OFD1, as a Ciliary Protein, Exhibits Neuroprotective Function in Photoreceptor Degeneration Models

<div><p><i>Ofd1</i> is a newly identified causative gene for Retinitis pigmentosa (RP), a photoreceptor degenerative disease. This study aimed to examine Ofd1 localization in retina and further to investigate its function in photoreceptor degeneration models. Ofd1 localization in rat retina was examined using immunofluorescence. N-methyl-N-nitrosourea (MNU)-induced rats and Royal College of Surgeons (RCS) rats were used as photoreceptor degeneration models. The expression pattern of Ofd1, other ciliary associated genes and Wnt signaling pathway genes were examined in rat models. Furthermore, pEGFP-Ofd1-CDS and pSUPER-Ofd1-shRNA were constructed to overexpress and knockdown the expression level in 661W and R28 cells. MNU was also used to induce cell death. Cilia formation was observed using immunocytochemistry (ICC). Reactive oxygen species (ROS) were detected using the 2', 7'-Dichlorofluorescin diacetate (DCFH-DA) assay. Apoptosis genes expression was examined using qRT-PCR, Western blotting and fluorescence-activated cell sorting (FACS). Ofd1 localized to outer segments of rat retina photoreceptors. Ofd1 and other ciliary proteins expression levels increased from the 1^st and 4^th postnatal weeks and decreased until the 6^th week in the RCS rats, while their expression consistently decreased from the 1^st and 7^th day in the MNU rats. Moreover, Wnt signaling pathway proteins expression was significantly up-regulated in both rat models. Knockdown of Ofd1 expression resulted in a smaller population, shorter length of cell cilia, and lower cell viability. Ofd1 overexpression partially attenuated MNU toxic effects by reducing ROS levels and mitigating apoptosis. To the best of our knowledge, this is the first study demonstrating Ofd1 localization and its function in rat retina and in retinal degeneration rat models. Ofd1 plays a role in controlling photoreceptor cilium length and number. Importantly, it demonstrates a neuroprotective function by protecting the photoreceptor from oxidative stress and apoptosis. These data have expanded our understanding of Ofd1 function beyond cilia, and we concluded that ofd1 neuroprotection could be a potential treatment strategy in retina degeneration models.</p></div

Ofd1, ciliary associated genes and Wnt signaling pathway genes expression in RCS rat retina.

<p>RCS rats were sacrificed between 1 and 6 postnatal weeks. One-week-old (1w) RCS rats were used as the control. (A) <i>Ofd1</i> mRNA expression level increased between the 1st and 4th weeks, and decreased after the 4th week, as determined by qRT-PCR. N = 4. (B) Expression level of Ofd1, Lca5 and Follistatin in RCS rat retina determined using Western blotting analysis. (C) Quantified results of Ofd1, Lca5 and Follistatin protein levels in RCS rats by QuantityOne software. Ofd1 showed the same expression pattern at the mRNA level. Lca5 and Follistatin expression presented the same pattern as Ofd1. (D) Expression level as determined by qRT-PCR of ciliary associated genes <i>Lca5</i>, <i>Fam161a</i>, <i>Rpgrip1</i>, <i>Cep290</i> and Wnt signaling pathway genes: <i>Axin2</i> and <i>Follistatin</i>. N = 4. *P<0.05, **P<0.01. For quantification results, three replicates were performed but only one band for three representative samples per group was shown.</p

Ofd1, ciliary associated genes and Wnt signaling pathway genes expression in MNU induced retinal degeneration rat model retina.

<p>Five-week-old SD rats were injected with 50mg/kg body weight of MNU and saline, and then sacrificed on the 1 day, 3 days and 7 days post injection stages (N = 6). (A) MNU rat model was established as the ERG amplitude decreased after injection on day 1, day 3 and became flat after 7days, compared to normal SD rats. Single stimulus intense: 0.06325 (cd×s/m2); stimulus frequency: 0.5Hz. (B) Ofd1 mRNA expression in the MNU rat model was detected using qRT-PCR. Ofd1 expression decreased since the 1st day after injection and continuously decreased with time until the 7th day. (C) Protein level of Ofd1, Lca5 and Follistatin was determined using Western blotting analysis. (D) Quantified results of the protein levels of Ofd1, Lca5 and Follistatin in the MNU rat model using QuantityOne software. (E) mRNA level of ciliary associated genes and Wnt signaling pathway genes was determined using qRT-PCR. N = 6. *P<0.05, **P<0.01. For quantification results, three replicates were performed but only one band for three representative samples per group was shown.</p

Ofd1 localization in rat retina and expression in retinal cell lines.

