Energy Adaptive Response During Parthanatos Is Enhanced by PD98059 and Involves Mitochondrial Function but Not Autophagy Induction

Biochimica et Biophysica Acta 1843 (2014) 531–543

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Energy adaptive response during parthanatos is enhanced by PD98059 and involves mitochondrial function but not autophagy induction

Chen-Tsung Huang a, Duen-Yi Huang a, Chaur-Jong Hu b,DeanWub,1, Wan-Wan Lin a,c,⁎,1 a Department of Pharmacology, College of Medicine, National Taiwan University, Taiwan b Department of Neurology, Shuang Ho Hospital, Taipei Medical University, New Taipei City, Taiwan c Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan article info abstract

Article history: Parthanatos is a programmed necrotic demise characteristic of ATP (adenosine triphosphate) consumption due Received 7 October 2013 to NAD+ (nicotinamide adenine dinucleotide) depletion by poly(ADP-ribose) polymerase 1 (PARP1)-dependent Received in revised form 27 November 2013 poly(ADP-ribosyl)ation on target proteins. However, how the bioenergetics is adaptively regulated during Accepted 2 December 2013 parthanatos, especially under the condition of macroautophagy deficiency, remains poorly characterized. Here, Available online 8 December 2013 we demonstrated that the parthanatic inducer N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) triggered ATP depletion followed by recovery in mouse embryonic fibroblasts (MEFs). Notably, Atg5−/− MEFs showed great sus- Keywords: Parthanatos ceptibility to MNNG with disabled ATP-producing capacity. Moreover, the differential energy-adaptive −/− ATP consumption responses in wild-type (WT) and Atg5 MEFs were unequivocally worsened by inhibition of AMP-activated pro- − − AMPK tein kinase (AMPK), sirtuin 1 (SIRT1), and mitochondrial activity. Importantly, Atg5 / MEFs disclosed diminished SIRT1 SIRT1 and mitochondrial activity essential to the energy restoration during parthanatos. Strikingly, however, Mitochondrion parthanatos cannot be exasperated by bafilomycin A1 and MNNG neither provokes microtubule-associated pro- PD98059 tein 1A/1B-light chain 3 (LC3) lipidation and p62 elimination, suggesting that parthanatos does not induce autophagic flux. Intriguingly, we reported unexpectedly that PD98059, even at low concentration insufficient to inhibit MEK, can promote mitochondrial activity and facilitate energy-restoring process during parthanatos, without modulating DNA damage responses as evidenced by PARP1 activity, p53 expression, and γH2AX (H2A histone family, member X (H2AX), phosphorylated on Serine 139) induction. Therefore, we propose that Atg5 deficiency confers an infirmity to overcome the energy crisis during parthanatos and further underscore the deficits in mitochondrial quality control, but not incapability of autophagy induction, that explain the vulnerability in Atg5- deficient cells. Collectively, our results provide a comprehensive energy perspective for an improved treatment to alleviate parthanatos-related tissue necrosis and disease progression and also provide a future direction for drug development on the basis of PD98059 as an efficacious compound against parthanatos. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Among all PARP superfamily, PARP1 is the most abundant protein localized in the nucleus that could immediately respond to the Parthanatosisaparticulartypeof programmed necrosis charac- DNA damage, such as alkylation or double-strand breaks, to promote terized by the death signal poly(ADP-ribose) (PAR) polymers, DNA repair machinery through NAD+-dependent PARylation on accep- poly(ADP-ribose) polymerase (PARP)-mediated PAR accumulation, tor proteins, such as histones and PARP1 itself [5,6]. However, if the ATP and NAD+ depletion, mitochondrial perturbation, and apoptosis- DNA damage is irreparable, PARP1 over-activation will culminate in inducing factor (AIF) nuclear translocation from mitochondria [1–4]. PARP1-dependent parthanatic cell death. Widespread as an environ- mental mutagen, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) is Abbreviations: 3AB, 3-aminobenzamide; 7AAD, 7-aminoactinomycin D; AIF, such a commonly used DNA-alkylating agent that potently initiates apoptosis-inducing factor; AMPK, AMP-activated protein kinase; DPI, diphenyliodonium; parthanatic cell death in a caspase-independent manner [3]. In our DXR, doxorubicin; FACS, fluorescence-activated cell sorting; FCCP, trifluorocarbonylcyanide previous study, we have confirmed that MNNG is able to induce AIF phenylhydrazone; LC3, microtubule-associated protein 1A/1B-light chain 3; LDH, lactate nuclear translocation and necrotic cell death in mouse embryonic dehydrogenase; MAPK, mitogen-activated protein kinase; MEFs, mouse embryonic fi fibroblasts; MNNG, N-methyl-N′-nitro-N-nitrosoguanidine; mtDNA, mitochondrial DNA; broblasts (MEFs) [7]. Moreover, the cell death was prevented by mTORC1, mammalian target of rapamycin complex 1; PAR, poly(ADP-ribose); PARP1, PARP1 inhibitors such as 3-aminobenzamide (3AB) and 3,4-dihydro-5 poly(ADP-ribose) polymerase 1; PGC1α, peroxisome proliferator-activated receptor (PPAR) [4-(1-piperindinyl) butoxy]-1(2H)-isoquinoline (DPQ), implicating gamma coactivator 1 alpha; SIRT1, sirtuin 1; WT, wild-type MNNG-induced cell death due to PARP1 over-activation [7]. ⁎ Corresponding author at: No 1, Sec 1, Jen-Ai Road, Taipei, Taiwan. Tel.: +886 2 Parthanatos has been implicated in several pathological condi- 23123456x88315; fax: +886 2 23513716. E-mail address: [email protected] (W.-W. Lin). tions, such as neurodegenerative disorders including Parkinson's 1 Equal contribution of Drs. D. Wu and W.W. Lin for correspondence. and Alzheimer's diseases, glutamate excitotoxicity, stroke, ischemia–

