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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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scAAV5-mediated transfer of the mature Edn2 cDNA at PN8 increases mutant PR survival.(A) Micrographs showing ONL thickness in individual retinas at PN14–15. (a) Injection of the scAAV5-smCBA-matEdn2 vector into WT retinas at PN8 did not alter retinal morphology at PN15. (b) Injection of the scAAV5-smCBA-matEdn2 vector at PN8 increased PR ONL thickness of Pde6brd1/rd1 retinas by 31% (n = 9; *p<0.05) at PN15, and (c) of Pde6brd1/rd1; Edn2−/− retinas at PN14 by 18% (n = 5; *p<0.05). (d) In contrast, injection of the scAAV5-smCBA-preproEdn2 vector at PN8 had no effect on PR ONL thickness of Pde6brd1/rd1 retinas (n = 6;p>0.05) at PN15, although (e) this vector improved PR survival in Pde6brd1/rd1; Edn2−/− retinas by 14% (n = 6; *p<0.05). (B) A bar graph summarizing the effects of AAV vectors expressing Edn2 cDNAs, injected at PN8, on mutant PR survival in Pde6brd1/rd1 and Pde6brd1/rd1; Edn2−/− retinas at PN14–15. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Black bar = 25 µm). Error bars indicate SEM.
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pone-0058023-g005: scAAV5-mediated transfer of the mature Edn2 cDNA at PN8 increases mutant PR survival.(A) Micrographs showing ONL thickness in individual retinas at PN14–15. (a) Injection of the scAAV5-smCBA-matEdn2 vector into WT retinas at PN8 did not alter retinal morphology at PN15. (b) Injection of the scAAV5-smCBA-matEdn2 vector at PN8 increased PR ONL thickness of Pde6brd1/rd1 retinas by 31% (n = 9; *p<0.05) at PN15, and (c) of Pde6brd1/rd1; Edn2−/− retinas at PN14 by 18% (n = 5; *p<0.05). (d) In contrast, injection of the scAAV5-smCBA-preproEdn2 vector at PN8 had no effect on PR ONL thickness of Pde6brd1/rd1 retinas (n = 6;p>0.05) at PN15, although (e) this vector improved PR survival in Pde6brd1/rd1; Edn2−/− retinas by 14% (n = 6; *p<0.05). (B) A bar graph summarizing the effects of AAV vectors expressing Edn2 cDNAs, injected at PN8, on mutant PR survival in Pde6brd1/rd1 and Pde6brd1/rd1; Edn2−/− retinas at PN14–15. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Black bar = 25 µm). Error bars indicate SEM.

Mentions: To examine the role of the increased PR expression of Edn2 on PR survival in IPD, we augmented the endogenous increase in Edn2 expression in PRs in Pde6brd1/rd1 mice using AAV vectors (Fig. 5). We used 1X PBS injection as the control injection in the contralateral eye, because when we compared ONL thickness in 1X PBS injected retinas to AAV-Gfp injected Pde6brd1/rd1 retinas, we observed no difference. The introduction of AAV-matEdn2 at PN8 in WT retinas did not alter PR morphology or survival at PN15 (n = 6; p>0.05) (Fig. 5A(a)). Remarkably, the injection of AAV-matEdn2 increased retinal ONL thickness in Pde6brd1/rd1 retinas at PN15 by 31% (n = 9; p<0.05) compared to the injection of 1X PBS in the contralateral eye (Fig. 5A(b)). In contrast, the AAV-preproEdn2 vector failed to improve PR survival in Pde6brd1/rd1 retinas (n = 6; p>0.05) (Fig. 5A(d)), Fig. 5B), suggesting that ECE enzyme sites may be saturated in this context, in which the endogenous increase in Edn2 expression is combined with the production of prepro Edn2 from the AAV vector. We also restored the PR expression of Edn2 in Pde6brd1/rd1; Edn2−/− mice using the AAV vectors. In this instance, the injection of either the matEdn2 or preproEdn2 vectors increased ONL thickness at PN14 and PN15, by 18% (n = 5; p<0.05) (Fig. 5A(c)) and 14% (n = 6; p<0.05) (Fig. 5A(e)), respectively. The effects of the AAV vectors on PR survival are summarized in Fig. 5B. Overall, these findings provide evidence that the increase in Edn2 expression is a protective response that increases the survival of mutant PRs, at least in Pde6brd1/rd1 retinas.


