Simultaneous activation of mitophagy and autophagy by staurosporine protects against dopaminergic neuronal cell death

Neuroscience Letters 561 (2014) 101–106

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Simultaneous activation of mitophagy and autophagy by staurosporine protects against dopaminergic neuronal cell death Ji-Young Ha a , Ji-Soo Kim a , Seo-Eun Kim a , Jin H. Son a,b,∗ a b

Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans university, Seoul, South Korea Department of Brain and Cognitive Sciences, Brain Disease Research Institute, Ewha W. University, Seoul, South Korea

h i g h l i g h t s • • • •

Staurosporine co-activates autophagy and mitophagy during dopaminergic cell death. Genetic/pharmacological blockades of mitophagy and autophagy increased cell death. Both mitophagy and autophagy exert a significant neuroprotective effect. This model is useful to study mechanism underlying their crosstalk with cell death.

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Article history: Received 8 October 2013 Received in revised form 28 November 2013 Accepted 27 December 2013 Keywords: Autophagy Mitophagy Staurosporine Apoptosis Parkinson’s disease Dopaminergic neuron

a b s t r a c t Abnormal autophagy is frequently observed during dopaminergic neurodegeneration in Parkinson’s disease (PD). However, it is not yet firmly established whether active autophagy is beneficial or pathogenic with respect to dopaminergic cell loss. Staurosporine, a common inducer of apoptosis, is often used in mechanistic studies of dopaminergic cell death. Here we report that staurosporine activates both autophagy and mitophagy simultaneously during dopaminergic neuronal cell death, and evaluate the physiological significance of these processes during cell death. First, staurosporine treatment resulted in induction of autophagy in more than 75% of apoptotic cells. Pharmacological inhibition of autophagy by bafilomycin A1 decreased significantly cell viability. In addition, staurosporine treatment resulted in activation of the PINK1–Parkin mitophagy pathway, of which deficit underlies some familial cases of PD, in the dopaminergic neuronal cell line, SN4741. The genetic blockade of this pathway by PINK1 null mutation also dramatically increased staurosporine-induced cell death. Taken together, our data suggest that staurosporine induces both mitophagy and autophagy, and that these pathways exert a significant neuroprotective effect, rather than a contribution to autophagic cell death. This model system may therefore be useful for elucidating the mechanisms underlying crosstalk between autophagy, mitophagy, and cell death in dopaminergic neurons. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Recently, an emerging role for dysregulated autophagy has been suggested in the pathophysiology of dopaminergic neuronal loss in Parkinson’s disease (PD) [13–15]. However, it is not clear whether active autophagy is beneficial with respect to dopaminergic neuronal death in patients and animal models of PD. Either neuroprotective autophagy or autophagic cell death could affect dopaminergic neuronal degeneration, depending on the cause and cellular context.

∗ Corresponding author at: Department of Brain & Cognitive Sciences, Ewha W. University, Science Building C, Room C307, 11-1, Daehyun-dong, Seodaemon-gu, Seoul 120-750, South Korea. Tel.: +82 2 3277 4504; fax: +82 2 3277 3760. E-mail addresses: [email protected], [email protected] (J.H. Son). 0304-3940/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.12.064

Autophagy is an evolutionarily conserved process that controls turnover of proteins and organelles through lysosomal degradation. Autophagy can be divided into several major types, including macroautophagy, chaperone-mediated autophagy (CMA), and mitophagy [3]. Macroautophagy is often referred to as autophagy (in this report, the term autophagy is used to represent non-selective macroautophagy). Alterations in major types of autophagy during degeneration of dopaminergic neurons have been well-documented in PD. For example, ultrastructural examination of degenerating nigral dopaminergic neurons in PD patients has revealed characteristics of both apoptosis and autophagy [1]. The first pathogenic role of autophagy was suggested by the finding that ␣-synuclein is degraded by macroautophagy and CMA [17,22,23]. Furthermore, ␣-synuclein overexpression impairs macroautophagy by mislocalizing Atg9 and decreasing omegasome formation [24]. Additionally, mutations in other

