Cerebral ischemia leads to translation arrest, and the inability to recover from this state correlates with neuronal cell death following ischemia/reperfusion injury. While each stage of translation (initiation, elongation, and termination) can be regulated, targeting translation initiation serves as an efficient and expeditious means of translation arrest. Ischemia reduces expression of eukaryotic initiation factors 2 and 4 (eIF2 and eIF4) which are essential for formation of the translation initiation complex;specifically, eIF2B mediates the exchange of GDP bound to eIF2 for GTP, a key regulatory step for the control of translation initiation in eukaryotes, and eIF4E functions to bin an mRNA cap and bring it to the ribosome. However, all of the mechanisms underlying ischemia-induced suppression of eIF proteins are not known. MicroRNAs (miRNAs) are small, non-coding RNAs which regulate post-transcriptional gene expression by repressing or degrading mRNA targets. Using real-time quantitative PCR profiling of miRNAs, we uncovered a universal ischemia-induced miRNA profile aimed at silencing eIF proteins in both male and female brain. Our central hypothesis is that this common subset of ischemia-induced miRNAs represses eIF2 and eIF4, disrupting the translation initiation complex and resulting in translation arrest. This proposal is innovative as it is the first to directly examine a role for miRNA- mediated regulation of eIFs as a mechanism underlying translation arrest following cerebral ischemia.
Aim 1 will determine the effects of ischemia on miRNA and eIF2B and eIF4E expression, and on translation arrest. We hypothesize that ischemia-induced miRNAs suppress eIF2B and eIF4E expression, resulting in translation arrest. Studies will be conducted in mice in vivo to confirm the effects of ischemia on (a) induced expression of candidate miRNAs and suppression of eIF2B and eIF4E protein in cerebral cortex, (b) the regional and cellular distribution of candidate miRNAs and eIF2B and eIF4E mRNA and protein in brain, and (c) translation arrest in cerebral cortex using polysome profiling.
Aim 2 will clarify the role of miRNAs on eIF2B and eIF4E expression, translation arrest and neuronal cell death. We hypothesize that miRNAs directly repress eIF2B and eIF4E, which disrupts mRNA translation and ultimately leads to cell death in response to ischemia. We will use in vitro studies to (a) functionally assess the ability of candidate miRNAs to regulate eIF2B and eIF4E expression in a luciferase reporter assay system, (b) examine effects of miRNA mimics and inhibitors on endogenous eIF2B and eIF4E expression and translation arrest in cultured neurons, and (c) examine effects of miRNA mimics and inhibitors, and of direct manipulation of eIF2B and eIF4E expression, on neuronal cell death following oxygen- glucose deprivation. The proposed work is significant as new insights will be gained about miRNAs in the brain's universal response to injury relative to translation arrest and how this might influence brain health and disease in men and women. Our findings could also lead to development of novel stroke therapies based on miRNAs and their potential for regulating translation initiation in ischemic brain.
The proposed research is relevant to public health because it will address gaps in our knowledge regarding the role of microRNAs as key effectors of translation arrest and neuronal cell death following stroke. Our findings will also establish whether microRNAs and/or eukaryotic translation initiation factors could serve as novel targets for the development of neuroprotective strategies to prevent, treat, and promote recovery from ischemic brain injury. The proposed studies are also relevant to the mission of NINDS to reduce the burden of neurological diseases through research on the causes and treatment of stroke.