SNRK, a member of the SNF1 family, is related to low K(+)-induced apoptosis of cultured rat cerebellar granule neurons

Abstract

When cerebellar granule neurons obtained from 11-day-old rats were cultured first in high K(+) medium for 4 days, followed by culture in low K(+) medium, the neurons underwent apoptosis and died. This cell death was prevented by actinomycin D, an inhibitor of RNA synthesis. Commitment time of the protective effect of RNA synthesis inhibition on the cell death was examined by adding actinomycin D at various time points after the switch to the low K(+) medium. More than 50% of the cells died when actinomycin D was added 3 h after changing to the low K(+) medium. To identify what kinds of newly synthesized genes are involved in regulation of the low K(+)-induced death, we performed PCR-based differential subtraction analysis using RNA prepared from the cultured neurons 0 and 3 h after changing to low K(+) medium. We isolated a clone that showed an increase in its mRNA level after changing to the low K(+) medium. This clone encoded the 3' untranslated region of SNRK, a serine/threonine kinase. Tissue distribution analysis showed that the mRNA was expressed mainly in the brain and testis. Developmental analysis in the brain showed that the mRNA expression increased in an age-dependent manner until P28, and was slightly decreased in adults. In situ hybridization analysis showed that the mRNA was expressed throughout the brain. The mRNA was shown to be expressed in neurons by double staining with anti-MAP2 antibody. In addition, anti-N-terminal SNRK antibody stained the nuclei of cultured rat cerebellar granule neurons. These results suggested that SNRK may be involved in regulation of low K(+)-induced apoptosis of cultured cerebellar granule neurons.