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familial PD-associated molecules, such as DJ-1, Parkin, PTENinduced putative kinase 1 (PINK1), leucine-rich repeat kinase 2 (LRRK2), and Fbxo7 have been shown to affect various autophagy pathways. For instance, functional deficiency of DJ-1 leads to increased autophagic flux [12]. Parkin mutations, together with PINK1 mutations, may disrupt mitophagy, leading to the accumulation of damaged mitochondria [8,18]. Cells transfected with LRRK2 G2019S mutant exhibit a significant increase in autophagic vacuoles [17]. Fbxo 7 knockdown caused the defective Parkin translocation to mitochondria during mitophagy [4]. However, it is still unclear whether specific activation of autophagy during the cell death process plays any protective role in dopaminergic neurons. Staurosporine is a commonly used inducer of apoptosis through caspase-dependent and caspase-independent mechanisms in mammalian cells and has been widely applied in mechanistic studies of apoptosis [2,11,19]. Although staurosporine-induced cell death frequently accompanies increased numbers of autophagosomes by crosstalk with apoptotic machinery [29,31] and mitochondrial damage including mitochondrial membrane potential ( m ) dissipation [5,21], the physiological significance of these phenomena has not yet been defined. Staurosporineinduced apoptosis was often used as a model system to elucidate mechanisms underlying dopaminergic neuronal degeneration and protective function of familial PD genes. For instance, knockdown of PINK1, a central player in mitophagy and linked with familial PD [28], resulted in increased apoptosis in human dopaminergic neurons [25]. Moreover, overexpression of PINK1 reduced the staurosporine-induced caspase 3 activity in SHSY5Y cells [16], suggesting a potential protective effect of PINK1 against staurosporine–induced cell death in dopaminergic neurons. However, it has not yet been firmly established whether autophagy and/or mitophagy is activated by staurosporine treatment in dopaminergic neurons, and whether these processes protect against neuronal cell death under these conditions. In the present study, we first demonstrate that staurosporine induces both autophagy and mitophagy during apoptotic cell death in dopaminergic neuronal cells. We further show that staurosporine treatment results in induction of autophagy in many apoptotic cells, and that inhibition of autophagy significantly increased cell death. The PINK1–Parkin mitophagy pathway was also found to be activated by staurosporine, and its genetic blockade by PINK1 null mutation resulted in an increase in staurosporine-induced cell death. Therefore, our data indicate that staurosporine induces simultaneous activation of both mitophagy and autophagy in dopaminergic neurons, and that these pathways play important neuroprotective roles during cell death.

2. Materials and methods 2.1. Materials and plasmids Staurosporine, bafilomycin A1 and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Antibodies for caspase 3, phospho mTOR/mTOR, LC3 and Parkin were from Cell Signaling (Danvers, MA, USA) and other antibodies for TOM20 and GAPDH were from Santa Cruz Biotechnology (Dallas, TX, USA). The pECFP-mito vector was purchased from Clontech (Mountain View, CA, USA) and vectors expressing mCherryParkin and Su9-RFP (mitochondria-targeted DsRed) were kind gifts from Dr. Xiao-Ming Yin and Katsuyoshi Mihara, respectively.

