Mechanisms and consequences of impaired glutathione homeostasis in the aging Lens

The age-related decline of lens glutathione is strongly correlated with cataract formation in the human and in experimental mouse models of glutathione depletion, as recently confirmed in our own studies. The age- related impairment of lens glutathione biosynthesis enzyme activity has been documented, though the mechanisms underlying this age-related change are poorly understood. In preliminary studies we made the paradigm shifting observation that the lens glutathione biosynthesis key enzyme, gamma glutamylcysteine ligase, catalytic subunit (Gclc) is subject to posttranslational truncation, and that this truncated proteoform accumulates with age, particularly in the lens cortical and core fiber region. Further studies revealed that the truncation is linked to the presence of a caspase-3 and 6 like cleavage motif triggering a C-terminal 13kD truncation and accumulation of a N-terminal 60kD stable proteoform (herein named Gclc60). The latter appears to engage in strong interaction with cell cytoskeleton proteins and profound cytoplasmic distribution discrepancies compared to the full length Gclc protein. We hypothesize that truncation of Gclc is a key event in the pathogenesis of age-related cataract, and that understanding the mechanism of formation of Gclc60 and the molecular biological consequences of its accumulation are important goals for the design of novel anti-cataract therapy. Accordingly, the three Specific Aims of this application are Aim 1: to determine the mechanism of Gclc truncation and its impact on lens GSH homeostasis. In particular, we will test the hypothesis that Gclc60 will suppresses GSH synthesis resulting in lowered GSH content and will identify the proteases that are responsible for age-related cleavage. In Aim 2, we will determine the in vivo effects on GCLC truncation and Gclc60 accumulation by testing a cleavage resistant knockin (KI) mouse model. In particular we hypothesize that KI mouse lens will significantly retain its GSH levels and biological functions during aging. In Aim3, we will determine the biological effects resulting from Gclc60 interaction with cytoskeletal proteins, hypothesizing that Gclc accumulation results in cytoskeletal matrix disorganization. ! 1!