Establishment of a transgenic mouse line for monitoring exposure history to electrophilic stress

<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>KEAP1‐NRF2 system is a major response mechanism against electrophilic stress. In normal condition, NRF2 is ubiquitinated by the KEAP1‐CUL3 ubiquitin E3 ligase complex in the cytoplasm, resulting in degradation through the proteasome. On the exposure to electrophilic stress, KEAP1 is inactivated leading to nuclear translocation of NRF2 and inducible expression of antioxidant proteins and detoxification enzymes. NRF2 activation caused by electrophilic phytochemicals has been shown beneficial for various human disorders including neurodegeneration such as Alzheimer’s disease. However, due to the transient and fluctuating nature of the NRF2 activation, it has not been easy to detect the electrophilic response, <jats:italic>i.e</jats:italic>., NRF2 activation, at a single‐cell resolution.</jats:p></jats:sec><jats:sec><jats:title>Method</jats:title><jats:p>In order to establish an <jats:italic>in vivo</jats:italic> detection system of NRF2 activation at a single‐cell resolution, we generated a unique mouse model by utilizing Neh2 domain of NRF2. Neh2 domain is located at the N‐terminus of Nrf2 and known as a degron mediating ubiquitination and degradation of NRF2 in KEAP1 dependent manner. We first established a transgenic mouse line expressing Neh2‐Cre recombinase fusion protein ubiquitously under the regulation of mouse <jats:italic>Mafg</jats:italic> gene regulatory region (MGRD‐Neh2‐Cre mouse). Secondly, we crossed the transgenic mouse with Rosa‐CAG‐LSL‐tdTomato mouse and obtained MGRD‐Neh2‐Cre:Rosa‐CAG‐LSL‐tdTomaato (Neh2‐Cre:tdTomato) mouse. In this mouse, Neh2‐Cre was expected to be stabilized under electrophilic stress and subsequently induce expression of tdTomato by recombining the Rosa locus.</jats:p></jats:sec><jats:sec><jats:title>Result</jats:title><jats:p>Although a single dose of an electrophilic compound, CDDO‐Im, did not induce tdTomato expression in Neh2‐Cre:tdTomato mice, repeated administration of CDDO‐Im gradually increase the number of cells expressing tdTomato in various organs. Furthermore, the mice continuously treated with 6‐methylsulfinylhexyl Isothiocyanate (6‐MSITC) in drinking water for 4‐5 months, which is a weak electrophile compared with CDDO‐Im, exhibited tdTomato expression in various organs including brain. These results suggest that 6‐MSITC as well as CDDO‐Im activates KEAP1‐NRF2 system in the central nervous system.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>We consider that the established Neh2‐Cre:tdTomato mouse is especially useful to monitor the cumulative weak response to chronic/repetitive electrophilic stimuli. Our study suggests that 6‐MSITC as well as CDDO‐Im can be applied for therapeutic approaches of aging‐related neurodegenerative disorders by activating NRF2 in the central nervous system.</jats:p></jats:sec>