Molecular approach to ameliorate the age-dependent loss of Clock protein Bmal1 and Nrf2-antioxidant response in eye lens: Implications for eye lens health span

About This Grant

Abstract In age-related cataracts (ARC), increasing levels of oxidative stress due to dysfunction of Nrf2 (NFE2-related factor)-mediated antioxidant defense is widely recognized, but much remains unexplored. Nrf2 is known to extend health/life span by maintaining redox homeostasis. However, with advancing age Nrf2 antioxidant function deteriorates, resulting in etiopathobiology. A better understanding of the molecular regulation of the age-dependent dysfunction of Nrf2-antioxidant pathway and its link to aging-related pathobiology and thereby, finding the way(s) to ameliorate the Nrf2-antioxidant pathway can offer an opportunity to prevent ARC. Recently, our in-vitro/in-vivo/ex-vivo studies with aging lens/lens epithelial cells (LECs) discovered that the anti- aging drug, Metformin (Met) is efficacious in activating the deteriorated Nrf2/ARE (antioxidant response element) antioxidant pathway via AMPK activation, resulting in lens/LECs protection. Notably, we identified that Nrf2 and antioxidant genes, like Prdx6, can be synergistically activated via Bmal1 (brain and muscle Arnt-like protein 1)/E-Box and Nrf2/ARE activities. Pursuing this line, we found that Met negatively regulates both the expression/activity of BTB and CNC homology 1 (Bach1), an inhibitor of Nrf2 at ARE site(s), and revives Nrf2/ARE pathway. Preliminary data also showed that Bmal1 or Nrf2 deficiency and increase of Bach1 levels in aging lenses/LECs attenuate the Nrf2/ARE pathway. We envisioned that with aging, within the eye lens, increased oxidative stress due to deterioration of Bmal1/Nrf2/ARE pathway creates a toxic milieu promoting disease state, ARC. The molecular pathways underlying this process, however, remain elusive. Using Met as a tool, the overall goal of this proposal is to determine the mechanisms underlying dysregulation of the AMPK/Bmal1/Nrf2/ARE axis gene networks axis in aging eye lenses/ARC, and to examine the transcriptional reprogramming of this axis and its contribution in abating aging pathobiology in response to Met by using state- of-the-art RNA-Seq with biochemical approaches. Our initial data using each of these factors also showed that; (i) Bach1 overexpression inhibits Nrf2 activation of antioxidants transcription, while Met treatment revives the process; (ii) Cotreatment with an AMPK inhibitor blocks Met activation of Bmal1/Nrf2/ARE pathway; (iii) Met instillation in aging mice eye, retards the lens pathobiology in-vitro/ex-vivo/in-vivo. These promising data support the hypothesis that the dysregulation of Bmal1/Nrf2 gene networks can be revived by Met. We will test the hypothesis via three specific aims: (1) Uncover the causes and the mechanisms of Bmal1/Nrf2 antioxidant pathway dysregulation in aging/oxidative stress in vitro. (2) Determine the mechanism(s) of Met- mediated revival of Bmal1/Nrf2/antioxidant pathway and other target genes, and attenuation of lens opacity in- vitro. (3) Determine the mechanisms underlying Met-mediated activities in preventing lens opacity in a mouse model(s) of ARC in-vivo. The mechanisms of pathological signaling and role of Met uncovered in this proposal should have a translational impact on increasing aging-related blinding diseases and improving public health.