Kijin Jang and Hans Tómas Björnsson
Introduction: Targeted temperature management (TTM) is the process of actively lowering core temperature (32°C-35°C) to attenuate neurological damage after catastrophic events. The mild hypothermia response (MHR) is an evolutionarily conserved mammalian cytoprotective program which is thought to mediate the neuroprotective effect of TTM. Currently, only a few key genes such as SP1 and RBM3 have been robustly shown to be part of the MHR. This study aims to identify previously unreported MHR regulators.
Methods: We selected 16 genes from a list of 1,100 candidate genes identified from a prior large CRISPR-Cas9 forward screen. We knocked down each of these genes using siRNA and read out impact using mild hypothermia indicators (MHIs) which fluoresce upon activation of key MHR genes SP1 and RBM3, respectively. Genes that showed promising results (7), were further evaluated by RT-qPCR of the endogenous locus.
Results: The knockdown of 7 of the 16 genes showed a significant (p<0.05) increase of SP1-MHI fluorescence at 37°C indicating a potential repressive role for SP1. Of these, 2 genes (CALHM2, TMEM66) showed a significant (p<0.05) increase in RBM3-MHI fluorescence suggesting these are shared regulators for both genes. Finally, 3 genes showed corresponding expression changes at the endogenous locus. Conclusion: This study identified two novel regulators of the MHR through screening of candidates from a large CRISPR-Cas9 screen. Further evaluation of the genes will lead to a deeper understanding of the MHR and may lead to therapeutic targets for treatment development for catastrophic neurological damage.
