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Gene profiling of postnatal Mfrprd6 mutant eyes reveals differential accumulation of Prss56, visual cycle and phototransduction mRNAs.

Soundararajan R, Won J, Stearns TM, Charette JR, Hicks WL, Collin GB, Naggert JK, Krebs MP, Nishina PM - PLoS ONE (2014)

Bottom Line: In Mfrprd6 eyes, a significant 1.5- to 2.0-fold decrease was observed among transcripts of genes linked to retinal degeneration, including those involved in visual cycle (Rpe65, Lrat, Rgr), phototransduction (Pde6a, Guca1b, Rgs9), and photoreceptor disc morphogenesis (Rpgrip1 and Fscn2).Levels of RPE65 were significantly decreased by 2.0-fold.In summary, the Mfrprd6 allele causes significant postnatal changes in transcript and protein levels in the retina and RPE.

View Article: PubMed Central - PubMed

Affiliation: The Jackson Laboratory, Bar Harbor, Maine, United States of America.

ABSTRACT
Mutations in the membrane frizzled-related protein (MFRP/Mfrp) gene, specifically expressed in the retinal pigment epithelium (RPE) and ciliary body, cause nanophthalmia or posterior microphthalmia with retinitis pigmentosa in humans, and photoreceptor degeneration in mice. To better understand MFRP function, microarray analysis was performed on eyes of homozygous Mfrprd6 and C57BL/6J mice at postnatal days (P) 0 and P14, prior to photoreceptor loss. Data analysis revealed no changes at P0 but significant differences in RPE and retina-specific transcripts at P14, suggesting a postnatal influence of the Mfrprd6 allele. A subset of these transcripts was validated by quantitative real-time PCR (qRT-PCR). In Mfrprd6 eyes, a significant 1.5- to 2.0-fold decrease was observed among transcripts of genes linked to retinal degeneration, including those involved in visual cycle (Rpe65, Lrat, Rgr), phototransduction (Pde6a, Guca1b, Rgs9), and photoreceptor disc morphogenesis (Rpgrip1 and Fscn2). Levels of RPE65 were significantly decreased by 2.0-fold. Transcripts of Prss56, a gene associated with angle-closure glaucoma, posterior microphthalmia and myopia, were increased in Mfrprd6 eyes by 17-fold. Validation by qRT-PCR indicated a 3.5-, 14- and 70-fold accumulation of Prss56 transcripts relative to controls at P7, P14 and P21, respectively. This trend was not observed in other RPE or photoreceptor mutant mouse models with similar disease progression, suggesting that Prss56 upregulation is a specific attribute of the disruption of Mfrp. Prss56 and Glul in situ hybridization directly identified Müller glia in the inner nuclear layer as the cell type expressing Prss56. In summary, the Mfrprd6 allele causes significant postnatal changes in transcript and protein levels in the retina and RPE. The link between Mfrp deficiency and Prss56 up-regulation, together with the genetic association of human MFRP or PRSS56 variants and ocular size, raises the possibility that these genes are part of a regulatory network influencing postnatal posterior eye development.

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RPE65 protein expression in RPE cells from B6 (C57BL/6J) and homozygous Mfrprd6 mice.(A) Western blot analysis of RPE65 protein extracted from RPE cells of B6 (C57BL/6J) and homozygous Mfrprd6 mice. There was a 2.0-fold decrease in RPE65 protein in Mfrprd6/Mfrprd6 (Lane 2) relative to the B6 control (Lane 1), whereas in Rpe65tvrm148/Rpe65tvrm148 mutant, RPE65 protein was undetected (Lane 3). β-Actin loading confirms equal protein loading in all lanes (1–3). (B) Quantitation of RPE65 protein in RPE cells of B6 (C57BL/6J) and Mfrprd6/Mfrprd6 mice. Student's T test was used to calculate statistical significance (* P<0.05 relative to B6 control).
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pone-0110299-g004: RPE65 protein expression in RPE cells from B6 (C57BL/6J) and homozygous Mfrprd6 mice.(A) Western blot analysis of RPE65 protein extracted from RPE cells of B6 (C57BL/6J) and homozygous Mfrprd6 mice. There was a 2.0-fold decrease in RPE65 protein in Mfrprd6/Mfrprd6 (Lane 2) relative to the B6 control (Lane 1), whereas in Rpe65tvrm148/Rpe65tvrm148 mutant, RPE65 protein was undetected (Lane 3). β-Actin loading confirms equal protein loading in all lanes (1–3). (B) Quantitation of RPE65 protein in RPE cells of B6 (C57BL/6J) and Mfrprd6/Mfrprd6 mice. Student's T test was used to calculate statistical significance (* P<0.05 relative to B6 control).

