Catherine Jomary

Association between the SERPING1 gene and age-related macular degeneration: a two-stage case–control study

BACKGROUND Age-related macular degeneration is the most prevalent form of visual impairment and blindness in developed countries. Genetic studies have made advancements in establishing the molecular cause of this disease, identifying mutations in the complement factor H (CFH) gene and a locus on chromosome 10 encompassing the HTRA1/LOC387715/ARMS2 genes. Variants in complement 3 (C3) and an HLA locus containing both factor B and C2 genes have also been implicated. We aimed to identify further genetic risk factors for this disease. METHODS We used a case-control study design in a UK sample of patients with age-related macular degeneration (n=479) and controls (n=479) and undertook a low-density screen of 32 genes using 93 single nucleotide polymorphisms (SNPs). Genes were selected as candidates on the basis of potential functional relevance to age-related macular degeneration. Significant initial findings were confirmed by replication in an independent US cohort of 248 unrelated patients with disease and 252 controls, and by high-density genotyping around association signals. FINDINGS The SNP variant rs2511989, located within intron six of the SERPING1 gene, showed highly significant genotypic association with age-related macular degeneration (uncorrected p=4.0x10(-5), corrected p=0.00372). We detected no evidence for association between disease and the other 31 candidate genes. The odds ratio for age-related macular degeneration in rs2511989 G/A heterozygotes compared with wild type G/G homozygotes was 0.63 (95% CI 0.47-0.84). A similar comparison of the A/A homozygotes with the wild type yielded an odds ratio of 0.44 (0.31-0.64). We replicated the observed genotypic association in a US cohort (p=0.008). Furthermore, a secondary high-density genotyping study across the SERPING1 gene region identified five additional SNP variants similarly associated with age-related macular degeneration (rs2244169, rs2511990, rs2509897, rs1005510, and rs2511988). INTERPRETATION Genetic variation in SERPING1 significantly alters susceptibility to age-related macular degeneration. SERPING1 encodes the C1 inhibitor, which has a crucial role in inhibition of complement component 1 (C1) and might implicate the classic pathway of complement activation in this disease.

Rescue of photoreceptor function by AAV-mediated gene transfer in a mouse model of inherited retinal degeneration

Knowledge of the mutations leading to inherited retinal degenerations provides a foundation for the development of somatic gene therapy in which potentially corrective genes are transferred to the target photoreceptor cells. Towards this end, we have evaluated the efficacy of a recombinant adeno-associated virus (AAV) vector to deliver and express the correct form of the cGMP phosphodiesterase-β (PDE-β) gene in the retinas of rd mice, which suffer rapid retinal degeneration due to recessive mutation in the endogenous gene. A truncated murine opsin promoter was used to drive expression of the PDE-β cDNA. Following intraocular injection of AAV.PDE-β, increased retinal expression of immunoreactive PDE protein was observed, including within photoreceptor cell bodies. Compared with age-matched controls, treated eyes showed increased numbers of photoreceptors and a two-fold increase in sensitivity to light as measured by in vitro electroretinography. These findings provide evidence that rescue of functional photoreceptor neurons can be achieved by somatic gene therapy.

Characterization of Cell Death Pathways in Murine Retinal Neurodegeneration Implicates Cytochrome c Release, Caspase Activation, and Bid Cleavage

Apoptosis is considered to be the final common pathway of photoreceptor cell death in different inherited retinal diseases. However, apoptosis encompasses diverse pathways of molecular interactions culminating in cellular demise. To begin dissecting these interactions, we have investigated key participants in the rd (retinal degeneration) model of retinal neurodegeneration. By Western blot analysis and immunocytochemistry, we found that cytochrome c release occurs in rd retinas concurrently with the activation of the proapoptotic protein Bid. Active forms of caspase-8 and the mitogen-activated protein kinase p38, both of which are capable of cleaving Bid, were detected in rd retinas at the peak time of photoreceptor death. In addition, the activated form of the cell death effector caspase-3 was detectable particularly at the photoreceptors in parallel with this peak degenerative phase. These data suggest that activation of both major apoptotic pathways occurs during photoreceptor degeneration in the rd mouse model of inherited blindness.

Modulated expression of secreted frizzled-related proteins in human retinal degeneration.

Inherited retinal degenerations such as retinitis pigmentosa (RP) are characterized by progressive loss of photoreceptors, apparently by apoptosis, and our recent report of increased secreted Frizzled-related protein-2 (SFRP2) in RP retinas suggests altered Wnt signalling may be a component of the degenerative process. The present study shows that levels of SFRP1, SFRP3 and SFRP5 mRNAs are also abnormal in RP, giving rise to idiosyncratic expression patterns. In highly degenerative retinas, the SFRP proteins localize mainly to the inner limiting membrane, but in a well-preserved retina SFRP1 and SFRP5 are notably localized to the surviving photoreceptors. Together with increased c-jun mRNA expression in all cases examined, these results support the notion that disruptions of Wnt network signalling are involved in retinal neurodegeneration.

Expression of αb-crystallin in a mouse model of inherited retinal degeneration

αB-CRYSTALLIN, which is abundantly expressed in the lens but also in a diversity of other tissues, functions as a stress-inducible molecular chaperone and is increased in brain neurodegenerative diseases. We compared retinal αB-crystallin expression in a model of inherited retinal degeneration, th e rd mouse, and controls. Northern and in sit u hybridization analysis showe αB-crystallin mRNA to have an altered spatio-temporal pattern with increased levels localized to glial cells in the degenerative state. Immunocytochemistry confirmed increased expression at Müller cells and astrocytes, together with transiently increased localization to the degenerating photoreceptors. These findings suggest that increase dαB-crystallin expression is associated with glial cell reaction to neuronal damage in the retina, and may comprise part of the retinas overall defensive response to the stress of apoptotic photoreceptor cell death.

