Shinsuke Umeda

Molecular composition of drusen and possible involvement of anti-retinal autoimmunity in two different forms of macular degeneration in cynomolgus monkey (Macaca fascicularis)

We have previously reported a cynomolgus monkey (Macaca fascicularis) pedigree with early onset macular degeneration that develops drusen at 2 yr after birth (1). In this study, the molecular composition of drusen in monkeys affected with late onset and early onset macular degeneration was both characterized. Involvement of anti‐retinalautoimmunity in the deposition of drusen and the pathogenesis of the disease was also evaluated. Funduscopic and histological examinations were performed on 278 adult monkeys (mean age=16.94 yr) for late onset macular degeneration. The molecular composition of drusen was analyzed by immunohistochemistry and/or direct proteome analysis using liquid chromatography tandem mass spectroscopy (LC‐MS/MS). Anti‐retinal autoantibodies in sera were screened in 20 affected and 10 age‐matched control monkeys by Western blot techniques. Immunogenic molecules were identified by 2D electrophoresis and LC‐MS/MS. Relative antibody titer against each antigen was determined by ELISA in sera from 42 affected (late onset) and 41 normal monkeys. Yellowish‐white spots in the macular region were observed in 90 (32%) of the late onset monkeys that were examined. Histological examination demonstrated that drusen or degenerative retinal pigment epithelium (RPE) cells were associated with the pigmentary abnormalities. Drusen in both late and early onset monkeys showed immunoreactivities for apolipoprotein E, amyloid P component, complement component C5, the terminal C5b‐9 complement complex, vitronectin, and membrane cofactor protein. LC‐MS/MS analyses identified 60 proteins as constituents of drusen, including a number of common components in drusen of human age‐related macular degeneration (AMD), such as annexins, crystallins, immunoglobulins, and complement components. Half of the affected monkeys had single or multiple autoantibodies against 38, 40, 50, and 60 kDa retinal proteins. The reacting antigens of 38 and 40 kDa were identified as annexin II and μ‐crystallin, respectively. Relative antibody titer against annexin II in affected monkeys was significantly higher than control animals (P<0.01). Significant difference was not observed in antibody titer against μ‐crystallin; however, several affected monkeys showed considerably elevated titer (360–610%) compared with the mean for unaffected animals. Monkey drusen both in late and early onset forms of macular degeneration had common components with drusen in human AMD patients, indicating that chronic inflammation mediated by complement activation might also be involved in the formation of drusen in these affected monkeys. The high prevalence of anti‐retinalautoantibodies in sera from affected monkeys demonstrated an autoimmune aspect of the pathogenesis of the disease. Although further analyses are required to determine whether and how autoantibodies against annexin II or μ‐crystallin relate to the pathogenesis of the disease, it could be hypothesized that immune responses directed against these antigens might trigger chronic activation of the complement cascade at the site of drusen formation.

Characterization of AOC2 gene encoding a copper-binding amine oxidase expressed specifically in retina

We have previously cloned a human, retina-specific, amine oxidase gene (RAO, gene symbol: AOC2), a member of the copper-binding amine oxidase super family. AOC2 shares sequence identity with the human kidney amine oxidase gene (KAO, gene symbol: AOC1) and the vascular adhesion protein-1 gene (VAP-1, gene symbol: AOC3). For further analysis of AOC2, the sequences surrounding the human AOC2 and the complete mouse and partial rat homologue of AOC2 were cloned for characterization. Real-time quantitative PCR, in situ hybridization, and immunohistochemistry were performed to determine the specific expression of AOC2 in the mouse retina and especially in the retinal ganglion cells. Our results demonstrated that the copper-binding motif and the enzyme active site of AOC1 and AOC3 were both conserved in mouse AOC2. The human and mouse AOC2 was flanked by two genes, the Psme3 gene for PA-28 gamma subunit and, surprisingly, the AOC3 gene. Rat AOC2 contained a stop codon that terminated the peptide length to 127 amino acids. The presence of human and rat AOC pseudogene in this region, in addition to the tandemly positioned two AOC genes, indicates the possibility of successful AOC3 replication to retina-specific AOC2 for human and mouse but unsuccessful for rat.

Immunohistochemical analysis of aldehyde-modified proteins in drusen in cynomolgus monkeys (Macaca fascicularis).

Protein modifications resulting from reactive aldehydes are thought to be involved in the pathogenesis of various degenerative diseases. Aged cynomolgus monkey (Macaca fascicularis) spontaneously develop drusen in the macula, consistent with the phenotype observed in early-stage age-related macular degeneration (AMD), indicating that this animal is an optimum model for AMD. In retinal sections from three monkeys with macular degeneration, regardless of their size, drusen were consistently positive with immunohistochemical labeling against protein modifications by 4-hydroxynonenal and 4-hydroxyhexenal, end products of non-enzymatic oxidation of n-6 and n-3 polyunsaturated fatty acids, respectively. Positive labeling for both modifications was observed in the ganglion cell layer, the inner nuclear layer, the outer nuclear layer, and the retinal pigment epithelium. However, no consistent differences in location or intensity of the labeling were observed between monkeys with normal macula and macular degeneration. The results suggest a possible association between drusen formation and protein modifications by aldehydes in the pathogenesis of AMD.