2.2. Cell culture The wild type dopaminergic neuronal cell line, SN4741, and PINK1 null cells were cultured in RF medium that contained Dulbecco’s modified Eagle’s medium (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone, South Logan, Utah, USA), 1% glucose, Penicillin (100 units/mL)–Streptomycin (100 g/mL) and l-glutamine (2 mM) and were maintained at 33 ◦ C as described previously [18,20]. 2.3. Isolation of mitochondria Mitochondria were prepared as previously described [18]. Briefly, cells were washed with ice-cold phosphate-buffered saline (PBS) and resuspended in ice-cold 10 mM Tris (pH7.6) containing a Roche Complete EDTA-Free protease inhibitor cocktail (Roche, Cat. #11873580001). The cells were disrupted mechanically by forcing cell suspension (5 × 106 cells/ml) back and forth (10–20 times) using a 1-ml sterile, disposable syringe with a 26 gauge needle, and ice-cold 1.5 M sucrose was immediately added to the lysate. The homogenate was centrifuged at 600 × g, 2 ◦ C for 10 min, and the supernatant was centrifuged at 14,000 × g, 2 ◦ C for 10 min to precipitate mitochondria. The mitochondrial pellet was washed 3 times with ice-cold 10 mM Tris (pH7.6) containing a protease inhibitor cocktail prior to lysis. 2.4. Analysis of cell death Cell viability was determined using the MTT assay, in combination with total cell counting using Trypan blue dye exclusion as previously described [18,20]. Briefly, thiazolyl blue tetrazolium bromide (Sigma) was dissolved in PBS and filter-sterilized to prepare a 5 mg/ml stock solution. After drug treatment, cells were incubated in RF medium containing 0.5 mg/ml MTT for 2 h. The resulting formazan crystals were dissolved in dimethyl sulfoxide and the absorbance was measured at 540 nm and 670 nm as a control. In order to quantify an apoptotic cell death, chromatin condensation was analyzed by confocal imaging of 4 ,6-diamidino2-phenylindole (DAPI) staining. After staurosporine treatment, culture slides were fixed and mounted using Vectashield aqueous mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA). Activation of caspase 3 was analyzed by immunoblot. 2.5. Immunoblot analysis Control and staurosporine-treated cells were washed with icecold PBS and lysed in ristocetin-induced platelet agglutination (RIPA) buffer containing a protease inhibitor cocktail and phosphatase inhibitors for 30 min on ice. The cells were scrapped on ice, and followed by centrifugation at 14,000 × g for 30 min at 4 ◦ C. The protein concentration was determined by the Bradford method using by bovine serum albumin (BSA) as a standard. After denaturation in 5× sample buffer and boiled at 90 ◦ C for 5 min, the protein samples (10–30 ␮g) were resolved by SDS-PAGE and transferred to pre-wetted polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were then incubated in blocking solution (5% non fat dry milk or 5% BSA in TBS with 0.1% Tween 20), for 1 h to inhibit non-specific binding. The primary antibodies were diluted in blocking solution and incubated at 4 ◦ C overnight with gentle shaking. The blots were then probed with appropriate secondary antibodies and visualized by ECL chemiluminescence (Amersham Biosciences, GE Healthcare, Buckinghamshire, UK) and exposed to X-ray films (AGFA, Greenville, SC, USA) as described previously [18,20].

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Fig. 1. Autophagy occurs in most dopaminergic neuronal cells undergoing staurosporine-induced apoptosis. (A) To monitor the occurrence of apoptosis and autophagy, temporal changes of active caspase 3, phospho-mTOR, and LC3-II were measured by immunoblot analysis during staurosporine treatment of SN4741 dopaminergic neuronal cells. Staurosporine treatment activated caspase 3, with concurrent induction of autophagy markers, phospho-mTOR and LC3-II. As levels of caspase 3 activation increased, levels of phospho-mTOR decreased and LC3-II increased, indicative of increased autophagy, as quantified (shown in the right side). In dopaminergic neuronal cells mitophagy usually occurs within 2 h after CCCP treatment. (B) Autophagy occurs in many apoptotic dopaminergic cells between 6 and 24 h after initiation of treatment. Accumulation of autophagosomes was analyzed by transfecting cells with a GFP-LC3 plasmid (green fluorescence). In many apoptotic cells with chromatin condensation, accumulation of autophagosomes was also evident at 6 h after the treatment. (C) Autophagosomes (AVs) were present in more than 75% of apoptotic cells with chromatin condensation (Chr. Cond.). Data are presented as means ± SEM from at least four independent experiments. ***Significant difference between control cells and staurosporine-treated cells, p < 0.005.

