Hallmarks of lens aging and cataractogenesis.

Lens homeostasis and transparency are dependent on the function and intercellular communication of its epithelia. While the lens epithelium is uniquely equipped with functional repair systems to withstand reactive oxygen species (ROS)-mediated oxidative insult, ROS are not necessarily detrimental to lens cells. Lens aging, and the onset of pathogenesis leading to cataract share an underlying theme; a progressive breakdown of oxidative stress repair systems driving a pro-oxidant shift in the intracellular environment, with cumulative ROS-induced damage to lens cell biomolecules leading to cellular dysfunction and pathology. Here we provide an overview of our current understanding of the sources and essential functions of lens ROS, antioxidative defenses, and changes in the major regulatory systems that serve to maintain the finely tuned balance of oxidative signaling vs. oxidative stress in lens cells. Age-related breakdown of these redox homeostasis systems in the lens leads to the onset of cataractogenesis. We propose eight candidate hallmarks that represent common denominators of aging and cataractogenesis in the mammalian lens: oxidative stress, altered cell signaling, loss of proteostasis, mitochondrial dysfunction, dysregulated ion homeostasis, cell senescence, genomic instability and intrinsic apoptotic cell death.

Postnatal Development of Spasticity Following Transgene Insertion in the Mouse ßIV Spectrin Gene (SPTBN4).

BACKGROUND: The L25 mouse line was generated by random genomic insertion of a lens-specific transgene. Inbreeding of L25 hemizygotes revealed an unanticipated spastic phenotype in the hind limbs. OBJECTIVE: The goals were to characterize the motor phenotype in the L25 mice and to map the transgene insert site within the mouse genome. METHODS: Six pairs of L25+/- mice were repeatedly mated. Beginning at weaning, all progeny were inspected for body weight and motor signs twice weekly until they displayed predefined ethical criteria for termination. The transgene insert site was determined by whole genome sequencing. Western blotting was used to compare the expression levels of beta-IV spectrin protein in the brain. RESULTS: Matings of hemizygous L25+/- × L25+/- mice yielded 20% (29/148) affected weanlings, identified by an abnormal retraction of the hind limbs when lifted by the tail, and a fine tremor. Affected mice were less mobile and grew more slowly than wild-type littermates. All affected mice required termination due to >15% loss of body weight (50% survival age 92 days). At the endpoint, mice showed varying degrees of spastic paresis or spastic paralysis localised to the hind limbs. Motor endplates remained fully innervated. Genome sequencing confirmed that the transgene was inserted in the locus of ßIV spectrin of L25 mice. Western blotting indicated that this random insertion had greatly reduced the expression of ßIV spectrin protein in the affected L25 mice. CONCLUSIONS: The results confirm the importance of ßIV spectrin for maintaining central motor pathway control of the hind limbs, and provide a developmental time course for the phenotype.

Nox4 Plays a Role in TGF-ß-Dependent Lens Epithelial to Mesenchymal Transition.

PURPOSE: Transforming growth factor-ß induces an epithelial to mesenchymal transition (EMT) in the lens, presented as an aberrant growth and differentiation of lens epithelial cells. Studies in other models of EMT have shown that TGF-ß-driven EMT is dependent on the expression of the reactive oxygen species (ROS)-producing enzyme nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase-4 (Nox4). We investigate the role of this enzyme in TGF-ß-induced lens EMT and determine whether it is required for this pathologic process. METHODS: Rat lens epithelial explants were used to investigate the role of Nox4 in TGF-ß-driven lens EMT. Nox1-4 expression and localization was determined by immunolabeling and/or RT-PCR. NADPH-oxidase-produced ROS were visualized microscopically using the fluorescent probe, dihydroethidium (DHE). VAS2870, a pan-NADPH oxidase inhibitor, was used to determine the specificity of Nox4 expression and its role in ROS production, and subsequently TGF-ß-driven EMT. RESULTS: We demonstrate, for the first time to our knowledge, in rat lens epithelial explants that TGF-ß treatment induces Nox4 (but not Nox1-3) expression and activity. Increased Nox4 expression was first detected at 6 to 8 hours following TGF-ß treatment and was maintained in explants up to 48 hours. At 8 hours after TGF-ß treatment, Nox4 was observed in cell nuclei, while at later stages in the EMT process (at 48 hours), Nox4 was predominately colocalized with α-smooth muscle actin. The inhibition of Nox4 expression and activity using VAS2870 inhibited EMT progression. CONCLUSIONS: Transforming growth factor-ß drives the expression of the ROS-producing enzyme Nox4 in rat lens epithelial cells and Nox4 inhibition can impede the EMT process.