Although persistent translation arrest correlates with the selective vulnerability of post-ischemic hippocampal CA1 neurons, the mechanism of persistent translation arrest is not fully understood. Using fluorescent in situ hybridization and immunofluorescence histochemistry, we studied colocalization of polyadenylated mRNAs [poly(A)] with the following mRNA binding factors: eIF4G (translation initiation factor), HuR (ARE-containing mRNA stabilizing protein), poly(A) binding protein (PABP), S6 (small ribosomal subunit marker), TIA-1 (stress granule marker), and TTP (processing body marker). We compared staining in vulnerable CA1 and resistant CA3 from 1 to 48 hr reperfusion, following 10 min global ischemia in the rat. In both CA1 and CA3 neurons, cytoplasmic poly(A) mRNAs redistributed from a homogenous staining pattern seen in controls to granular structures we term mRNA granules. The mRNA granules abated after 16 hr reperfusion in CA3, but persisted in CA1 neurons to 48 hr reperfusion. Protein synthesis inhibition correlated precisely with the presence of the mRNA granules. In both CA1 and CA3, the mRNA granules colocalized with eIF4G and PABP, but not S6, TIA-1 or TTP, indicating that they were neither stress granules nor processing bodies. Colocalization of HuR in the mRNA granules correlated with translation of HSP70, which occurred early in CA3 (8hr) and was delayed in CA1 (36 hr). Thus, differential compartmentalization of mRNA away from the 40S subunit correlated with translation arrest in post-ischemic neurons, providing a concise mechanism of persistent translation arrest in post-ischemic CA1.

A persistent translation arrest (TA) correlates precisely with the selective vulnerability of post‐ischemic neurons. Mechanisms of post‐ischemic TA that have been assessed include ribosome biochemistry, the link between TA and stress responses, and the inactivation of translational components via sequestration in subcellular structures. Each of these approaches provides a perspective on post‐ischemic TA. Here, we develop the notion that mRNA regulation via RNA‐binding proteins, or ribonomics, also contributes to post‐ischemic TA. We describe the ribonomic network, or structures involved in mRNA regulation, including nuclear foci, polysomes, stress granules, embryonic lethal abnormal vision/Hu granules, processing bodies, exosomes, and RNA granules. Transcriptional, ribonomic, and ribosomal regulation together provide multiple layers mediating cell reprogramming. Stress gene induction via the heat‐shock response, immediate early genes, and endoplasmic reticulum stress represents significant reprogramming of post‐ischemic neurons. We present a model of post‐ischemic TA in ischemia‐resistant neurons that incorporates ribonomic considerations. In this model, selective translation of stress‐induced mRNAs contributes to translation recovery. This model provides a basis to study dysfunctional stress responses in vulnerable neurons, with a key focus on the inability of vulnerable neurons to selectively translate stress‐induced mRNAs. We suggest a ribonomic approach will shed new light on the roles of mRNA regulation in persistent TA in vulnerable post‐ischemic neurons.

Following global brain ischemia and reperfusion, it is well-established that neurons undergo a translation arrest that is reversible in surviving neurons, but irreversible in vulnerable neurons. We previously showed a correlation between translation arrest in reperfused neurons and the presence of granular mRNA-containing structures we termed “mRNA granules.” Here we further characterized the mRNA granules in reperfused neurons by performing colocalization studies using fluorescent in situ hybridization for poly(A) mRNAs and immunofluorescence histochemistry for markers of organelles and mRNA binding proteins. There was no colocalization between the mRNA granules and markers of endoplasmic reticulum, cis- or trans-Golgi apparatus, mitochondria, microtubules, intermediate filaments, 60S ribosomal subunits, or the HuR ligands APRIL and pp32. The mRNA granules colocalized with the neuronal marker NeuN regardless of the relative vulnerability of the neuron type. RNA immunoprecipitation of HuR from the cytoplasmic fraction of 8 hr reperfused forebrains selectively isolated hsp70 mRNA suggesting the mRNA granules are soluble structures. Together, these results rule out several organelle systems and a known HuR pathway as being directly involved in mRNA granule function.

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.

Texture analysis provides a means to quantify complex changes in microscope images. We previously showed that cytoplasmic poly-adenylated mRNAs form mRNA granules in post-ischemic neurons and that these granules correlated with protein synthesis inhibition and hence cell death. Here we utilized the texture analysis software MaZda to quantify mRNA granules in photomicrographs of the pyramidal cell layer of rat hippocampal region CA3 around 1 hour of reperfusion after 10 min of normothermic global cerebral ischemia. At 1 hour reperfusion, we observed variations in the texture of mRNA granules amongst samples that were readily quantified by texture analysis. Individual sample variation was consistent with the interpretation that animal-to-animal variations in mRNA granules reflected the time-course of mRNA granule formation. We also used texture analysis to quantify the effect of cycloheximide, given either before or after brain ischemia, on mRNA granules. If administered before ischemia, cycloheximide inhibited mRNA granule formation, but if administered after ischemia did not prevent mRNA granulation, indicating mRNA granule formation is dependent on dissociation of polysomes. We conclude that texture analysis is an effective means for quantifying the complex morphological changes induced in neurons by brain ischemia and reperfusion.