Time-Dependent Changes in Apoptosis Upon Autophagy Inhibition in Astrocytes Exposed to Oxygen and Glucose Deprivation

Abstract

Recent studies have implicated the role of autophagy in brain ischemia pathophysiology. However, it remains unclear whether autophagy activation is protective or detrimental to astrocytes undergoing ischemic stress. This study evaluated the influence of ischemia-induced autophagy on cell death and the course of intrinsic and extrinsic apoptosis in primary cultures of rat cortical astrocytes exposed to combined oxygen-glucose deprivation (OGD). The role of autophagy was assessed by pharmacological inhibition with 3-methyladenine (3-MA). Cell viability was evaluated by measuring LDH release and through the use of the alamarBlue Assay. Apoptosis and necrosis were determined by fluorescence microscopy after Hoechst 33,342 and propidium iodide staining, respectively. The levels of apoptosis-related proteins were analyzed by immunoblotting. The downregulation of autophagy during OGD resulted in decreased cell viability and time-dependent changes in levels of apoptosis and necrosis. After short-term OGD (1, 4 h), cells treated with 3-MA showed higher level of cleaved caspase 3 compared with control cells. This result was consistent with an evaluation of apoptotic cell number by fluorescence microscopy. However, after prolonged exposure to OGD (8, 24 h), the number of apoptotic astrocytes (microscopically evaluated) did not differ or was even lower (as marked by caspase 3) in the presence of the autophagy inhibitor in comparison to the control. A higher level of necrosis was observed in 3-MA-treated cells compared to non-treated cells after 24 h OGD. The downregulation of autophagy caused time-dependent changes in both extrinsic (cleaved caspase 8, TNFα) and intrinsic (cleaved caspase 9) apoptotic pathways. Our results strongly indicate that the activation of autophagy in astrocytes undergoing ischemic stress is an adaptive mechanism, which allows for longer cell survival by delaying the initiation of apoptosis and necrosis.

Castanea sativa Mill. Bark Extract Protects U‐373 MG Cells and Rat Brain Slices Against Ischemia and Reperfusion Injury

“…Autophagy is a mechanism characterized by sequestration of cytosolic proteins and/or organelles in double membrane vesicles and their subsequent degradation by the cell's lysosomal system [Mizushima, ]. Autophagy may also determine the fate of the cell through its interactions with other cell death pathways, such as apoptosis, particularly when considering that the protein networks that control the initiation and execution of these two processes are highly interconnected [Rubinstein and Kimchi, ; Kasprowska et al, ]. The role of autophagy in the pathophysiology of cerebral ischemia is still unclear as it whether its activation is protective or detrimental to astrocytes undergoing ischemic stress.…”

Delta Opioid Peptide [d-Ala2, d-Leu5] Enkephalin (DADLE) Exerts a Cytoprotective Effect in Astrocytes Exposed to Oxygen-Glucose Deprivation by Inducing Autophagy

“…Primary culture of astrocytes was performed according to Juurlink 17 and Kasproska’s 13 methods. Newborn Wistar rats were purchased from the SLAC Animal Center (Shanghai, China).…”

“…OGD was performed as described previously 13 . Briefly, when cells reached approximately 80% confluency, the medium was replaced with DMEM free of glucose, FBS, and pyruvate.…”

Astrocyte, reactive astrocytes and self-regulative apoptosis in the neuroinflammation

“…[14] The ischemia-induced autophagy also influenced apoptosis of reactive astrocytes and down-regulation of autophagy caused time-dependent changes in extrinsic and intrinsic apoptotic pathways, indicating that autophagy in astrocytes might also act as an early adaptive response before initiation of apoptosis and necrosis. [15] Taken together, we hypothesize that apoptosis of reactive astrocytes may also possibly present a self-regulator for those over-activated astrocytes or functional balance between neurotrophic and inflammatory properties in neuroinflammation, while reactive astrocytes work actively as aparticipator in astrocyte-microglial communication and microglia-dominating inflammatory response. Nevertheless, one critical question regarding this self-regulatory apoptosis of reactive astrocytes in ameliorating the neuroinflammatory injury should still merit further extensive investigations to elucidate its exact roles in pathogenesis and disease progression of various neurological disorders.…”