Mentions: The parallel decrease in retina- and RPE-specific transcripts revealed by transcript analysis raised the possibility that the Mfrprd6 mutation might diminish retinal health by reducing the levels of RPE visual cycle proteins, which are critical for photoreceptor maintenance [23]. To test this possibility, we examined levels of the Rpe65 gene product RPE65, a 65 kDa RPE-specific isomerohydrolase that is essential for producing 11-cis retinal from all-trans-retinyl esters in the visual cycle [23], [24]. Western blot analysis of RPE/choroid/sclera lysates revealed decreased levels of RPE65 protein in Mfrprd6 mice compared to C57BL/6J controls (Fig. 4A). An Rpe65 mutant (Rpe65tvrm124/Rpe65tvrm124) was used to control for antibody specificity. No RPE65 protein was detected in lysates from the Rpe65 mutant mice, whereas a 65 kDa protein was detected in all other samples (Fig. 4A), thus confirming that the detected band is RPE65. Quantitation of the blot indicated a significant 2.0-fold decrease of RPE65 in the Mfrprd6 mutant eyes (Fig. 4B).


Gene profiling of postnatal Mfrprd6 mutant eyes reveals differential accumulation of Prss56, visual cycle and phototransduction mRNAs.

Soundararajan R, Won J, Stearns TM, Charette JR, Hicks WL, Collin GB, Naggert JK, Krebs MP, Nishina PM - PLoS ONE (2014)

RPE65 protein expression in RPE cells from B6 (C57BL/6J) and homozygous Mfrprd6 mice.(A) Western blot analysis of RPE65 protein extracted from RPE cells of B6 (C57BL/6J) and homozygous Mfrprd6 mice. There was a 2.0-fold decrease in RPE65 protein in Mfrprd6/Mfrprd6 (Lane 2) relative to the B6 control (Lane 1), whereas in Rpe65tvrm148/Rpe65tvrm148 mutant, RPE65 protein was undetected (Lane 3). β-Actin loading confirms equal protein loading in all lanes (1–3). (B) Quantitation of RPE65 protein in RPE cells of B6 (C57BL/6J) and Mfrprd6/Mfrprd6 mice. Student's T test was used to calculate statistical significance (* P<0.05 relative to B6 control).
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pone-0110299-g004: RPE65 protein expression in RPE cells from B6 (C57BL/6J) and homozygous Mfrprd6 mice.(A) Western blot analysis of RPE65 protein extracted from RPE cells of B6 (C57BL/6J) and homozygous Mfrprd6 mice. There was a 2.0-fold decrease in RPE65 protein in Mfrprd6/Mfrprd6 (Lane 2) relative to the B6 control (Lane 1), whereas in Rpe65tvrm148/Rpe65tvrm148 mutant, RPE65 protein was undetected (Lane 3). β-Actin loading confirms equal protein loading in all lanes (1–3). (B) Quantitation of RPE65 protein in RPE cells of B6 (C57BL/6J) and Mfrprd6/Mfrprd6 mice. Student's T test was used to calculate statistical significance (* P<0.05 relative to B6 control).
Mentions: The parallel decrease in retina- and RPE-specific transcripts revealed by transcript analysis raised the possibility that the Mfrprd6 mutation might diminish retinal health by reducing the levels of RPE visual cycle proteins, which are critical for photoreceptor maintenance [23]. To test this possibility, we examined levels of the Rpe65 gene product RPE65, a 65 kDa RPE-specific isomerohydrolase that is essential for producing 11-cis retinal from all-trans-retinyl esters in the visual cycle [23], [24]. Western blot analysis of RPE/choroid/sclera lysates revealed decreased levels of RPE65 protein in Mfrprd6 mice compared to C57BL/6J controls (Fig. 4A). An Rpe65 mutant (Rpe65tvrm124/Rpe65tvrm124) was used to control for antibody specificity. No RPE65 protein was detected in lysates from the Rpe65 mutant mice, whereas a 65 kDa protein was detected in all other samples (Fig. 4A), thus confirming that the detected band is RPE65. Quantitation of the blot indicated a significant 2.0-fold decrease of RPE65 in the Mfrprd6 mutant eyes (Fig. 4B).