2.6. Confocal fluorescence microscopy SN4741 cells were cultured on poly-l-lysine coated two-well slides for 24–36 h prior to transfection. FuGeneHD (Promega, Madison, WI, USA) was used for transient expression, according to the manufacturer’s recommended instructions. Transfected cells were fixed with 4% paraformaldehyde for 20 min and washed with PBS. The culture slides were then mounted and analyzed on a Zeiss LSM510 META laser scanning confocal microscope (Carl Zeiss, Germany). Image processing and analysis were performed with Zeiss LSM510 software version 2.3. 2.7. Statistical analysis Two-sample comparisons were performed using Student’s ttest, and multiple comparisons were made using one-way ANOVA followed by Bonferroni’s multiple comparison test. 3. Results 3.1. Staurosporine-induced apoptosis is accompanied by simultaneous induction of autophagy and mitophagy in dopaminergic neuronal cells We were interested in determining to what extent autophagy and/or mitophagy contributes to apoptotic cell death in

dopaminergic neurons, in which the types and roles of autophagy may vary depending upon the cause of cell death. Because staurosporine is widely used to induce apoptosis in animal cells including dopaminergic neurons, we first investigated whether staurosporine-induced apoptosis is accompanied by autophagy in the dopaminergic neuronal cell line SN4741. As expected, staurosporine treatment resulted in caspase 3 activation, which occurred concurrently with induction of autophagy markers, such as phospho-mTOR and LC3-II (Fig. 1A). In particular, levels of LC3-II began to increase within 2 h, when mitophagy frequently becomes obvious after CCCP treatment in SN4741 cells (our unpublished data), and continued to accumulate at levels proportional to the activation of caspase 3 between 6 and 30 h. Based on these data, we next assessed whether autophagy occurs in apoptotic dopaminergic cells. As demonstrated in Fig. 1B, autophagosomes stained with LC3-GFP were present in many apoptotic cells that exhibited chromatin condensation. Indeed, substantial number of autophagosomes were present in more than 75% of the apoptotic cells (Fig. 1C). Next we investigated whether the PINK1–Parkin mitophagy pathway is activated by staurosporine, as staurosporine has been reported to induce dissipation of the mitochondrial membrane potential during staurosporine-induced apoptosis in epithelial cells [5]. As expected, staurosporine treatment resulted in mitochondrial translocation of Parkin, evident as the colocalization of mCherry-Parkin (red fluorescence) and ECFP-Mito (cyan

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Fig. 2. Staurosporine induces mitophagy by driving mitochondrial translocation of Parkin in dopaminergic neuronal cells. (A) Because staurosporine was reported to affect on mitochondrial membrane potential (5), activation of the PINK1–Parkin mitophagy pathway by staurosporine was investigated in SN4741 dopaminergic neuronal cells. Staurosporine treatment induced mitochondrial translocation of Parkin, evident as colocalization of mCherry-Parkin (red fluorescence) and ECFP-Mito (cyan fluorescence) after 2 h. (B) The occurrence of mitophagy was further confirmed by colocalization of autophagosome (GFP-LC3; green fluorescence) and mitochondrial marker (Su9-RFP-Mito; red fluorescence) after 2 h of staurosporine treatment. (C) Mitochondrial translocation of Parkin was also confirmed by immunoblot analysis of isolated mitochondria from control cells (NT) and staurosporine-treated cells (ST) after 2 h. GAPDH and TOM20 were used as cytosolic and mitochondrial markers, respectively. The pECFP-mito (Clontech), mCherry-Parkin (fluorescence labeled Parkin from Dr. Xiao-Ming Yin) and Su9-RFP (mitochondria-targeted DsRed from Dr. Katsuyoshi Mihara). These are representative figures from at least four independent experiments.