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)

scAAV5-mediated transfer of the mature Edn2 cDNA at PN8 increases mutant PR survival.(A) Micrographs showing ONL thickness in individual retinas at PN14–15. (a) Injection of the scAAV5-smCBA-matEdn2 vector into WT retinas at PN8 did not alter retinal morphology at PN15. (b) Injection of the scAAV5-smCBA-matEdn2 vector at PN8 increased PR ONL thickness of Pde6brd1/rd1 retinas by 31% (n = 9; *p<0.05) at PN15, and (c) of Pde6brd1/rd1; Edn2−/− retinas at PN14 by 18% (n = 5; *p<0.05). (d) In contrast, injection of the scAAV5-smCBA-preproEdn2 vector at PN8 had no effect on PR ONL thickness of Pde6brd1/rd1 retinas (n = 6;p>0.05) at PN15, although (e) this vector improved PR survival in Pde6brd1/rd1; Edn2−/− retinas by 14% (n = 6; *p<0.05). (B) A bar graph summarizing the effects of AAV vectors expressing Edn2 cDNAs, injected at PN8, on mutant PR survival in Pde6brd1/rd1 and Pde6brd1/rd1; Edn2−/− retinas at PN14–15. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Black bar = 25 µm). Error bars indicate SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585171&req=5

pone-0058023-g005: scAAV5-mediated transfer of the mature Edn2 cDNA at PN8 increases mutant PR survival.(A) Micrographs showing ONL thickness in individual retinas at PN14–15. (a) Injection of the scAAV5-smCBA-matEdn2 vector into WT retinas at PN8 did not alter retinal morphology at PN15. (b) Injection of the scAAV5-smCBA-matEdn2 vector at PN8 increased PR ONL thickness of Pde6brd1/rd1 retinas by 31% (n = 9; *p<0.05) at PN15, and (c) of Pde6brd1/rd1; Edn2−/− retinas at PN14 by 18% (n = 5; *p<0.05). (d) In contrast, injection of the scAAV5-smCBA-preproEdn2 vector at PN8 had no effect on PR ONL thickness of Pde6brd1/rd1 retinas (n = 6;p>0.05) at PN15, although (e) this vector improved PR survival in Pde6brd1/rd1; Edn2−/− retinas by 14% (n = 6; *p<0.05). (B) A bar graph summarizing the effects of AAV vectors expressing Edn2 cDNAs, injected at PN8, on mutant PR survival in Pde6brd1/rd1 and Pde6brd1/rd1; Edn2−/− retinas at PN14–15. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Black bar = 25 µm). Error bars indicate SEM.
Mentions: To examine the role of the increased PR expression of Edn2 on PR survival in IPD, we augmented the endogenous increase in Edn2 expression in PRs in Pde6brd1/rd1 mice using AAV vectors (Fig. 5). We used 1X PBS injection as the control injection in the contralateral eye, because when we compared ONL thickness in 1X PBS injected retinas to AAV-Gfp injected Pde6brd1/rd1 retinas, we observed no difference. The introduction of AAV-matEdn2 at PN8 in WT retinas did not alter PR morphology or survival at PN15 (n = 6; p>0.05) (Fig. 5A(a)). Remarkably, the injection of AAV-matEdn2 increased retinal ONL thickness in Pde6brd1/rd1 retinas at PN15 by 31% (n = 9; p<0.05) compared to the injection of 1X PBS in the contralateral eye (Fig. 5A(b)). In contrast, the AAV-preproEdn2 vector failed to improve PR survival in Pde6brd1/rd1 retinas (n = 6; p>0.05) (Fig. 5A(d)), Fig. 5B), suggesting that ECE enzyme sites may be saturated in this context, in which the endogenous increase in Edn2 expression is combined with the production of prepro Edn2 from the AAV vector. We also restored the PR expression of Edn2 in Pde6brd1/rd1; Edn2−/− mice using the AAV vectors. In this instance, the injection of either the matEdn2 or preproEdn2 vectors increased ONL thickness at PN14 and PN15, by 18% (n = 5; p<0.05) (Fig. 5A(c)) and 14% (n = 6; p<0.05) (Fig. 5A(e)), respectively. The effects of the AAV vectors on PR survival are summarized in Fig. 5B. Overall, these findings provide evidence that the increase in Edn2 expression is a protective response that increases the survival of mutant PRs, at least in Pde6brd1/rd1 retinas.

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