The role of oxidative stress in photoreceptor degeneration

Reactive oxygen species (ROS) are constitutively produced by mitochondria and represent the major cellular source of oxidative stress. ROS are capable of attacking molecules such as DNA, proteins and lipids, and of compromising either the structural and functional integrity or the survival of cells. Mitochondria also play a key role in apoptosis, the major mechanism of cell death in retinitis pigmentosa (RP), which is a diverse group of inherited human retinal dystrophies associated with progressive degeneration of photoreceptor cells. Mutations in numerous genes have been implicated in RP, which have distinct pathophysiological mechanisms and lead to retinal degeneration at different rates. The aim of this thesis was to investigate the role of oxidative stress in disease progression using mouse models of human RP.The mouse mutants retinal degeneration 1 (rdl/rdl), atypical retinal degeneration 1 (atrdl/atrdl), rhodopsin knockout (R/?c/ ~) and peripherin/retinal degeneration slow (rds/rds) were firstly investigated for evidence of oxidative damage by analysis of oxidative stress markers. Secondly, the mutants were crossed to a superoxide dismutase 2 heterozygous mouse (Sod2+/~), with decreased mitochondrial antioxidant activity, to examine the effect on disease progression. Thirdly, mutants were treated with a mitochondrially targeted ubiquinone derivative (MitoQ), which is a powerful antioxidant, to try and slow the rate of retinal degeneration. MitoQ was administered orally during pregnancy and for an extended postnatal period and uptake, toxicity, breeding behaviour and survival were assessed. Rates of photoreceptor degeneration were estimated by morphometric and apoptosis assays, while the cellular redox status was assessed by glutathione assays and by measuring the activities of the mitochondrial enzymes NADH:ubiquinone oxidoreductase (complex I), which is oxidative stress-sensitive, compared with citrate synthase, which is oxidative stressinsensitive.All retinal degeneration mutants were found to show significantly reduced complex I activities, while citrate synthase was unchanged, indicating mitochondrial oxidative stress. Rates of photoreceptor degeneration were unchanged either by crossing to a Sod2+' genetic background or by MitoQ administration. Only the rds/rds mutant, with the slowest rate of degeneration, showed a significant increase in complex I activity after MitoQ administration. Although mitochondrial oxidative stress is shown to be present in all of the retinal degeneration mutants, altering the oxidative status of the retina had no effect on photoreceptor survival

Disease mechanisms in late-onset retinal macular degeneration associated with mutation in C1QTNF5

Late-onset retinal macular degeneration (L-ORMD) is an autosomal dominant condition resembling age-related macular degeneration (AMD) in which a key pathological feature is a thick extracellular sub-retinal pigment epithelial (RPE) deposit. L-ORMD is caused by mutation in the C1QTNF5 (CTRP5) short-chain collagen gene, but the disease mechanism is unknown. Here, we first show that wild-type C1QTNF5 is secreted, whereas mutant C1QTNF5 is misfolded and retained within the endoplasmic reticulum (ER). Secondly, the ER retained mutant protein has a shorter half-life than wild-type C1QTNF5 and is preferentially degraded by proteasomes. Thirdly, C1QTNF5 is shown to interact with the membrane-type frizzled related protein (MFRP), on the basis of yeast two-hybrid, protein pull-down and co-immunoprecipitation assays and RPE co-localization. These data suggest that L-ORMD is due to insufficient levels of secreted C1QTNF5, compromised RPE cell function resulting from ER retention of the mutant protein or both mechanisms

Evidence of severe mitochondrial oxidative stress and a protective effect of low oxygen in mouse models of inherited photoreceptor degeneration

The role of oxidative stress within photoreceptors (PRs) in inherited photoreceptor degeneration (IPD) is unclear. We investigated this question using four IPD mouse models (Pde6b(rd1/rd1), Pde6b(atrd1/atrd1), Rho(-/-) and Prph2(rds/rds)) and compared the abundance of reduced glutathione (GSH) and the activity of mitochondrial NADH:ubiquinone oxidoreductase (complex I), which is oxidative stress sensitive, as indirect measures of redox status, in the retinas of wild type and IPD mice. All four IPD mutants had significantly reduced retinal complex I activities (14-29% of wild type) and two showed reduced GSH, at a stage prior to the occurrence of significant cell death, whereas mitochondrial citrate synthase, which is oxidative stress insensitive, was unchanged. We orally administered the mitochondrially targeted anti oxidant MitoQ in order to reduce oxidative stress but without any improvement in retinal complex I activity, GSH or rates of PR degeneration. One possible source of oxidative stress in IPDs is oxygen toxicity in the outer retina due to reduced consumption by PR mitochondria. We therefore asked whether a reduction in the ambient O-2 concentration might improve PR survival in Pde6b(rd1/rd1) retinal explants either directly, by reducing reactive oxygen species formation, or indirectly by a neuroprotective mechanism. Pde6b(rd1/rd1) retinal explants cultured in 6% O-2 showed 31% less PR death than normoxic explants. We conclude that (i) mitochondrial oxidative stress is a significant early feature of IPDs; (ii) the ineffectiveness of MitoQ may indicate its inability to reduce some mediators of oxidative stress, such as hydrogen peroxide; and (iii) elucidation of the mechanisms by which hypoxia protects mutant PRs may identify novel neuroprotective pathways in the retina