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Dinusha Rajapakse, Katherine Peterson, Maria M Campos, Graeme Wistow; Amelotin, an enamel protein is expressed in retinal pigment epithelium and localizes to hydroxyapatite deposits in dry age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3700.
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Drusen containing hydroxyapatite (HAP) spherules and nodules are implicated in the development and progression of age-related macular degeneration (AMD). It has been documented that sub-RPE deposit growth is associated with the precipitation of HAP and the binding of proteins to HAP surface, but the proteins involved in the mineralization process is yet to be identified. We have previously shown amelotin (AMTN), a tooth enamel protein is expressed in an RPE cell-culture model. Here, using human donor eye tissue we investigate AMTN expression in AMD.
Six normal and 22 AMD pairs of eyes from male and female donors with an age range of 65–96 years were obtained through National Disease Research Interchange. One of each pair was fixed in formaldehyde and the other was frozen. Eyes were genotyped for the major AMD-related polymorphism Y420H of complement factor H. For confocal microscopy, eyes were cut and sectioned through the macula. Immunohistochemistry was performed to identify AMD eyes with lesions and categorize for types of drusen. BaseScope® in situ hybridization probes were used for detection of AMTN mRNA and an anti-Amelotin antibody for AMTN protein. Xylenol Orange, Alizarin Red S and Bone-Tag 680RD were used to stain HAP.
In situ hybridization showed that AMTN is expressed in RPE of donor eyes with dry AMD, particularly in regions with soft drusen containing HAP spherules or nodules. AMTN was not found in hard drusen, normal RPE, or donor eyes diagnosed with wet AMD. Immunofluorescence labelling showed AMTN in RPE surrounding soft drusen. Furthermore, isolated drusen with large calcified spherules and nodules from tissue cross-sections showed heterogeneous HAP staining, with intense staining of the crusts whereas AMTN was localized in a ring around the HAP structures.
Our findings confirm that AMTN is expressed in diseased human RPE, specifically in dry AMD and suggest that AMTN due to its function as a promoter of HAP mineralization in enamel formation, may also be a key protein in the organization of HAP mineralization and deposition of sub-RPE deposits implicated in clinical AMD progression. AMTN provide a new possibility for therapeutic intervention, thus further experiments are needed to understand the mechanism of function of AMTN in HAP deposition in AMD.
This is a 2020 ARVO Annual Meeting abstract.
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