fluorescence) as did treatment with the uncoupler CCCP (Fig. 2A). Further evidences for staurosporine-induced mitophagy were provided by the colocalization of autophagosome (GFP-LC3; green fluorescence) and damaged mitochondria (Su9-RFP-Mito; red fluorescence) (Fig. 2B) as well as immunoblot analysis of Parkin translocation to the mitochondria (Fig. 2C), in response to staurosporine treatment which suggest that staurosporine-mediated activation of mitophagy occurs through the PINK1-Parkin pathway. 3.2. Genetic blockade of mitophagy increases cell death We next investigated whether induction of mitophagy has a protective role during staurosporine-induced cell death by analyzing the effects of staurosporine in PINK1 null dopaminergic neuronal cells. The genetic ablation of PINK1 resulted in significantly higher cell death than that of wild type dopaminergic cells in response to both CCCP (20–100 nM) (Fig. 3A) and staurosporine (5–100 nM) (Fig. 3B). Consistent with these results, staurosporine treatment resulted in a significantly higher level of activation of caspase 3 in PINK1 null dopaminergic cells compared to wild type cells (Fig. 3C). 3.3. Pharmacological inhibition of autophagy increases staurosporine-induced cell death Because mitophagy appeared to exert a protective role during dopaminergic cell death, we next examined whether induction of autophagy has a similar protective function by pharmacologically inhibiting autophagy using bafilomycin A1 under the sublethal dose (less than 100 nM) in SN4741 cells. As shown in Fig. 3D, inhibition of autophagy decreased its protective effects moderately in two different concentrations (10 and 50 nM) of bafilomycin A1 during staurosporine-induced cell death of dopaminergic neuronal cells.

4. Discussion The physiological significance of active autophagy during dopaminergic neuronal loss in PD patients and models is controversial. Our current data clearly suggest that both active mitophagy and autophagy exert significant neuroprotective effect against dopaminergic neuronal cell death induced by staurosporine. Furthermore, the staurosporine model may represent a useful system to study the mechanism underlying crosstalk between autophagy, mitophagy and apoptosis in dopaminergic neurons. Alteration in autophagy in degenerating dopaminergic neurons of PD patients and PD models have been well-documented. Coexistence of autophagy and apoptosis was first revealed in nigral dopaminergic neurons from PD patients [1]. Later, PD-related toxins [9,10,30] and mutations in familial PD-associated genes were shown to affect autophagy pathways [4,7,12,17,25]. Although autophagy appears to primarily play a protective role in dopaminergic neurons, this process has also been suggested to have a paradoxical role in cell death [6,9,26,27,30]. Because the physiological contribution of each type of autophagy to dopaminergic cell death depends on the cellular context, employing a model in which both autophagy and mitophagy occur concurrently with cell death is ideal for analyzing the precise physiological role of each pathway. In the present staurosporine-induced dopaminergic cell death model, staurosporine induces the simultaneous activation of autophagy and mitophagy, accompanied by apoptosis (Figs. 1A and 2A). In this model, activation of caspase-3 and parallel induction of autophagy markers (i.e. LC3-II) increased as cell death increased. Moreover, evidence of autophagy (accumulation of autophagosome) was present in more than 75% of the apoptotic cells (Fig. 1C), suggesting that autophagy may have an important physiological role during apoptosis. This notion was further substantiated by demonstrating that pharmacological inhibition of autophagy using bafilomycin A1 significantly increased the rate

