Translation arrest and ribonomics in post-ischemic brain: layers and layers of players.

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.

Plasma Lactate Levels Increase during Hyperinsulinemic Euglycemic Clamp and Oral Glucose Tolerance Test.

Insulin resistance, which plays a central role in the pathogenesis of type 2 diabetes (T2D), is an early indicator that heralds the occurrence of T2D. It is imperative to understand the metabolic changes that occur at the cellular level in the early stages of insulin resistance. The objective of this study was to determine the pattern of circulating lactate levels during oral glucose tolerance test (OGTT) and hyperinsulinemic euglycemic clamp (HIEC) study in normal nondiabetic subjects. Lactate and glycerol were determined every 30 minutes during OGTT and HIEC on 22 participants. Lactate progressively increased throughout the HIEC study period (P < 0.001). Participants with BMI < 30 had significantly higher mean M-values compared to those with BMI ≥ 30 at baseline (P < 0.05). This trend also continued throughout the OGTT. In addition, those with impaired glucose tolerance test (IGT) had significantly higher mean lactate levels compared to those with normal glucose tolerance (P < 0.001). In conclusion, we found that lactate increased during HIEC study, which is a state of hyperinsulinemia similar to the metabolic milieu seen during the early stages in the development of T2D.

mRNA redistribution during permanent focal cerebral ischemia.

Translation arrest occurs in neurons following focal cerebral ischemia and is irreversible in penumbral neurons destined to die. Following global cerebral ischemia, mRNA is sequestered away from 40S ribosomal subunits as mRNA granules, precluding translation. Here, we investigated mRNA granule formation using fluorescence in situ histochemistry out to 8 h permanent focal cerebral ischemia using middle cerebral artery occlusion in Long Evans rats with and without diabetes. Neuronal mRNA granules colocalized with PABP, HuR, and NeuN, but not 40S or 60S ribosomal subunits, or organelle markers. The volume of brain with mRNA granule-containing neurons decreased exponentially with ischemia duration, and was zero after 8 h permanent focal cerebral ischemia or any duration of ischemia in diabetic rats. These results show that neuronal mRNA granule response has a limited range of insult intensity over which it is expressed. Identifying the limits of effective neuronal stress response to ischemia will be important for developing effective stroke therapies.

Polyadenylated mRNA staining reveals distinct neuronal phenotypes following endothelin 1, focal brain ischemia, and global brain ischemia/ reperfusion.

OBJECTIVES: Most work on ischemia-induced neuronal death has revolved around the relative contributions of necrosis and apoptosis, but this work has not accounted for the role of ischemia-induced stress responses. An expanded view recognizes a competition between ischemia-induced damage mechanisms and stress responses in the genesis of ischemia-induced neuronal death. An important marker of post-ischemic stress responses is inhibition of neuronal protein synthesis, a morphological correlate of which is the compartmentalization of mRNA away from ribosomes in the form of cytoplasmic mRNA granules. METHODS: Here we assessed the generality of this mRNA granule response following either 10 or 15 minutes global brain ischemia and 1 hour reperfusion, 4 hours focal cerebral ischemia alone, and endothelin 1 intraventricular injection. RESULTS: Both global and focal ischemia led to prominent neuronal cytoplasmic mRNA granule formation in layer II cortical neurons. In addition, we report here new post-ischemic cellular phenotypes characterized by the loss of nuclear polyadenylated mRNA staining in cortical neurons following endothelin 1 treatment and 15 minutes global ischemia. Both mRNA granulation and loss of nuclear mRNAs occurred in non-shrunken post-ischemic neurons. DISCUSSION: Where cytoplasmic mRNA granules generally appear to mark a protective response in surviving cells, loss of nuclear mRNAs may mark cellular damage leading to cell atrophy/death. Hence, staining for total mRNA may reveal facets of the competition between stress responses and damage mechanisms at early stages in post-ischemic neurons.