Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Novel molecular mechanisms for Prph2-associated pattern dystrophy

doi: 10.1096/fj.201901888R

Figure Lengend Snippet: C213Y Prph2 is expressed at very low levels. A, The C213Y mutation was introduced into exon 2 of the Prph2 gene in order to drive properly regulated expression of the C213Y Prph2 allele. B, qRT-PCR was performed on P30 retinal cDNAs from WT and Prph2C/C mice using primers that recognize both WT and C213Y Prph2 transcripts. Results were compared to cDNA isolated from liver as control. C-D, Levels of Prph2 (C) and Rom1 (D) proteins from retinal extracts of the indicated genotypes were analyzed. Band intensities were normalized to their actin levels and plotted as percent of the WT in bar graphs. E, Immunoblot of untreated and PNGase treated retinal extracts from WT, Prph2N/N, and Prph2C/C probed for Prph2 and Rom1. Prph2N/N is a knockin model for the non-glycosylated form of Prph2 and is used as a control. Each experiment contains N = 4–8 independent retinas per genotype. Data are plotted as mean ± SEM. * P < .05, **P < .01, ***P < .001, and ****P < .0001 by one-way ANOVA with Tukey’s post-hoc comparison

Article Snippet: The C213Y- Prph2 knockin mice were generated by inGenious Targeting Laboratory, Inc (Ronkonkoma, NY, USA).

Techniques: Mutagenesis, Expressing, Quantitative RT-PCR, Isolation, Control, Western Blot, Knock-In, Comparison

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Novel molecular mechanisms for Prph2-associated pattern dystrophy

doi: 10.1096/fj.201901888R

Figure Lengend Snippet: Antibodies used for immunofluorescence (IF), immunogold (IGL), and western blotting (WB)

Article Snippet: The C213Y- Prph2 knockin mice were generated by inGenious Targeting Laboratory, Inc (Ronkonkoma, NY, USA).

Techniques: Immunofluorescence, Western Blot, CRAfT Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Novel molecular mechanisms for Prph2-associated pattern dystrophy

doi: 10.1096/fj.201901888R

Figure Lengend Snippet: Primers for qRT-PCR

Article Snippet: The C213Y- Prph2 knockin mice were generated by inGenious Targeting Laboratory, Inc (Ronkonkoma, NY, USA).

Techniques: Sequencing

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Novel molecular mechanisms for Prph2-associated pattern dystrophy

doi: 10.1096/fj.201901888R

Figure Lengend Snippet: C213Y Prph2 leads to abnormalities in Prph2/Rom1 oligomerization. A, Reciprocal immunoprecipitation with anti-Prph2 (top panel) and anti-Rom1 (bottom panel) antibodies was performed on retinal extracts from P30 WT and Prph2C/C. B, Nonreducing SDS-PAGE/immunoblots were performed on whole retinal extract in the presence of NEM-containing buffer and were probed with antibodies against Prph2 (left), Rom1 (right), and actin (bottom) as a loading control. C-D, To analyze Prph2/Rom1 complexes, three independent (n = 3) retinal extracts were prepared from each of the indicated genotypes and run on a continuous 5% to 20% nonreducing sucrose gradient. Fractions were collected and analyzed using reducing SDS-PAGE and immunoblotting using antibodies specific for Prph2 (C) and Rom1 (D). The position of standard molecular weight markers is marked above the relevant fraction as demonstrated previously.26 Graphs plot mean (±SEM) amount of Prph2/Rom1 in each fraction as a % of total Prph2/Rom1. Arrows in (D) highlight small quantity of higher order Rom1 complexes in mutant retinas

Article Snippet: The C213Y- Prph2 knockin mice were generated by inGenious Targeting Laboratory, Inc (Ronkonkoma, NY, USA).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Control, Molecular Weight, Mutagenesis

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Novel molecular mechanisms for Prph2-associated pattern dystrophy

doi: 10.1096/fj.201901888R

Figure Lengend Snippet: Heterozygous C213Y Prph2 exerts dominant rod functional defect. Full-field ERGs were recorded under scotopic conditions at P30, P180, and P365. Shown are representative ERG wave forms from the indicated genotypes at P30 (A). B-D, Maximum scotopic a- and b-waves are plotted for P30 (B) P180 (C), and P365 (D). Data are presented as mean ± SEM from 5–7 mice per genotype. *P < .05, **P < .01, ***P < .001, and ****P < .0001 in one-way ANOVA with ANOVA with Tukey’s post-hoc comparison

Article Snippet: The C213Y- Prph2 knockin mice were generated by inGenious Targeting Laboratory, Inc (Ronkonkoma, NY, USA).

