Generation of a new mouse model of glaucoma characterized by reduced expression of the AP-2\u03b2 and AP-2\u03b4 proteins

Maria Monica Barzago,Mami Kurosaki,Enrico Garattini,Mineko Terao,Marco Bolis,Laura Nordio,Maddalena Fratelli,Chiara Giudice,Luciano Domenici,Elisa Cerri

doi:10.1038/s41598-017-11752-6

Abstract

We generated 6 transgenic lines with insertion of an expression plasmid for the R883/M xanthine dehydrogenase (XDH) mutant protein. Approximately 20% of the animals deriving from one of the transgenic lines show ocular abnormalities and an increase in intra-ocular pressure which are consistent with glaucoma. The observed pathologic phenotype is not due to expression of the transgene, but rather the consequence of the transgene insertion site, which has been defined by genome sequencing. The insertion site maps to chromosome 1qA3 in close proximity to the loci encoding AP-2β and AP-2δ, two proteins expressed in the eye. The insertion leads to a reduction in AP-2β and AP-2δ levels. Down-regulation of AP-2β expression is likely to be responsible for the pathologic phenotype, as conditional deletion of the Tfap2b gene in the neural crest has recently been shown to cause defective development of the eye anterior segment and early-onset glaucoma. In these conditional knock-out and our transgenic mice, the morphological/histological features of the glaucomatous pathology are surprisingly similar. Our transgenic mouse represents a model of angle-closure glaucoma and a useful tool for the study of the pathogenesis and the development of innovative therapeutic strategies.

Highlights

Glaucoma is one of the leading causes of blindness and it is characterized by a global prevalence of 3.5% in the population aged 40–80 years[1]
During the course of functional studies on the mouse molybdo-flavoenzyme, xanthine dehydrogenase (XDH)[12,13,14,15,16, 26], we generated 6 independent transgenic lines with stable insertion of an expression plasmid (Fig. 1A) containing the cDNA coding for a R883/M mutant of the XDH protein (Mut-XDH), which is characterized by acquisition of a different substrate specificity relative to the parental enzyme[21]
The histological findings were consistent with defective development of the anterior segment and filtration angle closure leading to glaucoma

Summary

Introduction

Glaucoma is one of the leading causes of blindness and it is characterized by a global prevalence of 3.5% in the population aged 40–80 years[1]. To evaluate the potential of the mutant protein to act as a dominant-negative factor and to silence the native enzyme in vivo, we generated Mut-XDH transgenic mice. The present study reports on the characterization of a Mut-XDH transgenic line of mice which, unexpectedly, is characterized by increased intra-ocular pressure (IOP) and post-natal development of ocular abnormalities consistent with angle-closure glaucoma. Overall our data support the idea that the glaucomatous phenotype is not caused by expression of Mut-XDH, but it is rather due to the serendipitous transgene insertion into a small region of chromosome 1 in close proximity to the Tfap2b gene coding for AP-2β. The evidence gathered from the characterization of our transgenic animal provides independent support to the idea that deficits in the expression of AP-2β cause angle-closure glaucoma in mice. Our study underlines the importance of defining the insertion site of a transgene into the mouse genome, as phenotypic traits observed in genetically engineered mice may not be always attributable to a direct action of the transgene itself

Methods

Results

Conclusion