NADPH oxidase plays a central role in cone cell death in retinitis pigmentosa

Abstract

Retinitis pigmentosa (RP) is a collection of diseases in which rod photoreceptors die from a variety of mutations. After rods die, the level of tissue oxygen in the outer retina becomes elevated and there is progressive oxidative damage to cones that ultimately triggers apoptosis. In this study, we investigated the hypothesis that NADPH oxidase (Nox) and/or xanthine oxidase serve as critical intermediaries between increased tissue oxygen and the generation of excessive reactive oxygen species that cause oxidative damage to cones. Apocynin, a blocker of Nox, but not allopurinol, a blocker of xanthine oxidase, markedly reduced the superoxide radicals visualized by hydroethidine in the outer retina in the retinal degeneration-1 (rd1(+/+)) model of RP. Compared to rd1(+/+) mice treated with vehicle, those treated with apocynin, but not those treated with allopurinol, had significantly less oxidative damage in the retina measured by ELISA for carbonyl adducts. Apocynin-treated, but not allopurinol-treated, rd1(+/+) mice had preservation of cone cell density, increased mRNA levels for m- and s-cone opsin, and increased mean photopic b-wave amplitude. In Q344ter mice, a model of dominant RP in which mutant rhodopsin is expressed, apocynin treatment preserved photopic electroretinogram b-wave amplitude compared to vehicle-treated controls. These data indicate that Nox, but not xanthine oxidase, plays a critical role in generation of the oxidative stress that leads to cone cell death in RP and inhibition of Nox provides a new treatment strategy.

Figures

Figure 1. Apocynin, an inhibitor of NADPH oxidase, reduces superoxide radicals in the retinas of rd1+/+ mice

Rd1+/+ mice were given daily intraperitoneal injections of vehicle or vehicle containing 10mg/kg apocynin between postnatal day (P) 11 and P14. At P14, wild type mice, untreated rd1 mice or those treated with vehicle or apocynin (n=4 for each) were given two intraperitoneal injections of 20 mg/kg hydroethidine thirty minutes apart and after 18 hours they were euthanized and ocular sections were examined by confocal microscopy as described in Methods. There was minimal fluorescence in the retinas of wild type mice (A–C), strong fluorescence primarily in the remaining outer nuclear layer of the retinas of rd1 mice (D–F) and vehicle treated rd1+/+ mice (G–I), and minimal fluorescence in the retinas of apocynin treated rd1+/+ mice (J–L) or rd1+/+ mice that were not injected with hydroethidine (M–O). This demonstrates that NADPH contributes to the accumulation of superoxide radicals that occurs in the outer retina of rd1+/+ mice after rods have degenerated. Scale bar=50 µm

Figure 2. Apocynin significantly reduces carbonyl content in the retinas of postnatal day (P) 25 rd1+/+ mice

Starting at P11, rd1+/+ mice were given daily intraperitoneal injections of vehicle or vehicle containing apocynin. Mice were euthanized at P25 and protein carbonyl content was measured by ELISA of retinal homogenates as described in Methods. The mean (±SEM) carbonyl content per mg retinal protein was significantly greater in vehicle-treated rd1+/+ mice compared to those treated with apocynin (*p<0.001 by unpaired Student’s t-test).

Figure 3. Apocynin promotes cone survival in rd1+/+ mice

Starting at P11, rd1+/+ mice were given daily intraperitoneal injections of vehicle or vehicle containing apocynin. Mice were euthanized at P25 and retinas were used for real time RT-PCR or stained with peanut agglutinin (PNA) and whole mounted as described in Methods. (A) Low magnification images of the superonasal quadrant of the retina show a higher density of cones in apocynin-treated rd1+/+ mice compared with vehicle treated rd1+/+ mice. The optic nerve is in the lower left hand corner of the images. Scale bar 200µm. (B) High magnification images of 0.0529 mm2 bins 0.5 mm superior, inferior, temporal, and nasal to the optic nerve show a higher density of cones in all 4 retinal quadrants of apocynin-treated rd1+/+ mice compared to vehicle-treated rd1 mice. Scale bar 50µm. (C) The level or mRNA for m-cone and s-cone opsins was significantly greater in apocynin-treated rd1+/+ mice compared to vehicle treated rd1+/+ mice (*p<0.05 by unpaired Welch’s t-test for m-cone opsin, **p<0.001 by unpaired Student’s t-test for s-cone opsin).

Figure 4. Apocynin promotes cone cell function in rd1+/+ mice

Starting at P11, rd1+/+ mice were given daily intraperitoneal injections of vehicle or vehicle containing 10 mg/kg apocynin. Low background photopic ERGs were done as described in Methods at P17. Representative waveforms are shown for each group and illustrate a substantially better waveform for apocynin-treated rd1+/+ mice compared to vehicle-treated rd1+/+ mice. The bars show mean (± SEM) photopic b-wave amplitude, which was significantly higher for apocynin-treated rd1+/+ mice compared to vehicle treated rd1+/+ mice (*p<5.0×10−4 by unpaired Student’s t-test).

Figure 5. Apocynin promotes cone cell function in Q344ter transgenic mice

Starting at P11, Q344ter transgenic mice were given daily intraperitoneal injections of vehicle or vehicle containing 10 mg/kg apocynin. Low background photopic ERGs were done at P20 and P25. Representative waveforms are shown for each group and illustrate a substantially better waveform in apocynin-treated Q344ter transgenic mice compared to vehicle-treated Q344ter mice at P25. The bars show mean (± SEM) photopic b-wave amplitude, which showed no significant difference between apocynin-treated and vehicle-treated Q344ter transgenic mice at P20 (P=0.49 by unpaired Student’s t-test), but a significantly higher b-wave amplitude apocynin-treated mice at P25 (*p<0.05 by unpaired Welch’s t-test).

Figure 6. The xanthine oxidase Inhibitor, allopurinol, does not reduce cone cell death in rd1+/+ mice

Starting at P11, rd1+/+ mice in a C57BL/6 background were given daily intraperitoneal injections of vehicle or vehicle containing 100 mg/kg allopurinol. (A) At P14, vehicle-treated and allopurinol treated rd1+/+ mice both showed strong fluorescence in the remaining outer nuclear layer of the retina 18 hours after intraperitoneal injection of hydroethidine. Scale bar=50 µm. (B) At P25, protein carbonyl content measured by ELISA was not significantly different in the retinas of allopurinol-treated rd1+/+ mice compared to vehicle- treated rd1+/+ mice (P=0.65 by unpaired Student’s t-test). (C) At P25, fluorescence confocal images of peanut agglutinin (PNA)-stained retinal flat mounts showed no difference in cone cell density in 0.0529 mm2 bins 0.5 mm superior, inferior, temporal and nasal to the center of the optic nerve for allopurinol-treated compared to vehicle-treated rd1+/+ mice. Scale bar=50 µm. (D) At P25, the relative expression level of m-cone and s-cone opsin mRNA per retina was not significantly different in allopurinol-treated versus vehicle-treated rd1+/+ mice (P=0.33 by unpaired Welch’s t-test for m-cone, P=0.98 by unpaired Student’s t-test for s-cone). (E) At P17, waveforms of low background photopic ERGs appeared the same for allopurinol- and vehicle-treated rd1+/+ mice and there was no significant difference in mean (± SEM) b-wave amplitude (P= 0.12 by unpaired Welch’s t-test).