title: Increased autophagy reduces endoplasmic reticulum stress after neonatal hypoxia-ischemia: role of protein synthesis and autophagic pathways.
abstract: The endoplasmic reticulum (ER) stress can result from several pathological conditions that perturb ER homeostasis and is characterized by accumulation of unfolded proteins in the ER lumen. To cope with ER stress, cells activate the unfolded protein response (UPR), a protein quality control mechanism aimed at restoring homeostasis. The present study was undertaken to characterize the UPR after neonatal hypoxia/ischemia (HI) and its crosstalk with autophagy. After HI, there was a significant increase of GRP78 and Hsp70 expression, phosphorylation of eIF2α, Xbp-1 mRNA splicing and CHOP expression, revealing severe ER stress and UPR. Increasing autophagy with rapamycin (Rap) significantly reduced the UPR. Rap did not further increase the eIF2α phosphorylation and p70S6 kinase (p70S6K) inactivation induced by HI. After autophagy activation, however, there was a clear co-localization between monodansylcadaverine (MDC)-positive autophagosome-like structures and the ribosomal protein S6 (RPS6), indicating the presence of ribosomes in autophagosomes (ribophagy). We found that the autophagy inhibitor 3-methyladenine administered after Rap treatment completely reverted the increased phosphorylation of eIF2α and p70S6K inactivation, and blocked the formation of autophagosome-like structures restoring the UPR. These results demonstrate that the UPR is strongly activated after neonatal HI. Over-activation of autophagy significantly reduces this response, highlighting the relevance of the cross-talk between ER and the autophagy machinery in this important pathological condition. Furthermore, the presence of ribosome subunits in autophagosome-like structures suggests that increased ribosome turnover through autophagy (ribophagy) may represent an additional mechanism involved in the neuroprotective effect observed after autophagy over-activation.
authors.affiliation: Department of Biomolecular Sciences, University of Urbino "Carlo Bo", Via S. Chiara 27, 61029 Urbino, Italy. Electronic address: silvia.carloni@uniurb.it.
authors.forename: Silvia
authors.initials: S
authors.lastname: Carloni
authors.affiliation: Department of Biomolecular Sciences, University of Urbino "Carlo Bo", Via S. Chiara 27, 61029 Urbino, Italy.
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Albertini
authors.affiliation: Department of Biomolecular Sciences, University of Urbino "Carlo Bo", Via S. Chiara 27, 61029 Urbino, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Galluzzi
authors.affiliation: Department of Paediatrics, Obstetrics and Reproductive Medicine, Policlinico Le Scotte, University of Siena, Viale M. Bracci 16, 53100, Siena, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Buonocore
authors.affiliation: Department of Paediatrics, Obstetrics and Reproductive Medicine, Policlinico Le Scotte, University of Siena, Viale M. Bracci 16, 53100, Siena, Italy.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Proietti
authors.affiliation: Department of Biomolecular Sciences, University of Urbino "Carlo Bo", Via S. Chiara 27, 61029 Urbino, Italy. Electronic address: walter.balduini@uniurb.it.
authors.forename: Walter
authors.initials: W
authors.lastname: Balduini
mesh_terms: D000818:Animals; D001343:Autophagy / Q000502:physiology*; D002452:Cell Count; D004721:Endoplasmic Reticulum / Q000378:metabolism; D059865:Endoplasmic Reticulum Stress / Q000502:physiology*; D020925:Hypoxia-Ischemia, Brain / Q000378:metabolism* / Q000503:physiopathology; D014176:Protein Biosynthesis / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D056811:Unfolded Protein Response / Q000502:physiology*
keywords: 3-Methyladenine; Autophagy; Endoplasmic reticulum (ER) stress; Hypoxia–ischemia; Rapamycin; Ribophagy