Altered ubiquitin signaling in mammalian lens development causes metabolic and mitochondrial dysfunction leading to cataractogenesis

Abstract

Purpose : Ubiquitin signaling participates in multiple critical functions in lens development and maintenance of lens transparency. Impaired ubiquitin signaling, via transgenic misexpression of a mutant ubiquitin protein (K6W-Ub) in the mouse, causes a severe congenital cataract, characterized by inappropriate calpain activation leading to altered calcium signaling, abrogation of lens fiber cell denucleation, and protein aggregation. We have undertaken a multiomics approach to evaluate the proteome and metabolome of K6W-Ub transgenic lenses to uncover the molecular mechanisms underlying cataractogenesis.

Methods : Lenses from E15.5, P1, or P30 K6W-Ub transgenic or WT littermates were analyzed via mass spectrometry to characterize the proteome. Computational molecular phenotyping (CMP) and ¹H-NMR were utilized to characterize the metabolome. Mitochondrial function was ascertained ultrastructurally via electron microscopy and using inhibitor studies.

Results : Proteomic analysis revealed that K6W-Ub expression led to an increase in enzymes involved in glycolysis, citric acid cycle, and amino acid metabolism, including the rate-limiting enzyme PGK1. Spatial analysis of the P1 K6W-Ub metabolome via CMP revealed an increase in levels of alanine and decreases in levels of taurine, glutathione, and glutamine. Aspartate and cysteine had abnormal variegated levels. P1 K6W-Ub mitochondria were increased in number and had unusual swollen cristae. Inhibitor studies using the depolarizing drug FCCP indicated that K6W-Ub expressing human lens epithelial cells were more sensitive to FCCP than WT-Ub expressing cells.

Conclusions : Altered ubiquitin signaling appears to alter glycolytic and oxidative metabolism in the lens, which leads to abnormal build-up or depletion of key glycolytic amino acid precursors. Mitochondrial function is impaired. Altered function of K6 on Ub also appears to alter mitophagic removal of damaged mitochondria. These metabolic alterations may contribute to cataract development. Analysis of the metabolome of the K6W-Ub cataractous lens shows significant overlap with human cataract samples, as well as with the aging mouse lens. Together, our findings illuminate multiple roles of ubiquitin signaling in the lens that extend beyond proteolysis and that may be common signatures of cataractogenesis.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.