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Breaking down autophagy and the Ubiquitin Proteasome System
Parkinsonism and Related Disorders xxx (2017) 1e4
Contents lists available at ScienceDirect
Parkinsonism and Related Disorders journal homepage: www.elsevier.com/locate/parkreldis
Breaking down autophagy and the Ubiquitin Proteasome System Shinae Jung 1, Yuhyun Chung 1, Young J. Oh* Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 July 2017 Accepted 25 July 2017
Autophagy is an evolutionarily conserved catabolic process that is involved in cellular homeostasis and stress responses. Although basal levels of autophagy are essential for cellular homeostasis, dysregulated autophagy is linked to neurodegeneration. Recent studies using genetic or neurotoxin-based models of Parkinson's disease (PD) detect autophagy. We demonstrate that neurotoxins induce autophagy in dopaminergic neuronal cell line and primary cultured neurons. Based on previous reports, including ones from our laboratory, which show that elevated reactive oxygen species (ROS) and cytosolic calcium are implicated in dopaminergic neurodegeneration, we reasoned that these triggers may play critical roles in determining dysregulated autophagy. Similarly, we have demonstrated that ROS-mediated signals play an essential role in 6-hydroxydopamine (6-OHDA)-induced apoptosis, whereas MPPþ causes elevations in cytosolic calcium and calpain activation. By using these experimental models, we specifically address the question as to whether an increase in ROS or cytosolic calcium governs abnormal flux of autophagy as well as the ubiquitin proteasome system (UPS). So far, our data support a notion that ROS and cytosolic calcium act on a distinct flux of autophagy and the UPS. Our data also raise the possibility of interplay between autophagy and other cell death modes (e.g., caspase- or calpain-dependent cell death) during dopaminergic neurodegeneration. © 2017 Published by Elsevier Ltd.
Keywords: Dopaminergic neurodegeneration Autophagy Reactive oxygen species Calcium
1. Parkinson's disease PD is the second most common neurodegenerative disease and its prevalence in the population over the age of 60 is 1e2%. PD is typically characterized by tremor at rest, rigidity, bradykinesia and postural instability. Clinical features are largely attributed to the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The cause of PD is still unclear but it is considered to be derived from the interplay between environmental factors and genetic susceptibility. Accumulating evidence shows that mitochondrial dysfunction, abnormal protein aggregation, oxidative stress and inflammation are involved in the pathogenesis. 2. Parkinson's disease and cell death Among 3 different types of cell death modes proposed [1], the majority of previous studies indicate that apoptosis is the most
* Corresponding author. Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, 134 Shinchon-dong, Seodaemoon-gu, Seoul 120-749, South Korea. E-mail address: [email protected] (Y.J. Oh). 1 Both authors contributed equally to this work.
dominant mechanism associated with dopaminergic neurodegeneration. Mutations in various genes linked to PD lead to apoptotic cell death. For example, LRRK2 interacts with Fasassociated protein with death domain (FADD) and induces caspase-8-dependent apoptosis [2]. Phosphorylation of p53 by LRRK2 leads to expression of p21(WAF1/CIP1) and apoptosis in neurons [3]. Wild type a-synuclein but not its mutants negatively regulates protein kinase C-d expression and suppress apoptosis [4]. Moreover, a-synuclein plays a key role in mitochondrial biology and reduces apoptotic cell death under oxidative stress [5]. Mutations in other PD-related genes including parkin and DJ-1 are associated with dopaminergic neurodegeneration via apoptotic mechanism. Given the biochemical changes reminiscent of those occurring in patients with PD, studies using toxin-based models are widely used to identify cell death modes and their associated mechanisms. The most widely used neurotoxins include 6-hydroxydopamine (6OHDA) and 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP; its active metabolite, MPPþ). More recently, studies have shown a potential link between rotenone and paraquat use and dopaminergic neurodegeneration. Similarly to MPTP, these toxins inhibit mitochondrial Complex I and consequently induce mitochondrial abnormality, oxidative stress and surge in cytosolic calcium. On the other hand, 6-OHDA kills dopaminergic neurons by inducing
