Rod-derived Cone Viability Factor-2 is a novel bifunctional-thioredoxin-like protein with therapeutic potential

<p>Abstract</p> <p>Background</p> <p>Cone degeneration is the hallmark of the inherited retinal disease retinitis pigmentosa. We have previously identified a trophic factor "Rod-derived Cone Viability Factor (RdCVF) that is secreted by rods and promote cone viability in a mouse model of the disease.</p> <p>Results</p> <p>Here we report the bioinformatic identification and the experimental analysis of RdCVF2, a second trophic factor belonging to the Rod-derived Cone Viability Factor family. The mouse RdCVF gene is known to be bifunctional, encoding both a long thioredoxin-like isoform (RdCVF-L) and a short isoform with trophic cone photoreceptor viability activity (RdCVF-S). RdCVF2 shares many similarities with RdCVF in terms of gene structure, expression in a rod-dependent manner and protein 3D structure. Furthermore, like RdCVF, the RdCVF2 short isoform exhibits cone rescue activity that is independent of its putative thiol-oxydoreductase activity.</p> <p>Conclusion</p> <p>Taken together, these findings define a new family of bifunctional genes which are: expressed in vertebrate retina, encode trophic cone viability factors, and have major therapeutic potential for human retinal neurodegenerative diseases such as <it>retinitis pigmentosa</it>.</p

Identification of an Alternative Splicing Product of the Otx2 Gene Expressed in the Neural Retina and Retinal Pigmented Epithelial Cells

International audienceTo investigate the complexity of alternative splicing in the retina, we sequenced and analyzed a total of 115,706 clones from normalized cDNA libraries from mouse neural retina (66,217) and rat retinal pigmented epithelium (49,489). Based upon clustering the cDNAs and mapping them with their respective genomes, the estimated numbers of genes were 9,134 for the mouse neural retina and 12,050 for the rat retinal pigmented epithelium libraries. This unique collection of retinal of messenger RNAs is maintained and accessible through a web-base server to the whole community of retinal biologists for further functional characterization. The analysis revealed 3,248 and 3,202 alternative splice events for mouse neural retina and rat retinal pigmented epithelium, respectively. We focused on transcription factors involved in vision. Among the six candidates suitable for functional analysis, we selected Otx2S, a novel variant of the Otx2 gene with a deletion within the homeodomain sequence. Otx2S is expressed in both the neural retina and retinal pigmented epithelium, and encodes a protein that is targeted to the nucleus. OTX2S exerts transdominant activity on the tyrosinase promoter when tested in the physiological environment of primary RPE cells. By overexpressing OTX2S in primary RPE cells using an adeno associated viral vector, we identified 10 genes whose expression is positively regulated by OTX2S. We find that OTX2S is able to bind to the chromatin at the promoter of the retinal dehydrogenase 10 (RDH10) gene

Rod-derived Cone Viability Factor-2 is a novel bifunctional-thioredoxin-like protein with therapeutic potential-1

<p><b>Copyright information:</b></p><p>Taken from "Rod-derived Cone Viability Factor-2 is a novel bifunctional-thioredoxin-like protein with therapeutic potential"</p><p>http://www.biomedcentral.com/1471-2199/8/74</p><p>BMC Molecular Biology 2007;8():74-74.</p><p>Published online 31 Aug 2007</p><p>PMCID:PMC2064930.</p><p></p> and the existing and predicted RdCVF and RdCVF2 proteins. The name, organism and accession number (in brackets) of each protein sequence are given (left). Identical (white text on black) small (A, D, G, P, S, T ; white text on green) hydrophobic (A, C, F, G, I, L, M, S, T, V, W, Y ; black text on yellow) polar (D, E, H, K, N, Q, R, S ; blue text) and charged (D, E, K, R ; white text on red) conserved residues are shown according to a conservation threshold of 85%. A consensus sequence is given below the multiple alignment in which s, h, p and c correspond to small, hydrophobic, polar and charged residues respectively. The secondary structures (β sheet and α helix) of the Crithidia fasciculata tryparedoxin I structure (1EWX) are given below the consensus sequence. The blue dashed rectangles indicate the three RdCVF(2) specific insertions. The green dashed rectangle shows the "cap" region absent in RdCVF(2)-S. The position of the human thioredoxin cleavage product (TRX80) is indicated (red triangle). Panel b displays the structure of the TRYX-I (1EWX) (left) mouse RdCVF-L (center) and mouse RdCVF2-L (right) models. Regions of TRYX-I backbone conserved in RdCVF(2)-L are colored in red. The "cap" region and the three specific insertions are depicted in green and blue respectively. The putative catalytic site (CXXC) is shown in yellow with a space-filling representation

Relative expression in cultured RPE with AAV-OTX2S versus AAV-GFP control transduced cells.

