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Gustavo D Aguirre, Louise M Downs, Sem Genini, Erin M. Scott, Simone Iwabe, William A Beltran; Photoreceptor development, degeneration and retinal gene expression in the canine NPHP5 Leber congenital amaurosis model. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5420.
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© ARVO (1962-2015); The Authors (2016-present)
A cytosine insertion in exon 10 of NPHP5 (IQCB1) results in an early onset, aggressive photoreceptor (PR) ciliopathy in dogs. This study characterized the kinetics of PR loss both functionally and structurally, and the expression profiles of PR-specific genes and of signaling pathways involved in PR degeneration.
Affected and control dogs were tested by full field ganzfeld ERGs recorded at 6, 14, and 33 wks of age. Retinas were embedded in OCT, and examined with H&E, TUNEL labeling and immunohistochemistry; the sections extended from the optic nerve to the ora serrata along the four cardinal meridians. Differential expression of selected genes in age-matched (5 wks) WT and mutant NPHP5 dogs was analyzed with a canine-specific profiling array. The ddCt method using GAPDH for normalization and an unpaired t-test (p<0.05 and fold change >2.0) were applied to identify differentially expressed genes.
Cone ERG loss was present by 6 wks of age, while rod responses were present but reduced ~60->95%. By 14 wks the rod signals were further reduced, and the ERG was extinguished at 33 wks. Cone OS were irregular and short at 6 weeks, and IS were broad and stunted; by 14 wks the OS further degenerated. A marked difference in ONL thickness between affected and control dogs was apparent at 33 weeks of age. At 42 wks, only remnants of cone somas were present next to the external limiting membrane, and rod degeneration was extensive. The proportion of dying photoreceptors, particularly rods, was highest at 6 weeks and reduced by 50% between 14 and 42 weeks of age. Downregulation of PR gene expression was present early, but was not uniform for all genes examined. Genes expressed in cones and rods (IRBP, NPHP5, PRPH2, RPGRIP1) or exclusively in rods (CNGA1, CNGB1, GRK1, PDE6B, RHO) were significantly downregulated. As is common in other early onset canine RD models, early downregulation of XIAP was present, but there was no uniform activation of members of the TNF gene superfamily.
Characterization of the disease metrics in terms of structure, cell death kinetics, ERG function and retinal gene expression profiles will be used to establish outcome measures to be used in forthcoming gene augmentation and other therapeutic interventions in this large animal ciliopathy model.
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