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Please cite this article in press as: S. Jung, et al., Breaking down autophagy and the Ubiquitin Proteasome System, Parkinsonism and Related Disorders (2017), http://dx.doi.org/10.1016/j.parkreldis.2017.07.026
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oxidative stress. We demonstrate that 6-OHDA induces ROSdependent apoptosis [6,7]. MPPþ-mediated cell death is not blocked by capsase inhibitors and is accompanied by necrotic features [8]. Recently, we have also raised the possibility that these neurotoxins can trigger cell death via a mechanism other than apoptosis and necrosis [9]. 3. Parkinson's disease and autophagy Chronic neurodegenerative diseases have features in common: abnormal protein aggregation and mitochondrial dysfunction. As autophagy is an intracellular degradation system whereby cellular components, including protein aggregates and damaged mitochondria, are degraded by the lysosomal pathway, dysregulation of autophagy is considered to be a major contributor to neurodegenerative diseases. Autophagy involves a double-membrane vesicle termed the autophagosome that eventually sequesters cytosolic cargos. In addition, autophagosome can selectively capture autophagic substrates such as damaged mitochondria (mitophagy), protein aggregates (aggrephagy) or invasive pathogens (xenophagy), through autophagy receptors that links the ubiquitinated cargos to LC3-coated phagophores. It is worth noting that both parkin and PINK1 play central roles in mitophagy. PINK1 acts as a sensor of damaged mitochondria and accumulates on the outer membrane of damaged mitochondria [10]. Under normal conditions, PINK1 is cleaved by mitochondrial processing peptidase and presenilins-associated rhomboid-like protein (PARL) after it is imported into the mitochondrial inner membrane (MIM) through its N-terminal mitochondrial targeting sequence. The resulting cleaved PINK1 is released into the cytosol and degraded by the UPS [11]. Upon mitochondrial depolarization, PINK1 accumulates on the mitochondrial outer membrane (MOM). Thereafter, parkin is recruited to the mitochondria in a PINK1-dependent manner [12]. PINK1 phosphorylates ubiquitin at Ser65 and this phosphorylated ubiquitin activates parkin in an allosteric way [13]. As a result, activated parkin recruits autophagic receptors (e.g., NDP52 and optineurin) to induce mitophagy [14]. a-synuclein is a substrate for the chaperone-mediated autophagy (CMA) pathway through the LAMP2A-containing complex [15]. Intriguingly, its pathogenic mutants tightly bind to LAMP2A, inhibiting uptake of CMA substrates into the lysosome. Other PD-related genes including LRRK2 and DJ-1 are also involved in dysregulation of macroautophagy. 4. Critical factors involved in neurodegenerative disorders including PD Under normal conditions, lower concentrations of ROS in cells play a physiological role as signaling molecules. On the other hand, higher concentrations of ROS have a deleterious effect on cells by disturbing several cellular homeostatic components [16]. When ROS levels are too high, cell undergoes ROS-dependent death. Excessive ROS are detected in the SNpc region of patients with PD and induce ROS-dependent apoptosis [17,18]. In support of this notion, we demonstrate that ROS directly cause a caspasedependent apoptosis that is blocked by antioxidant or caspase inhibitors [6e8]. In addition, 6-OHDA-induced production of excessive ROS can trigger autophagy as well. Consequently, cotreatment with antioxidants inhibits 6-OHDA-induced autophagy, suggesting that ROS are central for triggering autophagy. We are currently pursuing the extent to which glial cells contribute to 6-OHDAinduced autophagy [19]. Since calcium acts as a second messenger regulating a variety of cellular signaling pathways, its concentration in the cytosol is tightly controlled. However, increased cytosolic calcium
