Clusterin Enhances Photoreceptor Survival by Suppressing Neuronal Nitric Oxide Synthase (nNOS) Expression Levels in Rhodopsin S334ter-line 3 Retinitis Pigmentosa

Abstract

Purpose : Retinal environment changes, including oxidative stress, influence photoreceptor degeneration in Retinitis Pigmentosa (RP).^1,2 The production of reactive oxygen species causes oxidative damage to proteins, lipids, and DNA.³ Our hypothesis is that clusterin is involved in oxidative stress-induced cell death. We observed a robust preservation of rod photoreceptors in S334ter-line3 retinas with clusterin treatment.⁴ To explore biochemical pathways influencing oxidative stress, we examined nNOS expression levels before and after treatment of S334ter-line 3 retinas with either clusterin or an inhibitor of nNOS, N^G-nitro-L-arginine methyl ester (L-NAME), to determine if rod death is delayed and rod cell survival is prolonged.

Methods : After timed intravitreal injections to RP retinas of clusterin (10µg/ml) or intraperitoneal injection (IP) of L-NAME (100mg/kg), or saline, we studied the effect on their visual responses and nNOS expression levels. Physiological responses were measured using electroretinography (ERG) after treatment, followed by statistical analysis of immunohistochemistry and immunoblots with specific antibodies for nNOS and rhodopsin to investigate the impact of clusterin/L-NAME on nNOS expression and rod survival.

Results : Levels of nNOS are up-regulated in postnatal (P)15, P30, and P60 RP retinas compared to normal and clusterin treatment suppressed expression. Physiological responses using scotopic ERG reveal b-wave amplitudes from clusterin-treated retinas are significantly greater than from saline-treated RP retinas at P30 (P<0.0001) and P45 (P<0.05). In RP retinas, IP injection of L-NAME produced extended protection to delay rod photoreceptor cell death; however, clusterin treatment was more effective in rod survival (P<0.001).

Conclusions : Clusterin acts as a cytoprotective protein to slow the death of rod photoreceptors in RP retinas. Induction of nNOS expression in RP retinas and its down-regulation by clusterin treatment suggests it protects rod photoreceptors by suppressing the up-regulated nNOS levels. Moreover, nNOS/NO induction contributes to photoreceptor death and clusterin provides an important cellular defense mechanism.
¹LaVail MM, et al. Exp Eye Res. 2017.
²Cuenca N, et al. Prog Ret & Eye Res. 2014;43:17-75.
³Shen J, et al. J.Cell. Phy. 2005;203(3):457-64.
⁴Vargas A, et al. PloS one. 2017;12(8):e0182389.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.