Open Access Dissertation
Date of Award
Donald J. DeGracia
The final cause of death in most patients revived after cardiac arrest is ischemia and reperfusion (I/R) injury in the brain. Survival after brain I/R injury depends on the expression of new stress response proteins such as heat shock protein 70 (HSP70). Little is known about why recovering neurons are able to express new stress response proteins while neurons that will die can transcribe RNA but do not translated protein in early reperfusion. Previous studies suggested that the mRNA-binding protein HuR may regulate hsp70 mRNA in reperfused neurons through a novel cytoplasmic structure, the mRNA granule. To determine the roles of HuR and the mRNA granule in reperfused neurons and to characterize global translation regulation, we 1) prevented mRNA granule formation in reperfused neurons or induced mRNA granules in uninjured neurons by pharmacological manipulation polysomes, 2. studied potential HuR posttranscriptional regulation mechanisms of facilitated nuclear export and polysome association, and 3. performed translation state analysis of reperfused neurons from hippocampal subregions CA1 and CA3.
We found that mRNA granules can be prevented in reperfused neurons by locking mRNA onto polysomes with cycloheximide. In uninjured neurons, mRNA granules can be induced with puromycin, which promotes early translation termination and dissociation of ribosomes from mRNA. In hippocampal neurons after 10 minutes global brain ischemia and 8 hours reperfusion, HuR remained constant in nuclear and cytoplasmic fractions and unfractionated homogenate. Nucleocytoplasmic distribution of hsp70 mRNA was identical between ischemia-resistant CA3 and ischemia-vulnerable CA1 neurons at 8 hours reperfusion. HuR's distribution on polysome profiles was unchanged after 8 hours reperfusion, and HuR did not localize to polysomes in response to I/R injury. Translation state analysis of CA1 and CA3 neurons showed that the two regions have largely different mRNA populations on their polysomes at 8 hours reperfusion, and polysome-bound mRNA from both regions was enriched in the adenine and uridine rich element (ARE), a 3' untranslated region regulatory site.
In conclusion, polysome-associated mRNA is necessary for mRNA granule formation. HuR may regulate expression of stress response proteins, but does not do so by any known mechanism. Ischemia-vulnerable CA1 and ischemia-resistant CA3 neurons have different mRNA populations associated with polysomes at 8 hours reperfusion after global ischemia, but both are enriched in AREs.
Szymanski, Jeffrey J., "Investigation Of Posttranscriptional Regulation After Global Brain Ischemia And Reperfusion Injury" (2012). Wayne State University Dissertations. 705.