associated with certain pathological conditions activate calpains. Calpain-cleaved a-synuclein is accumulated in Lewy bodies in the brains of patients with PD [20]. Our previous studies demonstrated that MPPþ triggers surge of cytosolic calcium and, in turn, activates calpains [21]. Activated calpains cleave various cellular substrates including optineurin that is central for cargo selection and delivery to the autophagosome. Consequently, cotreatment with calcium chelators or overexpression of calcium-binding proteins rescues the cells from MPPþ-induced neurodegeneration. Similarly, MPPþ induces autophagy in dopaminergic neurons [9]. Similar autophagic features are found in rat brains which received stereotaxic injection of MPPþ or in mouse brain which had intraperitoneal injections of MPTP, suggesting a critical role of cytosolic calcium in dysregulating autophagy. 5. Interplay between autophagy and apoptosis The biggest question raised is “Does really autophagic cell death exist?”. Although it is still controversial, the following criteria must be met to clearly define autophagic cell death: (i) autophagic cell death must be independent of apoptosis and necrosis, (ii) increase in autophagic flux is detected in dying cells, (iii) cell death is blocked when autophagy is inhibited and (iv) autophagy can govern the final dismantling of cellular contents and hence execute a lethal pathway [22]. Nevertheless, it is quite clear that autophagy has a close connection with other cell death mechanisms, especially apoptosis. In many cases, autophagy and apoptosis are induced by the same stimuli and share various critical signaling molecules. Bcl2 family is extensively studied as regulators of autophagy and apoptosis. Under normal conditions, Beclin-1 is bound to Bcl-2/BclXL through interaction between Bcl-2 homology 3 (BH3) domain in Beclin-1 and BH3 binding pocket of Bcl-2/Bcl-XL. BH3-only proteins (e.g., NIX, NOXA, BAD, BID, and PUMA) can competitively disrupt the interaction between anti-apoptotic Bcl-2 family and Beclin-1 through binding to BH3-binding groove of Bcl-2/Bcl-XL. The dissociation of Beclin-1 from its inhibitors can induce autophagy. Thus, anti-apoptotic members of Bcl-2 family and pro-apoptotic BH3-only proteins are critically involved in regulation of autophagy. In addition, p53 [23], death associated protein kinase (DAPK) [24] and JNK [25] are associated with induction of autophagy and apoptosis, suggesting that interplay between the core molecular machinery controlling various types of cell death exist. When apoptosis is induced by intrinsic or extrinsic stimuli, mitochondria are damaged and apoptotic molecules (e.g. cytochrome c, Smac/DIABLO, EndoG, AIF) are released by mitochondrial outer membrane permeabilization (MOMP) [26]. It has been shown that autophagic clearance of the damaged mitochondria can prevent cells from developing apoptosis. Recent studies reveal that selective autophagy can directly degrade caspase-8 [27]. On the other hand, the proapoptotic role of autophagy is also proposed [28,29]. For example, autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila oogenesis [28]. Interestingly, constitutive generation of ROS from autophagosome and autolysosome is demonstrated in glutamate-induced oxidative cytotoxicity [29]. Consequently, inhibitors of autophagy and lysosomes reduce neuronal cell death in a rat ischemia model. 6. Roles of ROS and cytosolic calcium in governing dysregulation of autophagy Previously, we have demonstrated that dopaminergic neurotoxins act on distinct cell death pathways [6e8]. More specifically, 6-OHDA induces ROS-dependent caspase activation whereas MPPþ recruits a calcium-dependent calpain activation. Using neurotoxinbased models of PD, we have recently attempted to evaluate
Please cite this article in press as: S. Jung, et al., Breaking down autophagy and the Ubiquitin Proteasome System, Parkinsonism and Related Disorders (2017), http://dx.doi.org/10.1016/j.parkreldis.2017.07.026
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Fig. 1. A model for role of ROS and calcium in regulation of autophagy. Based on data obtained so far, we popose the following working hypothesis. In response to 6-OHDA or MPPþ, drug-induced surge of ROS and cytosolic calcium directly induces autophagy as determined by ultrastructural and biochemical analysis. Ultrastructural examination reveals the drug-induced formation of phagopore, autophagosome and autolysosome in degenerating dopaminergic neuronal cells. Both immunoblot analysis and immunocytochemistry also demonstrate changes in protein levels and distribution pattern of LC3 and p62 that are typical of autophagy. Although it is not clear whether and how a drug-induced activation of caspase or calpain is linked to dysregulation of autophagy, we strongly belive that they are all involved in executing dopaminergic neurodegeneration.
