Dysregulated autophagy is linked to BAX oligomerization and subsequent cytochrome c release in 6-hydroxydopmaine-treated neuronal cells
Graphical abstract
Introduction
Autophagy is the major intracellular degradation process and is responsible for the removal of damaged organelles and nonfunctional proteins by the lysosome [1]. When autophagy is induced, a double-membrane intermediate vesicle, called the autophagosome, forms and encapsulates the constituents targeted for degradation. The autophagosome subsequently fuses with the lysosome and forms an autolysosome, wherein degradation occurs. Autophagy has various physiological and pathophysiological roles in mammalian cells, including the maintenance of cellular homeostasis [2,3]. Because of its critical function, both deficient and excessive autophagy can lead to cell death [[4], [5], [6], [7], [8]]. Indeed, dysregulated autophagy is considered a major contributor underlying various neurodegenerative diseases including Parkinson disease (PD) [[9], [10], [11]]. Intriguingly, strong evidence including a relationship between autophagy and apoptosis, have been reported. Many stimulus that causes apoptosis can also trigger autophagy [12]. Furthermore, pro-apoptotic roles in autophagy also have also been proposed [[13], [14], [15]]. Therefore, it is worth characterizing the interplay between autophagy and apoptosis to verify the etiology of neurodegenerative disease. In previous studies, we demonstrated that reactive oxygen species (ROS)-mediated apoptosis is responsible for caspase-dependent neuronal cell death following 6-hydroxydopamine (6-OHDA) treatment [16,17]. Furthermore, we reported that abnormal induction of autophagic flux, downstream of 6-OHDA-triggered ROS, subsequently promotes caspase-dependent apoptosis.
In this study, we investigated how 6-OHDA-induced autophagy dysregulation impinges upon caspase-dependent apoptosis. We focused on the BCL-2 family of proteins that are factors regulating both autophagy and apoptosis [8]. Specifically, we focused on the pro-apoptotic BCL-2 family member BAX, which is mainly localized in the cytosol under normal conditions [18]. Following the induction of apoptosis, BAX is translocated to the outer membrane of the mitochondria and undergoes a major conformational change from inactive monomers to activated oligomers. BAX oligomerization is responsible for permeabilization of the mitochondrial outer membrane and the subsequent release of apoptosis-mediating proteins, including cytochrome c. Using the MN9D neuronal cell line [19,20], Atg5 knockout (KO) mouse embryonic fibroblast (MEFs), and primary cultures of cortical neurons, we found that 6-OHDA-induced autophagy regulated caspase-3-dependent apoptosis via BAX activation and oligomerization. Moreover, we demonstrated that BAX activation and oligomerization were significantly inhibited by pharmacological or genetic inhibition of autophagy.
Section snippets
Cell culture and drug treatment
Several cell types were used in this study (MN9D cells, MEFs, and cortical neurons) were maintained and used as previously described [8]. Two or three days after MN9D cells and MEFs were cultured, cells were exposed to drugs in N2 serum-free medium. For primary cultures of cortical neurons, cells were exposed to the indicated drugs at DIV 4. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Yonsei University (2017-10-647-01 and 2018-01-689-01).
6-OHDA-induced autophagy is linked to caspase-dependent apoptosis
To establish the levels of 6-OHDA-induced autophagy, MN9D cells were exposed to 6-OHDA, in combination with chloroquine (CQ; a lysosomal inhibitor) or 3-methyladenine (3-MA; a type III phosphatidylinositol 3-kinase (PI3K) inhibitor). The levels of autophagy and 6-OHDA-induced caspase-3 activation were investigated by immunoblotting (Fig. 1A–D). 6-OHDA treatment resulted in the appearance of LC3-II (microtubule-associated protein light chain 3, MAP1-LC3/LC3; Fig. 1A and B). Because LC3-II is
Discussion
Apoptosis is studied in the context of neurodegeneration [11,24]. We have demonstrated that 6-OHDA induces ROS-dependent apoptosis in the MN9D cells and in primary cultured neurons [16,17,[25], [26], [27]]. Recently, evidence suggests that abnormalities in autophagy can lead to neurodegeneration [7,[28], [29], [30], [31]]. Therefore, many hypotheses have been proposed to explain the potential crosstalk between autophagy and apoptosis [12]. Indeed, our laboratory has demonstrated that
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the Small Grant for Exploratory Research program (2018R1D1A1A02085731 to NY) through the National Research Foundation of Korea (NRF) grant funded by Ministry of Education and by the Brain Research Program (2017M37A1025369 to YJO) and by the Mid-Career Research Program (2019R1A2C1088793 to YJO) through the NRF grant funded by Ministry of Science and ICT.
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