd1/rd1 retinas, relative to WT Edn2 mRNA expression at PN12 and normalized to Gapdh mRNA (C), or rhodopsin mRNA (D). Edn2 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.

Mentions: To confirm that Edn2 transcripts are up-regulated in the Prph2rds/+, Tg(RHO P347S), and Pde6brd1/rd1 mouse models of IPD we studied, we measured retinal Edn2 mRNA abundance. As reported by Rattner and Nathans in other models [19], we found the Edn2 mRNA to be greatly increased vs. wild-type (WT) controls in Prph2rds/+ (32-fold), Tg(RHO P347S) (70-fold), and Pde6brd1/rd1 (72-fold) retinas, using qRT-PCR (for each model n = 3; p<0.005) (Fig. 1A).


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)

Retinal Edn2 mRNA is increased in several models of IPD.(A) qRT-PCR assays of the Edn2 mRNA. Edn2 was increased 32-fold, 70-fold, and 72-fold in the Prph2rds/+ (7 weeks), Tg(RHO P347S) (3 weeks) and Pde6brd1/rd1 (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. Edn2 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 Gapdh. (B) In situ hybridization of Edn2 mRNA in Prph2rds/+ and Tg(RHO P347S) retinas. Edn2 transcripts were undetectable in WT retina, but were detected exclusively in the ONL of the mutant PRs (arrowheads) in Prph2rds/+ and Tg(RHO P347S) retinas. (C,D) Temporal expression of Edn2 transcripts during PR degeneration in Pde6brd1/rd1 retinas, relative to WT Edn2 mRNA expression at PN12 and normalized to Gapdh mRNA (C), or rhodopsin mRNA (D). Edn2 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.
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pone-0058023-g001: Retinal Edn2 mRNA is increased in several models of IPD.(A) qRT-PCR assays of the Edn2 mRNA. Edn2 was increased 32-fold, 70-fold, and 72-fold in the Prph2rds/+ (7 weeks), Tg(RHO P347S) (3 weeks) and Pde6brd1/rd1 (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. Edn2 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 Gapdh. (B) In situ hybridization of Edn2 mRNA in Prph2rds/+ and Tg(RHO P347S) retinas. Edn2 transcripts were undetectable in WT retina, but were detected exclusively in the ONL of the mutant PRs (arrowheads) in Prph2rds/+ and Tg(RHO P347S) retinas. (C,D) Temporal expression of Edn2 transcripts during PR degeneration in Pde6brd1/rd1 retinas, relative to WT Edn2 mRNA expression at PN12 and normalized to Gapdh mRNA (C), or rhodopsin mRNA (D). Edn2 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.
Mentions: To confirm that Edn2 transcripts are up-regulated in the Prph2rds/+, Tg(RHO P347S), and Pde6brd1/rd1 mouse models of IPD we studied, we measured retinal Edn2 mRNA abundance. As reported by Rattner and Nathans in other models [19], we found the Edn2 mRNA to be greatly increased vs. wild-type (WT) controls in Prph2rds/+ (32-fold), Tg(RHO P347S) (70-fold), and Pde6brd1/rd1 (72-fold) retinas, using qRT-PCR (for each model n = 3; p<0.005) (Fig. 1A).

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