potentially distinct roles of ROS and cytosolic calcium in regulation of autophagy. After challenging with 6-OHDA or MPPþ, dysregulation of autophagy in MN9D cells and primary cultures of cortical neurons is monitored by morphological and biochemical analysis. We have reported that MPPþ results in impairment of the autophagic flux [9]. Recently, several attempts have been made to clearly demonstrate whether i) drug-induced surge of ROS and cytosolic calcium is linked to dysregulation of autophagy with an emphasis on excessive or impaired autophagic flux; ii) druginduced dysregulation of autophagy is directly linked to dopaminergic neurodegeneration; and iii) there exists an interplay between autophagy and other cell death mode (e.g., apoptosis, necrosis). Although it is still only a working hypothesis, we propose that both ROS and cytosolic calcium seem to be critical initiators of determining both excessive and impaired autophagy (Fig. 1). Although we have not clearly determined whether autophagic cell death is involved, we also propose that interplay between autophagy and other cell death modes may be directly linked to druginduced neurodegeneration. Conflict of interest The authors declare that they have no competing or conflict of interests. Acknowledgments This research was supported by the Brain Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning (2017M3C7A1025369 and 2016M3C7A1904394 to YJO). References [1] L. Galluzzi, I. Vitale, J.M. Abrams, E.S. Alnemri, E.H. Baehrecke, M.V. Blagosklonny, T.M. Dawson, V.L. Dawson, W.S. El-Deiry, S. Fulda, E. Gottlieb, D.R. Green, M.O. Hengartner, O. Kepp, R.A. Knight, S. Kumar, S.A. Lipton, X. Lu, F. Madeo, W. Malorni, P. Mehlen, G. Nunez, M.E. Peter, M. Piacentini, D.C. Rubinsztein, Y. Shi, H.U. Simon, P. Vandenabeele, E. White, J. Yuan, B. Zhivotovsky, G. Melino, G. Kroemer, Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell
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Contents lists available at ScienceDirect
Parkinsonism and Related Disorders journal homepage: www.elsevier.com/locate/parkreldis
Breaking down autophagy and the Ubiquitin Proteasome System Shinae Jung 1, Yuhyun Chung 1, Young J. Oh* Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 July 2017 Accepted 25 July 2017
Autophagy is an evolutionarily conserved catabolic process that is involved in cellular homeostasis and stress responses. Although basal levels of autophagy are essential for cellular homeostasis, dysregulated autophagy is linked to neurodegeneration. Recent studies using genetic or neurotoxin-based models of Parkinson's disease (PD) detect autophagy. We demonstrate that neurotoxins induce autophagy in dopaminergic neuronal cell line and primary cultured neurons. Based on previous reports, including ones from our laboratory, which show that elevated reactive oxygen species (ROS) and cytosolic calcium are implicated in dopaminergic neurodegeneration, we reasoned that these triggers may play critical roles in determining dysregulated autophagy. Similarly, we have demonstrated that ROS-mediated signals play an essential role in 6-hydroxydopamine (6-OHDA)-induced apoptosis, whereas MPPþ causes elevations in cytosolic calcium and calpain activation. By using these experimental models, we specifically address the question as to whether an increase in ROS or cytosolic calcium governs abnormal flux of autophagy as well as the ubiquitin proteasome system (UPS). So far, our data support a notion that ROS and cytosolic calcium act on a distinct flux of autophagy and the UPS. Our data also raise the possibility of interplay between autophagy and other cell death modes (e.g., caspase- or calpain-dependent cell death) during dopaminergic neurodegeneration. © 2017 Published by Elsevier Ltd.
