Brian Tulloch

Mutational hot spot within a new RPGR exon in X-linked retinitis pigmentosa

The gene RPGR was previously identified in the RP3 region of Xp21.1 and shown to be mutated in 10–20% of patients with the progressive retinal degeneration X-linked retinitis pigmentosa (XLRP). The mutations predominantly affected a domain homologous to RCC1, a guanine nucleotide exchange factor for the small GTPase Ran, although they were present in fewer than the 70–75% of XLRP patients predicted from linkage studies. Mutations in the RP2 locus at Xp11.3 were found in a further 10–20% of XLRP patients, as predicted from linkage studies. Because the mutations in the remainder of the XLRP patients may reside in undiscovered exons of RPGR, we sequenced a 172-kb region containing the entire gene. Analysis of the sequence disclosed a new 3′ terminal exon that was mutated in 60% of XLRP patients examined. This exon encodes 567 amino acids, with a repetitive domain rich in glutamic acid residues. The sequence is conserved in the mouse, bovine and Fugu rubripes genes. It is preferentially expressed in mouse and bovine retina, further supporting its importance for retinal function. Our results suggest that mutations in RPGR are the only cause of RP3 type XLRP and account for the disease in over 70% of XLRP patients and an estimated 11% of all retinitis pigmentosa patients.

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.

Biochemical Characterisation of the C1QTNF5 Gene Associated with Late-Onset Retinal Degeneration

Age-related macular degeneration (AMD) is the commonest cause of severe vision loss in adults, affecting up to 30% of the elderly population and accounting for 50–60% of new blind registration in western countries (Green and Enger, 1993; Seddon, 2001). It is characterised by a late-onset degeneration of the retinal macula and represents the advanced stage of a more common disorder, age-related maculopathy. There are two clinical subtypes of AMD, one is a “dry” form characterised by geographic atrophy, the other a “wet” form characterised by choroidal neovascularisation (CNV). This “wet” form represents only 10% of cases but accounts for about 90% of registered blindness (Ferris et al., 1984). The important early pathological features of AMD are the presence of both focal (drusen) and diffuse extracellular (basal) deposits in the macula, between the retinal pigment epithelium (RPE) and inner collagenous layer of Bruch’s membrane, a pentalaminar structure bounded by the basement membranes of RPE and choroidal capillary 1endothelium. These deposits lead to dysfunction and later death of RPE and associated photoreceptors. The nature of the proteins within the diffuse extracellular deposits have not been elucidated but the focal deposits (drusen) include >100 proteins, together with esterified and non-esterified cholesterol and other lipids and glycosaminoglycans (Crabb et al., 2002; Malek et al., 2003). Risk factors for AMD include age, sex, family history, APOE genotype, smoking, ethnicity and cardiovascular disease (Seddon, 2001). Genetic factors are implicated in AMD on the basis of twin and family studies but it appears to be a genetically complex disorder (Hammond et al., 2002).

Characterisation of a C1qtnf5 Ser163Arg Knock-In Mouse Model of Late-Onset Retinal Macular Degeneration

A single founder mutation resulting in a Ser163Arg substitution in the C1QTNF5 gene product causes autosomal dominant late-onset retinal macular degeneration (L-ORMD) in humans, which has clinical and pathological features resembling age-related macular degeneration. We generated and characterised a mouse “knock-in” model carrying the Ser163Arg mutation in the orthologous murine C1qtnf5 gene by site-directed mutagenesis and homologous recombination into mouse embryonic stem cells. Biochemical, immunological, electron microscopic, fundus autofluorescence, electroretinography and laser photocoagulation analyses were used to characterise the mouse model. Heterozygous and homozygous knock-in mice showed no significant abnormality in any of the above measures at time points up to 2 years. This result contrasts with another C1qtnf5 Ser163Arg knock-in mouse which showed most of the features of L-ORMD but differed in genetic background and targeting construct.

P4-167: Investigation of mitochondrial DNA damage in late-onset Alzheimer's disease

factor levels, respectively. Neurons were pre-treated for 2 days with estrogen at the low concentration of 10 pg/mL. Results: In our aging model of rat cortical neurons in common culture conditions, mitochondrial counts per neuron were found to be the same in middle-age (9-month) and old (24-month) neurons. Immunostaining for cytochrome C in individual mitochondria revealed an age-related deficit of this electron donor. Respiration measurements in middle-age and old neurons revealed no age-related changes in basal respiration, but after a glutamate stimulus, old neurons were unable to match the two-fold upregulation of respiration seen in middle-age neurons. Respiration in old neurons was inhibited by lower concentrations of potassium cyanide, suggesting a 29% deficit in functional enzyme in old compared to middle-age neurons. CCO activity in submitochondrial particles isolated from middle-age and old rat cortical neurons in culture showed no age-related differences. In addition to cytochrome C, CCO requires cardiolipin to function. Staining with nonylacridine orange revealed an age-related deficit in cardiolipin. Estrogen treatment of old neurons restored cardiolipin levels and upregulated respiration under glutamate stress. Attempts to induce mitochondrial turnover by neuronal multiplication also rejuvenated CCO activity in old neurons. Conclusions: These data suggest cytochrome C and cardiolipin levels are deficient in old neurons, preventing normal upregulation of respiration needed for oxidative phosphorylation in response to stress. Furthermore, the data suggest this deficit can be corrected with estrogen. These results provide a better understanding of the brain metabolic deficiencies that occur with age, possibly leading to susceptibility to Alzheimer’s Disease.