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Purpose Dysregulation of the alternative complement pathway is a major pathogenic mechanism in age-related macular degeneration. We investigated whether locally synthesized complement components contribute to AMD by profiling complement expression in postmortem eyes with and without AMD. Methods AMD severity grade 1 to 4 was determined by analysis of postmortem acquired fundus images and hematoxylin and eosin stained histological sections. TaqMan (donor eyes n = 39) and RNAscope/in situ hybridization (n = 10) were performed to detect complement mRNA. Meso scale discovery assay and Western blot (n = 31) were used to measure complement protein levels. Results The levels of complement mRNA and protein expression were approximately 15- to 100-fold (P < 0.0001-0.001) higher in macular retinal pigment epithelium (RPE)/choroid tissue than in neural retina, regardless of AMD grade status. Complement mRNA and protein levels were modestly elevated in vitreous and the macular neural retina in eyes with geographic atrophy (GA), but not in eyes with early or intermediate AMD, compared to normal eyes. Alternative and classical pathway complement mRNAs (C3, CFB, CFH, CFI, C1QA) identified by RNAscope were conspicuous in areas of atrophy; in those areas C3 mRNA was observed in a subset of IBA1+ microglia or macrophages. Conclusions We verified that RPE/choroid contains most ocular complement; thus RPE/choroid rather than the neural retina or vitreous is likely to be the key site for complement inhibition to treat GA or earlier stage of the disease. Outer retinal local production of complement mRNAs along with evidence of increased complement activation is a feature of GA.

Purpose Genome-wide association studies suggest a role for the complement system in age-related macular degeneration (AMD). We characterized ocular complement activation and evaluated a complement factor D (FD) neutralizing antibody. Methods Mice were treated with toll-like receptor (TLR) ligands, intravitreal injection (IVT), or corneal debridement. Levels of complement proteins and mRNA were measured. A FD neutralizing antibody was administered IVT into eyes of rabbits that were challenged with LPS (lipopolysaccharide) administered intravenously. Results Levels of C3 and factor B (FB) mRNA and protein in the eye were increased following intraperitoneal injection of TLR4 ligand LPS. Increased levels of C3 and FB breakdown products were observed in both eye tissues and plasma. Complement activation products were markedly reduced in C3-/- and Cfb-/- mice challenged with LPS. Ocular complement levels were also elevated in mice treated systemically with TLR2 and -3 ligands, injured by IVT injection or corneal debridement, or even in normal aging. IVT administration of a complement FD neutralizing antibody in rabbits inhibited LPS-induced complement activation in the posterior segment of the eye, but not in the anterior segment of the eye or in plasma. Conclusions Systemic TLR stimulation and eye tissue injury induced time-dependent alternative complement pathway activation in the eye. Ocular complement levels were also gradually elevated during aging. An anti-FD antibody IVT potently inhibited LPS-induced complement activation in the posterior segment of the eye. This study provides insights into the dynamic profile of ocular complement activation, which is valuable for complement research in eye diseases and for developing complement therapeutics for AMD.

Purpose We attempted to reproduce published studies that evaluated whether the following factors influence choroidal neovascularization (CNV) induced by laser photocoagulation in murine retinas: small interfering RNA (siRNA), cobra venom factor, complement factors C3 and C5, and complement receptor C5aR. In addition, we explored whether laser-induced CNV in mice was influenced by the vendor of origin of the animals. Methods Reagents or genotypes reported by others to influence CNV in this model were assessed using our standard procedures. Retrospective analyses of control or placebo mice in many experiments were done to evaluate whether the CNV area induced by laser photocoagulation varied according to vendor. Results Administration of the following agents did not have a substantial impact on the CNV induced by laser burns in mice: siRNA, low-molecular-weight inhibitor of the C5a receptor (PMX53), or cobra venom factor. Jackson Laboratory (JAX) mice lacking either C3 or C5 had increased neovascularization compared to non-littermate JAX wild-type controls. Taconic mice lacking C3 had reduced CNV compared to non-littermate Taconic wild-type control mice. A retrospective analysis of vehicle-treated wild-type C57BL/6 mice used as controls across 132 experiments conducted from 2007 to 2010 revealed that mice purchased from JAX or from Charles River produced less neovascularization than mice from Taconic. Conclusions We present our recommended methods for conducting experiments with the mouse laser-induced CNV model to enhance reproducibility and minimize investigator bias.

Purpose To determine the frequency of mutations in IMPDH1 among patients with autosomal dominant retinitis pigmentosa (RP), to characterize the clinical features of patients with the Asp226Asn mutation in this gene, and to compare these features with those found among patients with selected dominant mutations in other RP genes. Methods The coding sequence and the adjacent flanking intron sequences of all 14 coding exons were sequenced in 183 unrelated patients with dominant RP. The clinical findings evaluated included visual acuity, refractive error, visual field area measured with the Goldmann perimeter, final dark-adaptation threshold, full-field electroretinogram (ERG) amplitudes, cataract, and funduscopic bone spicule pigmentation. Results The mutation Asp226Asn was identified in 6 of the 183 unrelated patients with RP. One patient carried the novel, possibly pathogenic, change Lys238Glu. There was approximately a 100-fold variation in ERG amplitudes among patients of similar age with the Asp226Asn mutation. Patients had similar reductions of rod-plus-cone 0.5-Hz ERG amplitude and cone 30-Hz ERG amplitude. For a given amount of remaining visual field, there was a larger ERG amplitude in IMPDH1-carrying patients (average 0.5-Hz ERG/visual field ratio = 9.5 nV/deg(2)) compared with groups of patients with the RP1 mutation Arg677End (2.8 nV/deg(2)), the rhodopsin (RHO) mutation Pro23His (5.1 nV/deg(2)), or the RHO mutation Pro347Leu (1.7 nV/deg(2)). Conclusions IMPDH1 mutations account for approximately 2% of cases of dominant RP in North America. The most frequent mutation, Asp226Asn, appears to cause at least as much loss of rod function as cone function. Patients with this form of RP retain, on average, two to five times more ERG amplitude per unit of remaining visual area than patients with three other forms of dominant RP.