Keywords: Dopaminergic neurodegeneration Autophagy Reactive oxygen species Calcium
1. Parkinson's disease PD is the second most common neurodegenerative disease and its prevalence in the population over the age of 60 is 1e2%. PD is typically characterized by tremor at rest, rigidity, bradykinesia and postural instability. Clinical features are largely attributed to the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The cause of PD is still unclear but it is considered to be derived from the interplay between environmental factors and genetic susceptibility. Accumulating evidence shows that mitochondrial dysfunction, abnormal protein aggregation, oxidative stress and inflammation are involved in the pathogenesis. 2. Parkinson's disease and cell death Among 3 different types of cell death modes proposed [1], the majority of previous studies indicate that apoptosis is the most
* Corresponding author. Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, 134 Shinchon-dong, Seodaemoon-gu, Seoul 120-749, South Korea. E-mail address: [email protected] (Y.J. Oh). 1 Both authors contributed equally to this work.
dominant mechanism associated with dopaminergic neurodegeneration. Mutations in various genes linked to PD lead to apoptotic cell death. For example, LRRK2 interacts with Fasassociated protein with death domain (FADD) and induces caspase-8-dependent apoptosis [2]. Phosphorylation of p53 by LRRK2 leads to expression of p21(WAF1/CIP1) and apoptosis in neurons [3]. Wild type a-synuclein but not its mutants negatively regulates protein kinase C-d expression and suppress apoptosis [4]. Moreover, a-synuclein plays a key role in mitochondrial biology and reduces apoptotic cell death under oxidative stress [5]. Mutations in other PD-related genes including parkin and DJ-1 are associated with dopaminergic neurodegeneration via apoptotic mechanism. Given the biochemical changes reminiscent of those occurring in patients with PD, studies using toxin-based models are widely used to identify cell death modes and their associated mechanisms. The most widely used neurotoxins include 6-hydroxydopamine (6OHDA) and 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP; its active metabolite, MPPþ). More recently, studies have shown a potential link between rotenone and paraquat use and dopaminergic neurodegeneration. Similarly to MPTP, these toxins inhibit mitochondrial Complex I and consequently induce mitochondrial abnormality, oxidative stress and surge in cytosolic calcium. On the other hand, 6-OHDA kills dopaminergic neurons by inducing
http://dx.doi.org/10.1016/j.parkreldis.2017.07.026 1353-8020/© 2017 Published by Elsevier Ltd.
Please cite this article in press as: S. Jung, et al., Breaking down autophagy and the Ubiquitin Proteasome System, Parkinsonism and Related Disorders (2017), http://dx.doi.org/10.1016/j.parkreldis.2017.07.026
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oxidative stress. We demonstrate that 6-OHDA induces ROSdependent apoptosis [6,7]. MPPþ-mediated cell death is not blocked by capsase inhibitors and is accompanied by necrotic features [8]. Recently, we have also raised the possibility that these neurotoxins can trigger cell death via a mechanism other than apoptosis and necrosis [9]. 3. Parkinson's disease and autophagy Chronic neurodegenerative diseases have features in common: abnormal protein aggregation and mitochondrial dysfunction. As autophagy is an intracellular degradation system whereby cellular components, including protein aggregates and damaged mitochondria, are degraded by the lysosomal pathway, dysregulation of autophagy is considered to be a major contributor to neurodegenerative diseases. Autophagy involves a double-membrane vesicle termed the autophagosome that eventually sequesters cytosolic cargos. In addition, autophagosome can selectively capture autophagic substrates such as damaged mitochondria (mitophagy), protein aggregates (aggrephagy) or invasive pathogens (xenophagy), through autophagy receptors that links the ubiquitinated cargos to LC3-coated phagophores. It is worth noting that both parkin and PINK1 play central roles in mitophagy. PINK1 acts as a sensor of damaged mitochondria and accumulates on the outer membrane of damaged mitochondria [10]. Under normal conditions, PINK1 is cleaved by mitochondrial processing peptidase and presenilins-associated rhomboid-like protein (PARL) after it is imported into the mitochondrial inner membrane (MIM) through its N-terminal mitochondrial targeting sequence. The resulting cleaved PINK1 is released into the cytosol and degraded by the UPS [11]. Upon mitochondrial depolarization, PINK1 accumulates on the mitochondrial outer membrane (MOM). Thereafter, parkin is recruited to the mitochondria in a PINK1-dependent manner [12]. PINK1 phosphorylates ubiquitin at Ser65 and this phosphorylated ubiquitin activates parkin in an allosteric way [13]. As a result, activated parkin recruits autophagic receptors (e.g., NDP52 and optineurin) to induce mitophagy [14]. a-synuclein is a substrate for the chaperone-mediated autophagy (CMA) pathway through the LAMP2A-containing complex [15]. Intriguingly, its pathogenic mutants tightly bind to LAMP2A, inhibiting uptake of CMA substrates into the lysosome. Other PD-related genes including LRRK2 and DJ-1 are also involved in dysregulation of macroautophagy. 4. Critical factors involved in neurodegenerative disorders including PD Under normal conditions, lower concentrations of ROS in cells play a physiological role as signaling molecules. On the other hand, higher concentrations of ROS have a deleterious effect on cells by disturbing several cellular homeostatic components [16]. When ROS levels are too high, cell undergoes ROS-dependent death. Excessive ROS are detected in the SNpc region of patients with PD and induce ROS-dependent apoptosis [17,18]. In support of this notion, we demonstrate that ROS directly cause a caspasedependent apoptosis that is blocked by antioxidant or caspase inhibitors [6e8]. In addition, 6-OHDA-induced production of excessive ROS can trigger autophagy as well. Consequently, cotreatment with antioxidants inhibits 6-OHDA-induced autophagy, suggesting that ROS are central for triggering autophagy. We are currently pursuing the extent to which glial cells contribute to 6-OHDAinduced autophagy [19]. Since calcium acts as a second messenger regulating a variety of cellular signaling pathways, its concentration in the cytosol is tightly controlled. However, increased cytosolic calcium
associated with certain pathological conditions activate calpains. Calpain-cleaved a-synuclein is accumulated in Lewy bodies in the brains of patients with PD [20]. Our previous studies demonstrated that MPPþ triggers surge of cytosolic calcium and, in turn, activates calpains [21]. Activated calpains cleave various cellular substrates including optineurin that is central for cargo selection and delivery to the autophagosome. Consequently, cotreatment with calcium chelators or overexpression of calcium-binding proteins rescues the cells from MPPþ-induced neurodegeneration. Similarly, MPPþ induces autophagy in dopaminergic neurons [9]. Similar autophagic features are found in rat brains which received stereotaxic injection of MPPþ or in mouse brain which had intraperitoneal injections of MPTP, suggesting a critical role of cytosolic calcium in dysregulating autophagy. 5. Interplay between autophagy and apoptosis The biggest question raised is “Does really autophagic cell death exist?”. Although it is still controversial, the following criteria must be met to clearly define autophagic cell death: (i) autophagic cell death must be independent of apoptosis and necrosis, (ii) increase in autophagic flux is detected in dying cells, (iii) cell death is blocked when autophagy is inhibited and (iv) autophagy can govern the final dismantling of cellular contents and hence execute a lethal pathway [22]. Nevertheless, it is quite clear that autophagy has a close connection with other cell death mechanisms, especially apoptosis. In many cases, autophagy and apoptosis are induced by the same stimuli and share various critical signaling molecules. Bcl2 family is extensively studied as regulators of autophagy and apoptosis. Under normal conditions, Beclin-1 is bound to Bcl-2/BclXL through interaction between Bcl-2 homology 3 (BH3) domain in Beclin-1 and BH3 binding pocket of Bcl-2/Bcl-XL. BH3-only proteins (e.g., NIX, NOXA, BAD, BID, and PUMA) can competitively disrupt the interaction between anti-apoptotic Bcl-2 family and Beclin-1 through binding to BH3-binding groove of Bcl-2/Bcl-XL. The dissociation of Beclin-1 from its inhibitors can induce autophagy. Thus, anti-apoptotic members of Bcl-2 family and pro-apoptotic BH3-only proteins are critically involved in regulation of autophagy. In addition, p53 [23], death associated protein kinase (DAPK) [24] and JNK [25] are