Real-time analysis of dynamic compartmentalized GSH redox shifts and HO availability in undifferentiated and differentiated cells.

Background: Glutathione (GSH) is the most abundant, small biothiol antioxidant. GSH redox state (E) supports developmental processes, yet with disrupted GSH E, poor developmental outcomes may occur. The role of subcellular, compartmentalized redox environments in the context of redox regulation of differentiation is not well understood. Here, using the P19 neurogenesis model of cellular differentiation, kinetics of subcellular HO availability and GSH E were evaluated following oxidant exposure.

Methods: Stably transfected P19 cell lines expressing HO availability or GSH E sensors, Orp1-roGFP or Grx1-roGFP, respectively, targeted to the cytosol, mitochondria, or nucleus were used. Dynamic, compartmentalized changes in HO availability and GSH E were measured via spectrophotometric and confocal microscopy over 120 min following treatment with HO (100 μM) in both differentiated and undifferentiated cells.

Results: Generally, treated undifferentiated cells showed a greater degree and duration of both HO availability and GSH E disruption than differentiated neurons. In treated undifferentiated cells, HO availability was similar in all compartments. Interestingly, in treated undifferentiated cells, mitochondrial GSH E was most affected in both the initial oxidation and the rebound kinetics compared to other compartments. Pretreatment with an Nrf2 inducer prevented HO-induced effects in all compartments of undifferentiated cells.

Conclusions: Disruption of redox-sensitive developmental pathways is likely stage specific, where cells that are less differentiated and/or are actively differentiating are most affected.

General Significance: Undifferentiated cells are more susceptible to oxidant-induced redox dysregulation but are protected by chemicals that induce Nrf2. This may preserve developmental programs and diminish the potential for poor developmental outcomes.