<p>Relative expression in cultured RPE with AAV-OTX2S versus AAV-GFP control transduced cells.</p

Protein expression and cellular localization of OTX2 variants.

<p>(A) Protein expression of OTX2, OTX2L and OTX2S was detected by western blot analysis using polyclonal anti-OTX2 antibodies after transfection of HEK293 cells. HEK293 cells do not express OTX2 protein. The size of the proteins detected is 35, 36 and 33 kDa for OTX2, OTX2L and OTX2S respectively. HEK293 cells transfected with the empty vector, pcDNA3.1, were used as negative control. (B) Quantification of the expression performed using ImageJ. ACTB was used as control for normalization. OTX2L and OTX2S expression is 91% (<i>P</i> = 0.0111) and 24% (<i>P</i> < 0.0001) respectively of OTX2 expression (100%). Error bars represent standard deviation of the mean. (C) Nuclear localization of the OTX2 isoforms (red) in the nucleus of MSR 293 transfected cells using immunocytochemistry. Blue: DAPI. Scale bar 20 μm.</p

Gene expression profiles in primary RPE cells overexpressing OTX2S splicing variant.

<p>(A) Morphology of the primary pig RPE cells before infection. (B) Expression of GFP by RPE cells one week after infection of AAV2.1-GFP. (C) Expression of <i>Otx2S</i> in transduced pig primary RPE cells. (D) Western blotting analysis of pig RPE cells infected with recombinant AAV2.1 vectors as indicated. The asterisk points to OTX2S. (E) Quantitative RT-PCR for the indicated genes using mRNA isolated from primary RPE cells infected with adeno associated viruses (AAV2.1) expressing the <i>Otx2S</i> splice variant. AAV2.1 expressing the GFP was used as the non treated control (C). GAPDH was used as the internal control for normalization, the expression of which does not change between conditions. The expression of each gene was normalized by the expression in the GFP infected RPE cells and it is expressed as 2-ΔΔCt method. Each experiment was performed in triplicates.*, P < 0.05.</p

Normalization of mice and rat libraries.

<p>Normalization of mice and rat libraries.</p

OTX2S exerts a transdominant negative effect on the tyrosinase promoter.

<p>(A) The absence of effect of <i>Otx2S</i> on tyrosinase promoter in HEK293 co-transfected cells. (B) <i>Otx2S</i> has a transdominant activity of tyrosinase promoter on <i>Otx2</i> and <i>Otx2L</i> activity in primary RPE co-transfected cells. The inhibitory effect of <i>Otx2S</i> was found to be higher in <i>Otx2L</i> co-transfected assays. Each transfection was repeated three times with quadruplicates wells analyzed. Error bars represent standard deviation of the mean. *** <i>P</i> ≤ 0.001 (Two-way ANOVA Dunnett test).</p

Synergistic activation of the tyrosinase promoter by OTX2 isoforms and MITF.

<p>(A) Luciferase assays performed using mouse <i>Tyr</i> promoter construct -2226/+63 on HEK293 cells. (B) Luciferase assays performed using the mouse <i>Tyr</i> promoter construct on pig primary RPE cells. (C) Luciferase assays performed using mouse <i>Tyr</i> promoter construct on HEK293 cells using co-transfection with MITF. (D) Luciferase assays performed using mouse <i>Tyr</i> promoter construct on pig primary RPE cells using co-transfection with MITF. Each transfection was repeated three times with quadruplicates. Error bars represent Standard deviation of the mean. * <i>P</i> ≤ 0.05, ** <i>P</i> ≤ 0.01, *** <i>P</i> ≤ 0.001 (Two-way ANOVA Dunnett test).</p

Analysis of the protein sequence and the expression of <i>Otx2</i> splicing variants in retinal pigment epithelium and neural retina.

<p>(A) Sequence alignment of the protein region corresponding to the homeodomain of <i>Otx2</i> splicing variants found in the rat RPE library. (B, C, D) RT-PCR analysis was performed using the cDNA from RPE and NR of Long Evans rats. B) Primers amplifying all <i>Otx2</i> splice variants (<i>Otx2</i>, <i>Otx2L</i> and <i>Otx2S</i>: 247, 271 and 208 bp respectively). The RT-PCR product corresponding to <i>Otx2S</i> was validated by sequencing after gel extraction. (C) Touchdown PCR analysis using isoform-specific primers for the <i>Otx2</i>, <i>Otx2L</i> and <i>Otx2S</i>. (D) The relative expression of <i>Otx2</i> splicing variants was determined using specific primers. No significant contamination between the two tissues was observed by <i>Rpe65</i> and <i>Rho</i> expression. (E) Quantitative RT-PCR analysis of <i>Otx2</i> and <i>Otx2S</i> expression in a rat model of retinal detachment. The relative expression is expressed as 2^-ΔCt. <i>Actb</i> was used as the internal control for normalization.</p