Techniques: Functional Assay, Comparison

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Novel molecular mechanisms for Prph2-associated pattern dystrophy

doi: 10.1096/fj.201901888R

Figure Lengend Snippet: C213Y Prph2 exerts dominant cone functional defect. Full-field ERGs were recorded under photopic conditions (representative waveforms shown in A). B, Maximum amplitudes are shown at P30 recorded in response to white, green, and UV light. Maximum photopic b-wave amplitudes recorded in response to white light at P180 (C) and P365 (D) are plotted. Data are presented as mean ± SEM from 5 to 7 mice per genotype. *P < .05, **P < .01, ***P < .001, and ****P < .0001 in one-way ANOVA with ANOVA with Tukey’s post-hoc comparison

Article Snippet: The C213Y- Prph2 knockin mice were generated by inGenious Targeting Laboratory, Inc (Ronkonkoma, NY, USA).

Techniques: Functional Assay, Comparison

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Novel molecular mechanisms for Prph2-associated pattern dystrophy

doi: 10.1096/fj.201901888R

Figure Lengend Snippet: Fundus photographs of Prph2C/+ show an increase in retinal flecking at P180. Representative fundus images (top) and corresponding fluorescein angiograms (bottom) from the indicated genotypes were performed at P30 (A), P180 (B), and P365 (C). Black arrows denote flecking phenotype found in the Prph2C/+ as well as the Prph2−/−, and Prph2C/C. White arrows point to splotching, likely due to severe photoreceptor degeneration which occurs at later ages. Asterisks denote examples of splotches that align with the leaky vasculature observed in fluorescein angiograms. N = 6–8 eyes/group

Article Snippet: The C213Y- Prph2 knockin mice were generated by inGenious Targeting Laboratory, Inc (Ronkonkoma, NY, USA).

Techniques:

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Novel molecular mechanisms for Prph2-associated pattern dystrophy

doi: 10.1096/fj.201901888R

Figure Lengend Snippet: C213Y Prph2 is unable to support proper OS ultrastructure and leads to photoreceptor degeneration. Representative light microscopy (A) and transmission electron microscopy (B) from retinas of the indicated genotypes performed at P30. Arrows show highly malformed OSs in the Prph2C/C. C, ONL thickness and OS length were measured from the superior and inferior central retina and plotted as a mean ± SEM. D, Rhodopsin protein levels were assessed by immunoblot analysis (top) and quantified densitometrically on non-saturated blots. Graph shows rhodopsin levels normalized to actin and plotted as a % of WT. E, Mean rhodopsin levels are plotted as a ratio to ONL thickness (top) and OS length (bottom). F, Low (upper panels) and high (lower panels) magnification electron microscopy of the indicated genotypes showing improvements on the overall OS structures in the C213Y Prph2 retina in comparison with Prph2+/− at P180. Graphs show mean ± SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001 by one-way ANOVA (D) or two-way ANOVA (C) with Tukey’s post-hoc comparison. N = 3–4 retinas/genotype for histological analyses and 4–8 retinas per genotype for immunoblots. Scale bars: 20 μm (A), 10 μm (F-top), 2 μm (B, F-bottom). IS, inner segment; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium

Article Snippet: The C213Y- Prph2 knockin mice were generated by inGenious Targeting Laboratory, Inc (Ronkonkoma, NY, USA).

