RESUMEN
Prolonged translation arrest in post-ischemic hippocampal CA1 pyramidal neurons precludes translation of induced stress genes and directly correlates with cell death. We evaluated the 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 h of reperfusion after 10 min of global cerebral ischemia. There was no difference between CA1 and CA3 at 8 h of reperfusion in nuclear or cytoplasmic HuR protein or hsp70 mRNA, or HuR polysome association, suggesting that 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 posttranscriptional 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.
Asunto(s)
Isquemia Encefálica/metabolismo , Región CA1 Hipocampal/metabolismo , Región CA3 Hipocampal/metabolismo , Neuronas/metabolismo , ARN Mensajero/metabolismo , Daño por Reperfusión/metabolismo , Adenina/metabolismo , Animales , Western Blotting , Proteínas ELAV/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Masculino , Ratas , Ratas Long-Evans , Uridina/metabolismoRESUMEN
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.
Asunto(s)
Isquemia Encefálica/metabolismo , Neuronas/metabolismo , ARN Mensajero/metabolismo , Animales , Antígenos Nucleares/metabolismo , Isquemia Encefálica/complicaciones , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Proteínas ELAV/metabolismo , Hibridación Fluorescente in Situ , Infarto de la Arteria Cerebral Media/complicaciones , Masculino , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Ratas , Ratas Long-Evans , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Factores de TiempoRESUMEN
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 1h of reperfusion after 10min of normothermic global cerebral ischemia. At 1h 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.
Asunto(s)
Isquemia Encefálica/metabolismo , Poli A/metabolismo , Reperfusión/métodos , Animales , Muerte Celular , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Long-Evans , Programas Informáticos , Coloración y EtiquetadoRESUMEN
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.
Asunto(s)
Isquemia Encefálica/patología , Endotelina-1/administración & dosificación , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Fenotipo , ARN Mensajero/metabolismo , Daño por Reperfusión/patología , Estrés Fisiológico , Animales , Isquemia Encefálica/genética , Isquemia Encefálica/fisiopatología , Modelos Animales de Enfermedad , Endotelina-1/metabolismo , Masculino , Degeneración Nerviosa/fisiopatología , ARN Mensajero/genética , Ratas , Ratas Long-Evans , Daño por Reperfusión/genética , Daño por Reperfusión/fisiopatología , Estrés Fisiológico/genéticaRESUMEN
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.