Calcification of Bruch’s membrane and RPE-associated functional changes in the ENPP1 transgenic mice

Purpose : In the absence of pathology, calcification is restricted to bones and teeth. Calcium and phosphate are strictly regulated to avoid ectopic calcification. However, in diseases like age-related macular degeneration (AMD), pseudoxanthoma elasticum (PXE), and generalized arterial calcification in infancy (GACI) ectopic calcification of Bruch’s membrane can lead to vision loss. In GACI, ectopic calcification is the result of the mutations of the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). In this study, we investigate the effects of exon 9 deletion in the ENPP1 gene on Bruch’s membrane calcification, examining the surrounding ocular tissues to understand this pathological change.

Methods : Transgenic mice were generated by deleting the functional domain on exon 9 of ENPP1. Animals were aged for up to 6 months. Retinal changes were assessed by colour fundus photography (CFP, n=9), optical coherence tomography (OCT, n=5), and electroretinography (ERG, n=9). In addition, murine eyes were assessed for calcification using hydroxyapatite-specific fluorescent staining (Osteosense-680RD) and confocal microscopy. Transcriptional changes were assessed by RNA-sequencing. ENPP1-/- (transgenic) animals were compared to ENPP1+/- (heterozygous) and ENPP1+/+ (wildtype) littermate controls.

Results : A higher number of inflammation-like white lesions was identified in the transgenic animals using CFP, when compared to control animals (47±7 vs. 19±5). Retinal changes were assessed by OCT and showed no structural differences among the groups. ERG measurements showed no changes in either the scotopic a-wave or b-wave. However, transgenic animals showed a ~50% decreased c-wave amplitude compared to wildtype controls (p<0.01). Osteosense labelling confirmed that there is ectopic calcification in the Bruch’s membrane of mutant mice. RNA-seq analysis of the neurosensory retina showed no differential expression between the groups.

Conclusions : This study shows that the inactivation of ENPP1 through gene mutation causes multiple changes in the eyes of transgenic mice, including Bruch’s membrane calcification. The most likely cause of this calcification is changes in the RPE cells. We propose that the ENPP1 transgenic animals are a valuable pre-clinical model to study Bruch’s membrane calcification and the cellular and molecular changes leading to this pathological calcification in the eye.