Integrated computational and experimental approach to identify Nrf2-regulated molecular targets in cerebral ischemia

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of the cellular antioxidant response, playing an important role in protecting neurons from ischemic injury. The hippocampus exhibits region-specific vulnerability to ischemia, with CA1 neurons being highly susceptible, while CA2-3 and dentate gyrus (DG) neurons demonstrate greater resistance. Our previous work revealed higher basal and post-ischemia/reperfusion (I/R) Nrf2 activity in the resistant CA2-3,DG region compared to CA1. This study aimed to identify potential Nrf2-regulated genes that contribute to this regional neuroprotection in a gerbil model of global cerebral ischemia.

We used a combined computational and experimental approach. By utilizing the mouse Hipposeq database and Nrf2 target gene lists from the GSEA Molecular Signatures Database, we identified 15 candidate genes with predicted roles in the CA2-3,DG stress response. Quantitative real time-PCR and Western blot analysis were then used to validate expression patterns in the gerbil hippocampus following I/R.

The analysis confirmed distinct expression patterns. Although some genes, including MPP3, RET, and SHISA2, showed higher basal expression in CA2-3,DG, they were unexpectedly downregulated after I/R. In contrast, others, such as AIFM2, BRIP1, and CAMK1, were specifically upregulated in this region. Furthermore, some (GPC1) showed delayed upregulation or showed altered protein levels despite unchanged mRNA expression (FZD7, STC2).

These results emphasize the regional and time-dependent regulation of gene expression in the hippocampus after I/R. The identified up- and downregulated genes represent novel molecular targets whose pharmacological modulation could enhance endogenous neuroprotective pathways, revealing new therapeutic avenues for stroke.