Induction of functional photoreceptor phenotype by exogenous Crx expression in mouse retinal stem cells.

PURPOSE This study was undertaken to determine whether exogenous expression of the transcription factor Crx can promote the differentiation of mouse retinal stem cells (RSCs) into cells with a functional photoreceptor phenotype exhibiting light-sensitive properties. METHODS RSCs isolated from mouse ciliary epithelium and maintained in serum-free culture were genetically modified by electroporation to express exogenous epitope-tagged murine Crx. Changes in the expression of stem cell markers (homeodomain transcription factor Pax6; POU transcription factor Oct3/4; proliferating cell nuclear antigen [PCNA]); of neuronal markers (nestin, neuron-specific class III beta-tubulin [beta III Tub] and neurofilament [NF 200]); and of photoreceptor-specific markers (rhodopsin [Rho], cyclic nucleotide-gated cation channel-3 [CNG3], blue-cone opsin, and cGMP phosphodiesterase [PDE]); were evaluated during differentiation by immunocytochemistry and Western blot analysis. Phototransduction cascade activity was assessed by measuring light-induced hydrolysis of cyclic (c)GMP levels with a cGMP enzyme-linked immunoassay. RESULTS Transient Crx transgene expression was observed in 63% of RSCs. Expression of stem cell markers of proliferation and pluripotency Pax6, PCNA, and Oct3/4, was significantly decreased by exogenous Crx expression. Concomitantly, Crx induced expression of the analyzed neuron- and photoreceptor-specific markers. Light-induced cGMP hydrolysis was increased in RSCs expressing exogenous Crx, and inhibition of PDE resulted in elevated cGMP levels. CONCLUSIONS Crx halted proliferation of RSCs and induced them to differentiate into cells expressing photoreceptor-specific markers and displaying light-induced sensitivity characteristic of an activatable visual phototransduction cascade. This study demonstrates that Crx can successfully induce RSCs to differentiate into cells with functional photoreceptor phenotypes.

Comparison of clusterin gene expression in normal and dystrophic human retinas

To gain insight into the mechanisms underlying altered clusterin expression in retinal degeneration, the cellular distribution of clusterin mRNA in normal and in retinitis pigmentosa-affected retinas was compared using in situ hybridization. In contrast to the normal retina, where clusterin mRNA is localized in the inner nuclear and ganglion cell layers, a clustered distribution is observed throughout the dystrophic retina. The results indicate an expression of clusterin gene in normal retinal neurons and suggest that its altered regulation in neurodegeneration is not purely a glial cell phenomenon.

Abnormal distribution of retinal clusterin in retinitis pigmentosa

Increased expression of clusterin mRNA is associated with neurodegenerative states, including retinas affected by retinitis pigmentosa (RP). We have investigated the distribution of immunoreactive clusterin in normal and RP-affected retinas. Reactivity at the inner limiting membrane, plexiform layers, and photoreceptors in normal retina accords well with clusterins postulated role as a membrane protective agent. In RP-affected retina the organized distribution is lost and overall reactivity appears decreased. The changes in this case may reflect increased turnover or removal of clusterin, perhaps via interaction with components of the immune system.

Localization of tissue inhibitor of metalloproteinases-3 in neurodegenerative retinal disease.

TISSUE inhibitor of metalloproteinases-3 (TIMP-3) is one of a family of genes whose products are implicated in the regulation of remodelling of the extracellular matrix. The level of mRNA coding for TIMP-3 is increased in retinas affected by the photoreceptor degenerative disease, simplex retinitis pigmentosa (RP), and mutations in TIMP-3 are associated with an inherited form of macular dystrophy. Here we compare TIMP-3 protein expression in normal retina and in those affected by RP and by age-related macular degeneration. Immunoreactive TIMP-3 is present in normal retinal pigment epithelium, and in degenerative retinas particularly at Bruchs membrane and additionally in photoreceptor-retaining regions in simplex RP. The pattern suggests a role for TIMP-3 in normal retinal homeostasis, and, in the disease state, in the modulation of extracellular matrix metabolism and neovascularization.

Lack of causal relationship between clusterin expression and photoreceptor apoptosis in light-induced retinal degeneration.

Abstract: Induction of apoptosis in the retina leads to cellular death by molecular mechanisms that are not well understood. Clusterin expression is increased in tissues undergoing apoptosis, including retinal neurodegenerative states, but the causal relationships remain to be clarified. To gain insight into clusterins role in photoreceptor apoptosis, the cellular distribution of clusterin mRNA was compared with the pattern of apoptotic nuclear labelling in a rat model of light‐induced retinal degeneration. In control retinal sections, clusterin mRNA was localized to the retinal pigment epithelium cells, photoreceptor inner segments, inner nuclear layer, and ganglion cell layer. Clusterin expression decreased in photoreceptors and retinal pigment epithelium cells, which progressively degenerated, and increased in preserved inner nuclear layer, in proportion to the duration of light exposure in both cyclic light‐ and dark‐reared animals. These results suggest that clusterin is not causally involved in apoptotic mechanisms of photoreceptor death, but may relate to cytoprotective functions.