Bottom Line: In Mfrprd6 eyes, a significant 1.5- to 2.0-fold decrease was observed among transcripts of genes linked to retinal degeneration, including those involved in visual cycle (Rpe65, Lrat, Rgr), phototransduction (Pde6a, Guca1b, Rgs9), and photoreceptor disc morphogenesis (Rpgrip1 and Fscn2).Levels of RPE65 were significantly decreased by 2.0-fold.In summary, the Mfrprd6 allele causes significant postnatal changes in transcript and protein levels in the retina and RPE.

View Article: PubMed Central - PubMed

Affiliation: The Jackson Laboratory, Bar Harbor, Maine, United States of America.

ABSTRACT
Mutations in the membrane frizzled-related protein (MFRP/Mfrp) gene, specifically expressed in the retinal pigment epithelium (RPE) and ciliary body, cause nanophthalmia or posterior microphthalmia with retinitis pigmentosa in humans, and photoreceptor degeneration in mice. To better understand MFRP function, microarray analysis was performed on eyes of homozygous Mfrprd6 and C57BL/6J mice at postnatal days (P) 0 and P14, prior to photoreceptor loss. Data analysis revealed no changes at P0 but significant differences in RPE and retina-specific transcripts at P14, suggesting a postnatal influence of the Mfrprd6 allele. A subset of these transcripts was validated by quantitative real-time PCR (qRT-PCR). In Mfrprd6 eyes, a significant 1.5- to 2.0-fold decrease was observed among transcripts of genes linked to retinal degeneration, including those involved in visual cycle (Rpe65, Lrat, Rgr), phototransduction (Pde6a, Guca1b, Rgs9), and photoreceptor disc morphogenesis (Rpgrip1 and Fscn2). Levels of RPE65 were significantly decreased by 2.0-fold. Transcripts of Prss56, a gene associated with angle-closure glaucoma, posterior microphthalmia and myopia, were increased in Mfrprd6 eyes by 17-fold. Validation by qRT-PCR indicated a 3.5-, 14- and 70-fold accumulation of Prss56 transcripts relative to controls at P7, P14 and P21, respectively. This trend was not observed in other RPE or photoreceptor mutant mouse models with similar disease progression, suggesting that Prss56 upregulation is a specific attribute of the disruption of Mfrp. Prss56 and Glul in situ hybridization directly identified Müller glia in the inner nuclear layer as the cell type expressing Prss56. In summary, the Mfrprd6 allele causes significant postnatal changes in transcript and protein levels in the retina and RPE. The link between Mfrp deficiency and Prss56 up-regulation, together with the genetic association of human MFRP or PRSS56 variants and ocular size, raises the possibility that these genes are part of a regulatory network influencing postnatal posterior eye development.

Show MeSH
Related in: MedlinePlus