associated with induction of autophagy and apoptosis, suggesting that interplay between the core molecular machinery controlling various types of cell death exist. When apoptosis is induced by intrinsic or extrinsic stimuli, mitochondria are damaged and apoptotic molecules (e.g. cytochrome c, Smac/DIABLO, EndoG, AIF) are released by mitochondrial outer membrane permeabilization (MOMP) [26]. It has been shown that autophagic clearance of the damaged mitochondria can prevent cells from developing apoptosis. Recent studies reveal that selective autophagy can directly degrade caspase-8 [27]. On the other hand, the proapoptotic role of autophagy is also proposed [28,29]. For example, autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila oogenesis [28]. Interestingly, constitutive generation of ROS from autophagosome and autolysosome is demonstrated in glutamate-induced oxidative cytotoxicity [29]. Consequently, inhibitors of autophagy and lysosomes reduce neuronal cell death in a rat ischemia model. 6. Roles of ROS and cytosolic calcium in governing dysregulation of autophagy Previously, we have demonstrated that dopaminergic neurotoxins act on distinct cell death pathways [6e8]. More specifically, 6-OHDA induces ROS-dependent caspase activation whereas MPPþ recruits a calcium-dependent calpain activation. Using neurotoxinbased models of PD, we have recently attempted to evaluate
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Fig. 1. A model for role of ROS and calcium in regulation of autophagy. Based on data obtained so far, we popose the following working hypothesis. In response to 6-OHDA or MPPþ, drug-induced surge of ROS and cytosolic calcium directly induces autophagy as determined by ultrastructural and biochemical analysis. Ultrastructural examination reveals the drug-induced formation of phagopore, autophagosome and autolysosome in degenerating dopaminergic neuronal cells. Both immunoblot analysis and immunocytochemistry also demonstrate changes in protein levels and distribution pattern of LC3 and p62 that are typical of autophagy. Although it is not clear whether and how a drug-induced activation of caspase or calpain is linked to dysregulation of autophagy, we strongly belive that they are all involved in executing dopaminergic neurodegeneration.
potentially distinct roles of ROS and cytosolic calcium in regulation of autophagy. After challenging with 6-OHDA or MPPþ, dysregulation of autophagy in MN9D cells and primary cultures of cortical neurons is monitored by morphological and biochemical analysis. We have reported that MPPþ results in impairment of the autophagic flux [9]. Recently, several attempts have been made to clearly demonstrate whether i) drug-induced surge of ROS and cytosolic calcium is linked to dysregulation of autophagy with an emphasis on excessive or impaired autophagic flux; ii) druginduced dysregulation of autophagy is directly linked to dopaminergic neurodegeneration; and iii) there exists an interplay between autophagy and other cell death mode (e.g., apoptosis, necrosis). Although it is still only a working hypothesis, we propose that both ROS and cytosolic calcium seem to be critical initiators of determining both excessive and impaired autophagy (Fig. 1). Although we have not clearly determined whether autophagic cell death is involved, we also propose that interplay between autophagy and other cell death modes may be directly linked to druginduced neurodegeneration. Conflict of interest The authors declare that they have no competing or conflict of interests. Acknowledgments This research was supported by the Brain Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning (2017M3C7A1025369 and 2016M3C7A1904394 to YJO). References [1] L. Galluzzi, I. Vitale, J.M. Abrams, E.S. Alnemri, E.H. Baehrecke, M.V. Blagosklonny, T.M. Dawson, V.L. Dawson, W.S. El-Deiry, S. Fulda, E. Gottlieb, D.R. Green, M.O. Hengartner, O. Kepp, R.A. Knight, S. Kumar, S.A. Lipton, X. Lu, F. Madeo, W. Malorni, P. Mehlen, G. Nunez, M.E. Peter, M. Piacentini, D.C. Rubinsztein, Y. Shi, H.U. Simon, P. Vandenabeele, E. White, J. Yuan, B. Zhivotovsky, G. Melino, G. Kroemer, Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell
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Please cite this article in press as: S. Jung, et al., Breaking down autophagy and the Ubiquitin Proteasome System, Parkinsonism and Related Disorders (2017), http://dx.doi.org/10.1016/j.parkreldis.2017.07.026