HuR Function and Translational State Analysis Following Global Brain Ischemia and Reperfusion

Abstract

Prolonged translation arrest in post-ischemic hippocampal CA1 pyramidal neurons precludes translation of induced stress genes and directly correlates with cell death. We evaluated regulation of mRNAs containing adenine and uridine rich elements (ARE) by assessing HuR protein and hsp70 mRNA nuclear translocation, HuR polysome binding, and translation state analysis of CA1 and CA3 at 8 hr reperfusion after 10 min global cerebral ischemia. There was no difference between CA1 and CA3 at 8 hr of reperfusion in nuclear or cytoplasmic HuR protein or hsp70 mRNA, or HuR polysome association, suggesting neither mechanism contributed to post-ischemic outcome. Translation state analysis revealed that 28% and 58% of unique mRNAs significantly different between 8hR and NIC, in CA3 and CA1, respectively, were not polysome-bound. There was significantly greater diversity of polysome-bound mRNAs in reperfused CA3 compared to CA1, and in both regions ARE-containing mRNAs accounted for 4% – 5% of the total. These data indicate that post-transcriptional ARE-containing mRNA regulation occurs in reperfused neurons and contributes to post-ischemic outcome. Understanding the differential responses of vulnerable and resistant neurons to ischemia will contribute to the development of effective neuroprotective therapies.

Embryonic lethal abnormal vision proteins and adenine and uridine-rich element mRNAs after global cerebral ischemia and reperfusion in the rat

“…The full lists of differentially expressed probe sets for the experimental groups are provided in Supplemental File 3. Consistent with our previous finding, 31 only a percentage of altered transcripts were polysome bound. The percent of polysome-bound transcripts was 63% in CA3 and only 21% in CA1, of the respective total changed probe sets.…”

“…Normothermic global forebrain ischemia in male Long Evans rats, weighing 275-300 g, used the twovessel bilateral carotid artery occlusion and hypovolemic hypotension (2VO/HT) model of Smith et al, 30 as we previously described. 13,14,[31][32][33] …”

“…31,33 PMS was used to obtain total RNA to avoid nascent transcripts from nucleus and mitochondria. All samples had A 260 / A 280 > 1.8 and were shipped overnight on dry ice to Genome Explorations (Memphis, Tennessee) who performed all microarray procedures.…”

“…Microarrays were performed as previously described 31 with two major differences. First, the present study used polysome pellets to obtain polysomal RNA.…”

Alpha-7 Nicotinic Receptor Signaling Pathway Participates in the Neurogenesis Induced by ChAT-Positive Neurons in the Subventricular Zone

“…injection of 4% chloral hydrate. Then the brain was carefully dissected out, and the right SVZ was dissected from the frontal slice extending between the crossing of the anterior commissure and the rostral opening of the third ventricle as previously described [34–36]. We used the Membrane and Nuclear Protein Extraction Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol.…”