Techniques: Light Microscopy, Transmission Assay, Electron Microscopy, Western Blot, Comparison

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Novel molecular mechanisms for Prph2-associated pattern dystrophy

doi: 10.1096/fj.201901888R

Figure Lengend Snippet: C213Y Prph2 localizes abnormally to the inner segment and outer nuclear layer. A-H, Retinal sections from the indicated genotypes were immunofluorescently co-labeled with Prph2 (green) and Rom1, rhodopsin, S-opsin, and M-opsin (red) at P30. Nuclei in all sections were counterstained with DAPI (blue). White arrows show accumulation of Prph2 in the ONL and IS. Yellow arrows show mislocalization of rhodopsin and S-opsin in the ONL and IS. Red arrows show proper localization of Rom1 to the OS layer in the Prph2C/C. Panels A-H were captured at 63× with E-H showing enlarged regions from A to D. I, Shown is electron microscopy of retinal sections immunogold labeled for Prph2, Rom1, rhodopsin, S-opsin, and M-opsin at P30. Insets in all images show higher magnifications of the marked areas with black arrowheads. Scale bars: 10 μM (A-H), 500 nm (I). IS, inner segments; ONL, outer nuclear layer; OS, outer segments

Article Snippet: The C213Y- Prph2 knockin mice were generated by inGenious Targeting Laboratory, Inc (Ronkonkoma, NY, USA).

Techniques: Labeling, Electron Microscopy

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Novel molecular mechanisms for Prph2-associated pattern dystrophy

doi: 10.1096/fj.201901888R

Figure Lengend Snippet: WT Prph2 supplementation improves cellular phenotypes in the Prph2C/+. A, Shown are immunoblots and associated densitometric analysis of Prph2, Rom1, and rhodopsin relative to actin as a loading control from retinal extracts collected at P30. B, P30 retinal sections were immunofluorescently labeled for Prph2 (red) and rhodopsin or S-opsin (green). Nuclei are counterstained with DAPI (blue). Prph2 and S-opsin are mislocalized to the inner segment and outer nuclear layer of Prph2C/+ (white arrows) but properly localized to the OS in NMP/Prph2C/+ retinas. Immunofluorescence experiments were repeated three times using sections from 3–5 different animals. C, Light microscopy (upper) and electron microscopy (lower) taken from P30 retinas of the indicated genotypes. Scale bars: 20 μm (B, C-top) and 500 nm (C-bottom). INL, inner nuclear layer; IS, inner segment; ONL, outer nuclear layer; OS, outer segment. *P < .05, **P < .01, and ***P < .001 by one-way ANOVA with Tukey’s post-hoc comparison

Article Snippet: The C213Y- Prph2 knockin mice were generated by inGenious Targeting Laboratory, Inc (Ronkonkoma, NY, USA).

Techniques: Western Blot, Control, Labeling, Immunofluorescence, Light Microscopy, Electron Microscopy, Comparison

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Novel molecular mechanisms for Prph2-associated pattern dystrophy

doi: 10.1096/fj.201901888R

Figure Lengend Snippet: WT Prph2 supplementation does not rescue rod or cone function in the Prph2C/+. A, Full-field ERGs were recorded under scotopic or photopic conditions at P30, P180, and P365 from the indicated genotypes. Plotted are mean (±SEM) maximum scotopic a-, scotopic b-, and photopic b- wave amplitudes. N = 5–15 animals/genotype/age. Each symbol represents a different pairwise comparison: * WT vs. Prph2+/−, # WT vs. Prph2C/+, ^ WT vs. NMP/Prph2C/+, v Prph2+/− vs. Prph2C/+, + Prph2+/− vs. NMP/Prph2C/+, and $ Prph2C/+ vs. NMP/Prph2C/+. One symbol P < .05, two symbols P < .01, three symbols P < .001 and four symbols P < .0001 by one-way ANOVA with Tukey’s post-hoc comparison. B, Shown are representative fundus images (top) and fluorescein angiograms (bottom) from animals of the indicated genotypes captured at P180

Article Snippet: The C213Y- Prph2 knockin mice were generated by inGenious Targeting Laboratory, Inc (Ronkonkoma, NY, USA).

Techniques: Comparison

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Novel molecular mechanisms for Prph2-associated pattern dystrophy

doi: 10.1096/fj.201901888R

Figure Lengend Snippet: Cysteines are essential for the organization of the Prph2 D2 loop. Shown is a diagram highlighting the seven conserved D2 loop cysteines in Prph2 and their position with regard to the location of the Prph2/Rom1 interaction domains

Article Snippet: The C213Y- Prph2 knockin mice were generated by inGenious Targeting Laboratory, Inc (Ronkonkoma, NY, USA).

Techniques: