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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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FGF2 expression in Tg(RHO P347S) PRs returns to WT levels in the absence of EDN2.(A) qRT-PCR quantification of Edn2, Gm12541, Fgf2 and Gfap expression in Tg(RHO P347S) 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 Edn2+/+ (WT) retinas. Fold down-regulation in Tg(RHO P347S); Edn2−/− retinas vs. Tg(RHO P347S); Edn2+/+ 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 Gapdh mRNA. ND (not detected). (B) Immunostaining for FGF2 showed low levels of expression in all three retinal nuclear layers in Edn2+/+ and Edn2−/− retinas, but FGF2 expression increased significantly in the PRs of Tg(RHO P347S); Edn2+/+ retinas at PN21 (third top panel). In contrast, FGF2 staining was similar to WT retinas in Tg(RHO P347S); Edn2−/− retinas and, most notably, from PRs (fourth upper panel). GFAP expression in Müller cells was increased in Tg(RHO P347S); Edn2+/+ retinas (third bottom panel), but reduced in Tg(RHO P347S); Edn2−/− retinas (fourth bottom panel). GFAP expression in Tg(RHO P347S); Edn2−/− retinas was higher than in Edn2+/+ retinas. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Bar = 25 µm). Error bars indicate SEM.
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pone-0058023-g007: FGF2 expression in Tg(RHO P347S) PRs returns to WT levels in the absence of EDN2.(A) qRT-PCR quantification of Edn2, Gm12541, Fgf2 and Gfap expression in Tg(RHO P347S) 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 Edn2+/+ (WT) retinas. Fold down-regulation in Tg(RHO P347S); Edn2−/− retinas vs. Tg(RHO P347S); Edn2+/+ 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 Gapdh mRNA. ND (not detected). (B) Immunostaining for FGF2 showed low levels of expression in all three retinal nuclear layers in Edn2+/+ and Edn2−/− retinas, but FGF2 expression increased significantly in the PRs of Tg(RHO P347S); Edn2+/+ retinas at PN21 (third top panel). In contrast, FGF2 staining was similar to WT retinas in Tg(RHO P347S); Edn2−/− retinas and, most notably, from PRs (fourth upper panel). GFAP expression in Müller cells was increased in Tg(RHO P347S); Edn2+/+ retinas (third bottom panel), but reduced in Tg(RHO P347S); Edn2−/− retinas (fourth bottom panel). GFAP expression in Tg(RHO P347S); Edn2−/− retinas was higher than in Edn2+/+ retinas. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Bar = 25 µm). Error bars indicate SEM.

Mentions: Of the 20 genes whose expression is most reduced by the loss of EDN2, only Fgf2[9], [44], [45] has been shown to increase PR survival in IPDs, and only the loss of Gfap has been demonstrated to attenuate PR death due to retinal injury [14]. To investigate the role of Fgf2 and Gfap in the response of the Tg(RHO P347S) retina to the loss of EDN2 function, we first used qRT-PCR to confirm and to quantify the decrease in the expression of Fgf2 and Gfap in Tg(RHO P347S); Edn2−/− retinas compared to Tg(RHO P347S); Edn2+/+ retinas (Fig. 7A). We also quantified the expression of Gm12541 because the microarray analyses indicated that the dramatic increase in Gm12541 expression in Tg(RHO P347S); Edn2+/+ retinas was reduced in Tg(RHO P347S); Edn2−/− retinas (Fig. 6). The qRT-PCR analyses confirmed remarkable increases in the expression of Edn2 and Gm12541 in Tg(RHO P347S); Edn2+/+ vs. Edn2+/+ retinas (70.3- and 29.8-fold respectively), as well as a substantial increase in Fgf2 and Gfap expression (3.0- and 2.0-fold respectively; Fig. 7A). In the absence of EDN2 in Tg(RHO P347S); Edn2−/− retinas, the expression of Gm12541, Fgf2 and Gfap decreased significantly (from 1.6–2.6 fold;Fig. 7A).


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)

FGF2 expression in Tg(RHO P347S) PRs returns to WT levels in the absence of EDN2.(A) qRT-PCR quantification of Edn2, Gm12541, Fgf2 and Gfap expression in Tg(RHO P347S) 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 Edn2+/+ (WT) retinas. Fold down-regulation in Tg(RHO P347S); Edn2−/− retinas vs. Tg(RHO P347S); Edn2+/+ 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 Gapdh mRNA. ND (not detected). (B) Immunostaining for FGF2 showed low levels of expression in all three retinal nuclear layers in Edn2+/+ and Edn2−/− retinas, but FGF2 expression increased significantly in the PRs of Tg(RHO P347S); Edn2+/+ retinas at PN21 (third top panel). In contrast, FGF2 staining was similar to WT retinas in Tg(RHO P347S); Edn2−/− retinas and, most notably, from PRs (fourth upper panel). GFAP expression in Müller cells was increased in Tg(RHO P347S); Edn2+/+ retinas (third bottom panel), but reduced in Tg(RHO P347S); Edn2−/− retinas (fourth bottom panel). GFAP expression in Tg(RHO P347S); Edn2−/− retinas was higher than in Edn2+/+ retinas. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (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-g007: FGF2 expression in Tg(RHO P347S) PRs returns to WT levels in the absence of EDN2.(A) qRT-PCR quantification of Edn2, Gm12541, Fgf2 and Gfap expression in Tg(RHO P347S) 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 Edn2+/+ (WT) retinas. Fold down-regulation in Tg(RHO P347S); Edn2−/− retinas vs. Tg(RHO P347S); Edn2+/+ 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 Gapdh mRNA. ND (not detected). (B) Immunostaining for FGF2 showed low levels of expression in all three retinal nuclear layers in Edn2+/+ and Edn2−/− retinas, but FGF2 expression increased significantly in the PRs of Tg(RHO P347S); Edn2+/+ retinas at PN21 (third top panel). In contrast, FGF2 staining was similar to WT retinas in Tg(RHO P347S); Edn2−/− retinas and, most notably, from PRs (fourth upper panel). GFAP expression in Müller cells was increased in Tg(RHO P347S); Edn2+/+ retinas (third bottom panel), but reduced in Tg(RHO P347S); Edn2−/− retinas (fourth bottom panel). GFAP expression in Tg(RHO P347S); Edn2−/− retinas was higher than in Edn2+/+ retinas. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Bar = 25 µm). Error bars indicate SEM.
Mentions: Of the 20 genes whose expression is most reduced by the loss of EDN2, only Fgf2[9], [44], [45] has been shown to increase PR survival in IPDs, and only the loss of Gfap has been demonstrated to attenuate PR death due to retinal injury [14]. To investigate the role of Fgf2 and Gfap in the response of the Tg(RHO P347S) retina to the loss of EDN2 function, we first used qRT-PCR to confirm and to quantify the decrease in the expression of Fgf2 and Gfap in Tg(RHO P347S); Edn2−/− retinas compared to Tg(RHO P347S); Edn2+/+ retinas (Fig. 7A). We also quantified the expression of Gm12541 because the microarray analyses indicated that the dramatic increase in Gm12541 expression in Tg(RHO P347S); Edn2+/+ retinas was reduced in Tg(RHO P347S); Edn2−/− retinas (Fig. 6). The qRT-PCR analyses confirmed remarkable increases in the expression of Edn2 and Gm12541 in Tg(RHO P347S); Edn2+/+ vs. Edn2+/+ retinas (70.3- and 29.8-fold respectively), as well as a substantial increase in Fgf2 and Gfap expression (3.0- and 2.0-fold respectively; Fig. 7A). In the absence of EDN2 in Tg(RHO P347S); Edn2−/− retinas, the expression of Gm12541, Fgf2 and Gfap decreased significantly (from 1.6–2.6 fold;Fig. 7A).

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