Impaired Lens Development and Fiber Cell Disintegration in Transgenic Mice Expressing Acylpeptide Hydrolase

Abstract

Purpose:: Acylpeptide hydrolase (APH) catalyses the release of N-acetyl amino acids from proteins and peptides. There is a correlation between APH activity, crystallin breakdown and protein aggregates in vivo. To understand the role of APH in the lens, we generated transgenic mice over expressing APH.

Methods:: Minigenes that carry either the wild type (wt) active or mutant Ser₅₈₆→Ala (mt) inactive porcine liver APH cDNA were constructed using the modified αA-crystallin promoter (ΔenαA) vector. Transgenic mice were generated by pronuclear microinjection and genotyped by genomic DNA PCR. APH expression levels in transgenic mouse lenses were determined by SDS-PAGE and by protease activity analysis using Ac-Ala-p-nitroanilide as a substrate. The abnormalities in the transgenic mouse lens were analyzed by histology.

Results:: Seven transgenic lines expressing active APH (or wt-APH) and three expressing the inactive enzyme (or mt-APH) were established. Transgenic mice heterozygous for the transgene in 6 out of the 7 wt-APH lines developed cataracts. The onset and the severity of the cataracts correlated with the expression levels of the wt-APH transgene. Transgenic mice carrying the inactive mt-APH did not develop cataract despite having comparable levels of APH protein as in the wt-APH lens. At the level of gross morphology, the wt-APH lenses were smaller and broken down into a transparent anterior part and a cataractous posterior part. Histological analysis showed that the anterior portion of the transgenic lens was less affected whereas the posterior region was severely affected with degenerating fiber cells. Lens capsule rupture was seen at the posterior pole of the lens in severely affected wt-APH lines. Lens debris was present in the vitreous. The lens epithelial cells were abnormal and vacuolated. No apparent defects were found in the inactive mt-APH lenses.

Conclusions:: Overexpression of APH interferes with the proper formation of the lens structure and results in protein aggregation in lens fiber cells.

Keywords: transgenics/knock-outs • proteolysis • cataract