ABSTRACT
Title
Inhibition of rapamycin-induced autophagy causes necrotic cell death associated with Bax/Bad mitochondrial translocation
Authors
S. Carloni1, S. Girelli1, G. Buonocore2, M. Longini2, F. Proietti2 and W. Balduini1
1Dept. of Biomolecular Sciences, University of Urbino “Carlo Bo”, Urbino, Italy
2Dept. of Paediatrics, Obstetrics and Reproductive Medicine, University of Siena, Siena, Italy
1Dept. of Biomolecular Sciences, University of Urbino “Carlo Bo”, Urbino, Italy
2Dept. of Paediatrics, Obstetrics and Reproductive Medicine, University of Siena, Siena, Italy
Abstract
Rapamycin, a lipophilic macrolide antibiotic, has been found to reduce injury in different models of neurodegenerative disorders. We have previously shown that in neonatal rats subjected to hypoxia-ischemia (HI) the neuroprotective effect of rapamycin was associated with increased autophagy, and decreased caspase-3 activation (Carloni et al., 2008). The present study was undertaken to better investigate the modulation of the intrinsic apoptotic mitochondrial pathwayby rapamycin after HI in neonatal rats and its correlation to autophagy.
On postnatal-day 7 (PN7), newborn rats were subjected after anaesthesia to permanent ligation of the right common carotid artery followed by 2.5 h hypoxia. Rapamycin was administered at the dose of 0.5 ng in 0.5 µl 0.1% DMSO in to the right brain ventricle 30 minutes before the onset of the ischemic procedure. To assess how theanti-apoptotic effect of rapamycin was linked to the strong autophagy signal induced by the drug, a group of animals was treated with 3-methyladenine (3MA, 5 µL, 10 mM), that blocks the formation of autophagosomes in this model of neonatal HI,10 min after rapamycin injection.Animals were sacrificed 24 h after HI for biochemical studies.
Rapamycin administration caused a marked reduction of Bax and Bad translocation to mitochondria, cytochrome c release, and caspase-3 activation. PARP-1 cleavage and the number of terminal dUDP nick-end labeling (TUNEL)-positive cells were also reduced. Rapamycin also reduced the mitochondrial translocation of the tumor suppressor p53 and of the p53-upregulated modulator of apoptosis (PUMA). 3MA elicited again Bax and Bad translocation to the mitochondria but did not cause cytochrome c release and caspase-3 activation. After 3MA cells underwent to necrotic cell death.Thesedata indicate that rapamycin administered before HI prevents the apoptotic signaling taking place via the mitochondrial pathway and this effect is strongly correlated to its ability to induce autophagy.
Carloni et al. (2008). Neurobiol Dis. 32, 329-39.
On postnatal-day 7 (PN7), newborn rats were subjected after anaesthesia to permanent ligation of the right common carotid artery followed by 2.5 h hypoxia. Rapamycin was administered at the dose of 0.5 ng in 0.5 µl 0.1% DMSO in to the right brain ventricle 30 minutes before the onset of the ischemic procedure. To assess how theanti-apoptotic effect of rapamycin was linked to the strong autophagy signal induced by the drug, a group of animals was treated with 3-methyladenine (3MA, 5 µL, 10 mM), that blocks the formation of autophagosomes in this model of neonatal HI,10 min after rapamycin injection.Animals were sacrificed 24 h after HI for biochemical studies.
Rapamycin administration caused a marked reduction of Bax and Bad translocation to mitochondria, cytochrome c release, and caspase-3 activation. PARP-1 cleavage and the number of terminal dUDP nick-end labeling (TUNEL)-positive cells were also reduced. Rapamycin also reduced the mitochondrial translocation of the tumor suppressor p53 and of the p53-upregulated modulator of apoptosis (PUMA). 3MA elicited again Bax and Bad translocation to the mitochondria but did not cause cytochrome c release and caspase-3 activation. After 3MA cells underwent to necrotic cell death.Thesedata indicate that rapamycin administered before HI prevents the apoptotic signaling taking place via the mitochondrial pathway and this effect is strongly correlated to its ability to induce autophagy.
Carloni et al. (2008). Neurobiol Dis. 32, 329-39.