To identify pathogenic mutations in the guanylate cyclase-activating protein 1 (GCAP1) and GCAP2 genes and to characterize the biochemical effect of mutation on guanylate cyclase (GC) stimulation.The GCAP1 and GCAP2 genes were screened by direct sequencing for mutations in 216 patients and 421 patients, respectively, with various hereditary retinal diseases. A mutation in GCAP1 segregating with autosomal dominant cone degeneration was further evaluated biochemically by employing recombinant proteins, immunoblotting, Ca2+-dependent stimulation of GC, fluorescence emission spectra, and limited proteolysis in the absence and presence of Ca2+.A novel GCAP1 mutation, I143NT (substitution of Ile at codon 143 by Asn and Thr), affecting the EF4 Ca2+-binding loop, was identified in a heterozygote father and son with autosomal dominant cone degeneration. Both patients had much greater loss of cone function versus rod function; previous histopathologic evaluation of the father's eyes at autopsy (age 75 years) showed no foveal cones but a few, scattered cones remaining in the peripheral retina. Biochemical analysis showed that the GCAP1-I143NT mutant adopted a conformation susceptible to proteolysis, and the mutant inhibited GC only partially at high Ca2+ concentrations. Individual patients with atypical or recessive retinitis pigmentosa (RP) had additional heterozygous GCAP1-T114I and GCAP2 gene changes (V85M and F150C) of unknown pathogenicity.A novel GCAP1 mutation, I143NT, caused a form of autosomal dominant cone degeneration that destroys foveal cones by mid-life but spares some cones in the peripheral retina up to 75 years. Properties of the GCAP1-I143NT mutant protein suggested that it is incompletely inactivated by high Ca2+ concentrations as should occur with dark adaptation. The continued activity of the mutant GCAP1 likely results in higher-than-normal scotopic cGMP levels which may, in turn, account for the progressive loss of cones.

To measure the rate of progression of retinal degeneration in patients with retinitis pigmentosa due to dominant rhodopsin mutations and to determine whether the rate of progression correlates with the location of the altered amino acid in the rhodopsin molecule.Change in ocular function was observed for an average of 8.7 years in 140 patients. After censoring data to eliminate "ceiling" and "floor" effects, longitudinal rates of change were compared, after weighting by follow-up time and number of visits, with rates inferred from cross-sectional analyses of the data from baseline visits. Mean rates of change were compared among groups of patients with mutations affecting the globule, plug, or C-terminal region of the protein after adjusting for age, gender, and baseline function.Mean annual exponential rates of decline were 1.8% for visual acuity, 2.6% for visual field area, and 8.7% for ERG amplitude. The rates of visual acuity and ERG amplitude decline were significantly faster, and the rate of visual field area decline was significantly slower, than those inferred from baseline visits. Rates of acuity loss did not vary significantly with the region affected by the mutation. In contrast, the mean annual rate of field loss in the C terminus group (7.4%) was significantly faster than that in the globule (1.7%) or plug (1.1%) group. The mean annual rate of ERG decline was also significantly faster in the C terminus group (13.5%) than in the globule (8.5%) or plug (3.7%) groups and significantly faster in the globule group than in the plug group.Rates of decline in visual function for groups of patients with rhodopsin mutations cannot be accurately inferred from cross-sectional analyses of baseline visits. Average rates of decline of visual field area and ERG amplitude are fastest in patients with mutations affecting the C-terminal region.

To study the possible involvement of the rod (SLC24A1) and cone (SLC24A2) Na-Ca+K exchanger (NCKX) genes in retinal diseases.DNA was collected from unrelated patients with retinal disease, mainly from North America. A human genomic library was screened with the cone NCKX cDNA, and hybridizing clones were sequenced to determine the genomic organization of the SLC24A2 gene. The single-strand conformation polymorphism (SSCP) technique and direct sequencing were used to screen the patients' DNA for mutations in SLC24A1 and SLC24A2. The effect of selected missense changes on protein function was tested by measuring potassium-dependent Na-Ca exchange of the mutant proteins expressed in insect cells.Twenty-seven novel sequence changes were found in the rod NCKX gene, 21 of which are unlikely to be pathogenic, because they did not cosegregate with the disease or did not affect conserved regions of the protein. Of the remaining six, two were frameshift mutations found in one patient each. If translated, these alleles would encode nonfunctional proteins. Three of the six possibly pathogenic mutations were missense changes located in conserved regions, and their protein functions were assayed. Only one (Ile992Thr) had a significantly low level of exchanger function, but it was found in two unrelated patients who were heterozygotes with different retinal diseases, and this mutation could not be unequivocally associated with either disease. The last of the six changes is likely to create a new splice acceptor site. The genomic organization of the cone NCKX gene was determined, and it contained 11 exons with a few splice variants. Fifteen novel sequence changes were identified in the cone exchanger gene in patients with a cone dysfunction or degeneration. Only three of these sequence changes, all missense changes found in heterozygous patients, were considered possibly pathogenic. Functional analysis showed only a slight reduction in the activity of the corresponding mutant proteins.Although variant alleles of the rod and cone NCKX genes were found, none could be definitively associated with a specific retinal disease. The human phenotype associated with mutant exchanger alleles remains unknown.