Olaparib significantly delays photoreceptor loss in a model for hereditary retinal degeneration

Abstract

The enzyme poly-ADP-ribose-polymerase (PARP) mediates DNA-repair and rearrangements of the nuclear chromatin. Generally, PARP activity is thought to promote cell survival and in recent years a number of PARP inhibitors have been clinically developed for cancer treatment. Paradoxically, PARP activity is also connected to many diseases including the untreatable blinding disease Retinitis Pigmentosa (RP), where PARP activity appears to drive the pathogenesis of photoreceptor loss. We tested the efficacy of three different PARP inhibitors to prevent photoreceptor loss in the rd1 mouse model for RP. In retinal explant cultures in vitro, olaparib had strong and long-lasting photoreceptor neuroprotective capacities. We demonstrated target engagement by showing that olaparib reduced photoreceptor accumulation of poly-ADP-ribosylated proteins. Remarkably, olaparib also reduced accumulation of cyclic-guanosine-monophosphate (cGMP), a characteristic marker for photoreceptor degeneration. Moreover, intravitreal injection of olaparib in rd1 animals diminished PARP activity and increased photoreceptor survival, confirming in vivo neuroprotection. This study affirms the role of PARP in inherited retinal degeneration and for the first time shows that a clinically approved PARP inhibitor can prevent photoreceptor degeneration in an RP model. The wealth of human clinical data available for olaparib highlights its strong potential for a rapid clinical translation into a novel RP treatment.

Figure 1. Olaparib rescues rd1 photoreceptors in short-term retinal explant cultures.

(a) Immunohistochemical staining revealed a dose-dependent effect of olaparib treatment with 100 nM as the most protective concentration and toxic effects of high concentrations. (b) Quantification of photoreceptor rows. (c) Quantification of the percentage of TUNEL positive cells. Bar graphs represent means ± SEM. n(wt, untreated) = 6; n(wt, 0.1 μM olaparib) = 6; n(rd1, untreated) = 9; n(rd1, 0.01 μM olaparib) = 5; n(rd1, 0.1 μM olaparib) = 9; n(rd1, 1 μM olaparib) = 7; n(rd1, 10 μM olaparib) = 7; n(rd1, 20 μM olaparib) = 4; n(rd1, 50 μM olaparib) = 6. **p < 0.01 by Kruskal-Wallis test for multiple analysis; scale bar is 20 μm.

Figure 2. Effect of PARP inhibition with olaparib on PARylation and cGMP levels in rd1 retinal cultures.

(a,b) The higher the concentration of olaparib, the smaller was the percentage of PAR positive cells. (c) Western blot analysis confirmed the previously reported strong PARylation difference between rd1 and wildtype (wt) retinas. Furthermore, there was a strong reduction of PARylation in rd1 cultures treated with 100 nM olaparib compared to control cultures. (d) Immunohistochemical stainings showed an increase of cGMP level in rd1 compared to wt. (e) Quantification of the cGMP signal revealed a significant reduction due to treatment with olaparib. Bar graphs represent means ± SEM. n(wt, untreated) = 6; n(wt, 0.1 μM olaparib) = 6; n(rd1, untreated) = 9; n(rd1, 0.01 μM olaparib) = 5; n(rd1, 0.1 μM olaparib) = 9; n(rd1, 1 μM olaparib) = 7; n(rd1, 10 μM olaparib) = 7; n(rd1, 20 μM olaparib) = 4; n(rd1, 50 μM olaparib) = 6; n(WB, in vivo, rd1) = 1; n(WB, in vivo, wt) = 1; n(WB, in vitro, rd1, untreated) = 3; n(WB, in vitro, rd1, olaparib) = 3. **p < 0.01 by Kruskal-Wallis test for multiple analysis; scale bars in (a and d) are 20 μm.

Figure 3. Long-term protective effects of olaparib treatment in rd1^TN-XL retinal cultures at P17.

(a) Immunohistochemical staining, at P17, showed improved photoreceptor viability in 0.1 μM olaparib treated cultures, when compared to untreated (untr.) control. (b) The number of photoreceptor rows was significantly increased after treatment. (c) There were less TUNEL positive cells in olaparib-treated cultures. (d) Cone density (EGFP signal) remained unchanged after treatment. Bar graphs represent means ± SEM. n(rd1, untreated) = 5; n(rd1, 0.1 μM olaparib) = 5. **p < 0.01 by two-sample Komolgorov-Smirnov test; scale bar is 20 μm.

Figure 4. Olaparib does not affect increased DNA methylation.

(a) Co-localization of TUNEL, 5mC, and 5hmC on rd1 retina at P13. (b) Of the TUNEL positive cells (n = 69), about half were also 5mC positive and nearly all cells were 5hmC positive. All 5mC positive cells (n = 29) were also TUNEL positive, whereas of all 5hmC positive cells (n = 57), there was a small fraction (n = 6) that did not show TUNEL signal. (c) Quantification of co-localization, in rows, e.g. of all TUNEL positive cells 50.6 ± 11.4 were 5mC and 72.2 ± 4.7 were 5hmC positive. (d) Immunohistochemical staining showed an increase of both 5mC and 5hmC levels in rd1 retinal cultures compared to wildtype (wt). (e) Quantification of histological methylation signal. In rd1 cultures there was no significant reduction after treatment with olaparib (5mC: p = 0.3517; 5hmC: p = 0.7858). Bar graphs represent means ± SEM. n(rd1 P13) = 4; n(wt, untreated) = 6; n(wt, olaparib) = 6; n(rd1, untreated) = 10; n(rd1, olaparib) = 8. Tested by two-sample Komolgorov-Smirnov test. Scale bars in (a and d) are 20 μm.

Figure 5. Olaparib protects rd1 photoreceptors in vivo.

After a single intravitreal olaparib injection at P11 rd1 animals where analyzed for cell death (TUNEL assay) and photoreceptor survival at P13 and P15. (a) Retinas from sham-injected rd1 animals display a high number of dying, TUNEL positive cells in the ONL. At P13, in treated animals cell death is reduced, an effect that is still apparent at P15. Note the decrease in the overall size of the rd1 ONL and the number of photoreceptor rows at P15. (b) A subset of rd1 photoreceptors showed a strong immunoreactivity for PARylated proteins, their numbers appeared lower in P13 and P15 treated animals. (c) Top panel: Quantification of TUNEL positive cells in untreated vs. treated animals at P13 and P15. At P13 there were significantly less photoreceptors dying in treated rd1 retina. Bottom panel: Quantification of PAR positive cells in untreated vs. treated animals at P13 and P15. (d) Quantitative analysis of photoreceptor survival along the dorso-ventral axis. While untreated P15 rd1 retina displayed 3-6 photoreceptor rows, olaparib treated rd1 animals had up to two rows more photoreceptors in the mid-periphery, dorsal retina. n(rd1, sham-injected P13) = 6; n(rd1, treated P13) = 5; n(rd1, sham-injected P15) = 4; n(rd1, untreated P15) = 5. Tested by unpaired, two-tailed Student’s t-test. Scale bars in (a and b) are 50 μm.