<p>(A) Representative confocal images of endogenous OFD1 with rod photoreceptor marker Rhodopsin, cone photoreceptor marker PNA (Peanut Agglutinin), and cilium transition zone marker Tmem231, respectively. Ofd1 immunostaining partially overlapped with photoreceptor outer segments of the retina in 2-week postnatal SD rat. Negative control: immunostaining without primary antibody incubation. Magnification 600× under confocal microscopy. Scale bar: 50μm. (B) Ofd1 mRNA expression level in retina cell lines: ARPE-19, R28 and rMC-1detected by qRT-PCR. (C) Ofd1 protein expression level rMC-1 examined using Western blotting analysis. These results confirmed that Ofd1 was expressed in these cell lines. ARPE-19: human retinal pigment epithelial immortalized cell. R28: rat retinal progenitor neuronal cell. rMC-1: immortalized retinal Müller cell. For quantification results, three replicates were performed but only one band for three representative samples per group was shown.</p

Ofd1 expression levels in cells affect cilia number and length.

<p>(A) shRNA-mediated (pSUPER-EGFP1-shRNA-Ofd1) down-regulation of Ofd1 in R28 cells determined using qRT-PCR and Western blotting analysis. Knockdown efficiency was 52%. *P<0.05. (B–C) Ciliated cell number and cilia length decreased after shRNA mediated down-regulation of Ofd1 in R28 cells (shRNA), compared with pSUPER-scramble shRNA (without EGFP) treated cells (control). *P<0.05. Unit: um. For quantification results, three replicates were performed but only one band for three representative samples per group was shown. (D) Representative image of Acetylated-α-tubulin immunostaining results. Acetylated-α-tubulin-cy3, marker of cilia axoneme, was used to demonstrate cilia in R28 cells. Cells were transfected with pSUPER-EGFP1-shRNA-Ofd1 for 48 h (shRNA), or pSUPER-scramble shRNA construct (without EGFP) as control. Magnification 600× under microscopy. Scale bar: 50μm. Enlarged: the magnified image of representative part in white box. White arrow: Acetylated-α-tubulin-cy3 staining presented cilia.</p

Ofd1 protection on photoreceptor via decreasing apoptosis.

<p>(A) Western blotting analysis to detect protein levels of Bax, Caspase3 and Bcl-2 in 661W cell. 661W cells with Ofd1 over-expression had lower expression of Bax (nearly half), lower Capsase3 expression (approximately 20% lower), and higher Bcl-2 expression (2-fold). (B) qRT-PCR to detect mRNA levels of apoptotic genes (<i>Bax</i>, <i>Caspase3</i> and <i>Bcl-2</i>) in 661W cell. (C-D) Cells apoptosis was analyzed flow cytometer in 661W cell. Q3: the percentage of early apoptotic cells. Q1: the percentage of late apoptotic cells. N: non-treated cells. MNU: MNU 500 ug/ml treated 661W for 10 mins. MNU+Empty Vector: after 661W transfected with pEGFP vector for 48h, MNU 500 ug/ml treated for 10 mins. MNU+Ofd1 CDS: after 661W transfected with pEGFP-Ofd1-CDS for 48h, MNU 500 ug/ml treated for 10 mins. (C) is the statistical result of (D), which shows percentage of apoptotic cells in 661W. Cell percentage undergoing apoptosis significantly increased after MNU treatment (approximately 35% increase), but returned back to normal levels after Ofd1 overexpression. For quantification results, three replicates were performed but only one band for three representative samples per group was shown.</p

Ofd1 protection of photoreceptors from oxidative stress via decreasing ROS production.

<p>(A) The intracellular oxidant DCFH of MNU-induced ROS in 661W cells observed by green fluorescence using Leica fluorescence microscope (with monochromatic CCD), and measured using fluorometer at wavelength of 488/525 nm. ROS production was stimulated 10 mins by 500 ug/ml MNU or ROSup as positive control. N: non-treated cells. MNU: MNU 500 ug/ml treated 661W for 10 mins. MNU+Empty Vector: after 661W transfected with pEGFP vector for 48h, MNU 500 ug/ml treated for 10 mins. MNU+Ofd1 CDS: after 661W transfected with pEGFP-Ofd1-CDS for 48h, MNU 500 ug/ml treated for 10 mins. Scale bar: 50μm. (B) Cell viability and proliferation detection in 661W cell using the MTT assay. Ofd1 expression was up- or down-regulated by plasmid transfection by Lipofectamine 2000. Ofd1-CDS was used to overexpress Ofd1, and Ofd1-shRNA was used to inhibit expression. After transfection for 36 h, 500 ug/ml MNU was added and incubated for additional 12 h. NC: normal control. * p<0.05. n = 6. (C) These results showed that the SOD and CAT activities decreased by 40% and 30% in MNU-treated cells (MNU), but was partially attenuated by Ofd1 overexpression (Ofd1-CDS+MNU). The difference between the MNU group and Ofd1-CDS+MNU group was significant. *: p < 0.05. n = 4. (D) Antioxidant enzymes amount or activity detection in RCS rat at 6 weeks (RCS) compared to age-matched normal SD rat as the control (NC), MNU-induced rat at 7 days (MNU) compared to samples obtained from age-matched no- treated rats as the control (NC). The two NC are the same, 6-week normal SD rat. * p<0.05. n = 4.</p