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Fig. 3. Either genetic blockade of mitophagy by a PINK1 null mutation or inhibition of autophagy by bafilomycin A1 increased cell death induced by staurosporine treatment. To investigate a potential protective role for mitophagy during staurospoine-induced cell death, wild type (WT) and PINK1 null dopaminergic neuronal cells were used. A significantly higher rate of cell death occurred in PINK1 null dopaminergic cells compared to WT cells when treated with the mitophagy inducing agents, CCCP (20–100 nM), (A) and staurosporine (5–100 nM) (B) after 24 h. Consistent with the observed higher rates of cell death in PINK1 null cells, staurosporine treatment resulted in higher levels of caspase 3 activation in PINK1 null dopaminergic cells compared to WT cells (C). (D) To determine whether induction of autophagy has a protective effect, the rate of staurosporine-induced cell death (ST, 100 nM) was determined in the presence of pharmacological inhibition of autophagy with bafilomycin A1. Bafilomycin A1 treatment increased cell death by 10–15% in two different concentrations (10 and 50 nM) under the sublethal doses of Bafilomycin A1 in SN4741 cells. All values are means ± SEM from at least four independent experiments. (C) ***Significant difference between WT and PINK1 null cells, p < 0.005. (D) ** and ***Significant differences between control and treated cells with p < 0.01 and p < 0.005, respectively.

of cell death in response to staurosporine (Fig. 3D). Thus, simultaneous induction of autophagy in the apoptotic cells appears to exert a substantial protective effect in dopaminergic neuronal cells. Mitophagy is activated through the PINK1-Parkin pathway in neuronal systems [28]. Importantly, this pathway is impaired in patients with familial PD carrying Parkin and PINK1 mutations. In human dopaminergic neurons, knockdown of PINK1 resulted in an increased staurosporine-induced apoptosis [25]. Consistent with this finding, overexpression of PINK1 reduced staurosporineinduced caspase 3 activity [16], suggesting a potential protective effect of PINK1 against staurosporine-induced cell death in dopaminergic neurons. However, it is not known whether the PINK1–Parkin mitophagy pathway is activated by staurosporine treatment in dopaminergic neurons, and, if so, whether activation of this pathway is neuroprotective against cell death. Our study demonstrates that treatment with staurosporine induces the mitochondrial translocation of Parkin, similar to CCCP treatment (Fig. 2A). Induction of mitophagy by staurosporine was further confirmed by demonstrating the colocalization of autophagosome and impaired mitochondria (Fig. 2), consistent with the activation of the PINK1-Parkin pathway by staurosporine in dopaminergic neurons. Furthermore, genetic blockade of mitophagy in dopaminergic neuronal cells carrying PINK1 null mutation resulted in a significantly higher rate of cell death and caspase 3 activation compared to control cells (Fig. 3C), suggesting that mitophagy play a protective role in the process of dopaminergic cell death.

Staurosporine has been widely applied for use in mechanistic studies of apoptosis [2,11,19]. However, staurosporine-induced cell death has been shown to be accompanied by an increase in the number of autophagosomes [29,31] and in mitochondrial damage [5,21], although the physiological and mechanistic significance of these changes have never been clearly defined in dopaminergic neurons. Thus, determination of the precise physiological role of autophagy and mitophagy during death of dopaminergic neurons, may provide significant insight into the identification of potential therapeutic targets and diagnostic marker in dopaminergic cell death. In this report, we, for the first time, describe the staurosporine-induced dopaminergic cell death model as an ideal system for investigation of the physiological functions of both autophagy and mitophagy during apoptotic cell death in dopaminergic neuronal cells. In summary, staurosporine treatment resulted in simultaneous activation of autophagy in many apoptotic cells, and inhibition of autophagy resulted in a significant increase in cell death. The PINK1–Parkin mitophagy pathway was also activated by staurosporine and genetic blockade of this pathway via a PINK1 null mutation also increased staurosporine-induced cell death. Therefore, together, these data indicate that staurosporine co-activates both mitophagy and autophagy in dopaminergic neurons and these pathways appear to play an important neuroprotective role during cell death. Furthermore, we propose that the staurosporine model represents an ideal system for investigation of the mechanism underlying crosstalk between autophagy, mitochondrial quality control and cell death in dopaminergic neurons.

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