Abstract
To examine the temporal relationship of cortical autophagic flux with delayed neuronal cell death after hypoxia-ischemia (HI) in neonatal piglets. HI was produced with 45-min hypoxia and 7-min airway occlusion in 3–5-day-old piglets. Markers of autophagic, lysosomal and cell death signaling were studied via immunohistochemistry, immunoblotting, and histochemistry in piglet brains. In vitro, autophagy was impaired in cultured mouse cortical neurons treated with chloroquine with or without rapamycin for 1 d in the presence of Z-VAD-fmk, cyclosporine A, or vehicle control, and cell viability was assessed with the MTT assay. In vivo, neuronal cell death of sensorimotor cortex was delayed by 1–2 days after HI, whereas LC3-II, Beclin-1, PI3KC3, ATG12-ATG-5, and p-ULK1 increased by 1.5–6 h. Autophagosomes accumulated in cortical neurons by 1 d owing to enhanced autophagy and later to decreased autophagosome clearance, as indicated by LC3, Beclin-1, and p62 accumulation. Autophagy flux impairment was attributable to lysosomal dysfunction, as indicated by low lysosomal-associated membrane protein 2, cathepsin B, and cathepsin D levels at 1 d. Ubiquitin levels increased at 1 d. Autophagosome and p62 accumulated predominantly in neurons at 1 d, with p62 puncta occurring in affected cells. Beclin-1 colocalized with markers of caspase-dependent and caspase-independent apoptosis and necrosis in neurons. In vitro, mouse neonatal cortical neurons treated with rapamycin and chloroquine showed increased autophagosomes, but not autolysosomes, and increased cell death that was attenuated by cyclosporine A. Neonatal HI initially increases autophagy but later impairs autophagosome clearance, coinciding with delayed cortical neuronal death.
Highlights
Autophagy can be divided into macro, micro, and chaperonemediated autophagy
Studies on rodent models of perinatal hypoxia-ischemia (HI) have indicated that cell death occurs along a ‘continuum’ of apoptosis to necrosis, and features of both these processes can be detected in varying proportions in different pathologies.[6,7]
We found that upstream regulators and mediators of autophagy were increased as early as 1.5–3 h after neonatal HI, leading to increased autophagy induction, which partially accounted for autophagosome accumulation in the early stages after HI
Summary
Macroautophagy (hereafter referred to as ‘autophagy’) helps in cellular homeostasis by mediating the bulk degradation and recycling of cytoplasmic organelles. This evolutionarily conserved, catabolic process is initiated by crescent-shaped structures called phagophores. We determined the levels of autophagy and autophagic flux after neonatal HI induced by asphyxial CA in newborn piglets, as the brain regions vulnerable to HI in neonatal piglets correspond to those in human term newborns.[14,15,16] We postulated that increased autophagy flux is an early event in this region, but that later lysosomal dysfunction impairs autophagosome clearance that precedes or coincides with delayed neuronal cell death signaling. We directly tested the effect of impaired autophagosme clearance in neurons in vitro
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