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.

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.

Developmental and tissue expression of Xenopus laevis RPGR.

PURPOSE: The present study examined the developmental and tissue expression of the retinitis pigmentosa GTPase regulator (RPGR) gene in Xenopus laevis. METHODS: The cDNA for X. laevis RPGR (XRPGR) was isolated from adult eye mRNA by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. The deduced peptide sequence was aligned with RPGR orthologues. Gene expression was examined by whole-mount in situ hybridization and RT-PCR. The localization of XRPGR in X. laevis photoreceptor cells and XTC-2 cells was determined by immunostaining. RESULTS: The XRPGR(ex1-19) isoform encodes a protein of 727 amino acids containing an RCC1 domain and a C-terminal isoprenylation anchorage motif. It shares 33% to 41% amino acid identity with human, mouse, and dog RPGR. The C-terminal exon of the alternatively spliced RPGR(ORF15) isoform is also conserved across species. XRPGR is expressed at the earliest stages of X. laevis development and persists into adulthood, where expression is highest in the eye. XRPGR is expressed in presumptive eye fields (stages 18 to 22), becoming largely restricted to the central retina (stages 28 to 40). XRPGR protein colocalizes with beta-tubulin at the X. laevis ciliary axoneme and with gamma-tubulin at centrosomes in XTC-2 cells. CONCLUSIONS: XRPGR is widely expressed throughout development but shows highest expression after the appearance of the eye primordium and persists in the eye into adulthood. The data are consistent with XRPGR expression in a single microtubular organelle-the centriole or basal body and associated ciliary transitional zone found in modified sensory cilia of photoreceptors and motile cilia.

Lifespan and mitochondrial control of neurodegeneration.

We examine the allometric (comparative scaling) relationships between rates of neurodegeneration resulting from equivalent mutations in a diverse group of genes from five mammalian species with different maximum lifespan potentials. In both retina and brain, rates of neurodegeneration vary by as much as two orders of magnitude and are strongly correlated with maximum lifespan potential and rates of formation of mitochondrial reactive oxygen and nitrogen species (RONS). Cell death in these disorders is directly or indirectly regulated by the intrinsic mitochondrial cell death pathway. Mitochondria are the main source of RONS production and integrate cellular stress signals to coordinate the intrinsic pathway. We propose that these two functions are intimately related and that steady-state RONS-mediated signaling or damage to the mitochondrial stress-integration machinery is the principal factor setting the probability of cell death in response to a diverse range of cellular stressors. This provides a new and unifying framework for investigating neurodegenerative disorders.