0167-4889/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbamcr.2013.12.001 532 C.-T. Huang et al. / Biochimica et Biophysica Acta 1843 (2014) 531–543 reperfusion injury, and diabetes mellitus [8–12].Manyeffortshavebeen (Thr202/Tyr204, 9101), p-JNK (Thr183/Tyr185, 9251), p-p38 (Thr180/ spent to understand this distinct type of cell death to hinder from exten- Tyr182, 9211), p53 (9282), p-AMPKα1 (Thr172, 2531), AMPKα1 sive tissue inflammation and disease progression [12]. Besides several (2532), p-GSK3β (Ser9, 9336), p-Akt (Ser473, 9271), Akt (9272), and parthanatic hallmarks already defined [2], mitogen-activated protein SQSTM1/p62 (5114) were purchased from Cell Signaling Technology kinase (MAPK) signaling pathways such as JNK and p38 have also been (Beverly, MA, USA); GSK3β (sc-9166), ERK1/2 (sc-292838), JNK1 implied to regulate the parthanatic responses [13,14]. Considering (sc-474), and p38 (sc-7149) were from Santa Cruz Biotechnology the energy depletion as an essential biochemical feature, enforced reple- (Santa Cruz, CA, USA); Atg5 antibody (ab54033-100) was purchased tion of metabolic intermediates has been utilized to prevent from from Abcam; β-actin antibody (MAB1501) was purchased from parthanatic cell death in neurons and astrocytes [15,16]. Despite the Upstate Biotechnology; PAR antibody (551813) was purchased current progress in our understandings about parthanatos, the need from BD Pharmigen; γH2AX antibody (05-636) was ordered from for efficacious PARP inhibitors of better therapeutic potential is emerg- Merck Millipore; LC3 antibody (PM036) was purchased from ing [17]. Currently, the crystal structure of a DNA double-strand break in MBL. Normal rabbit IgG and horseradish peroxidase-conjugated anti- complex with human PARP1 domains obligatory for activation has been bodies (sc-2004) were purchased from Santa Cruz Biotechnology. resolved [18], which may facilitate the process in designing novel Enhanced chemiluminescence (ECL) reagent was ordered from Perkin PARP1 inhibitors. Elmer. Macroautophagy (hereafter termed autophagy) is highly coordi- nated self-digesting process involving autophagosomal engulfment 2.2. Cell culture and lysosomal degradation of damaged proteins or organelles for recycling to maintain intracellular energy homeostasis under normal Atg5+/+ and Atg5−/− MEFs were kindly provided by Dr. Noburu physiological condition or in response to certain stress stimuli [19,20]. Mizushima (Tokyo Medical and Dental University). Primary WT and Derangement of autophagy has a pathogenic contribution to several PARP1−/− MEFs were isolated from fresh 129S WT and 129S PARP1−/− diseases, like infection, cancer, neurodegeneration, heart diseases, and mice, respectively. HeLa cells were purchased from the American Type – aging [21 25]. Culture Collection (Manassas, VA, USA). MEFs and HeLa cells were Currently, however, less is known about the regulation of energy cultured in high-glucose DMEM supplemented with 10% heat- turnover during parthanatos. Considering autophagy is a self-protective inactivated FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. The scheme in response to stress for energy preservation and parthanatos cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. is a cell death process associated with energy depletion, we were inter- ested in understanding the role of autophagy in energy regulation during 2.3. Intracellular ATP assay parthanatos. Moreover, inspired by the concomitant occurrences of autophagy dysregulation and parthanatos in neurodegenerative and Intracellular ATP was determined by CellTiter-Glo® Luminescent Kit cardiovascular disorders, we speculated that autophagy defects (G7571) from Promega according to the manufacturer's protocol. In may amplify the severity of parthanatic cell death. In this study, brief, 5 × 104 cells were seeded per well in a 24-well plate for 24 h we tried to elucidate the contribution of autophagy dependency as well followed by the treatment indicated. Medium was then replaced with as the elsewhere mentioned MAPK pathways to energy homeostasis the new one, and the reagent was added to lyze the cells. The plate and severity during parthanatos in MEFs. Here, we demonstrate for the − − was gently shaken for 2 min, placed stationary in the dark for 10 min, first time that Atg5 / MEFs are extremely susceptible to MNNG and and then 150 μl solution was transferred to a 96-well plate for lumines- that PD98059, a traditionally known MEK inhibitor, shows an unexpect- cence detection using LB96V MicroLumat Plus (American Laboratory ed protectiveness and saves the vulnerable MEFs during parthanatos in a Trading). With background subtraction, the values were normalized to MEK-independent manner. On the other hand, we also highlight the individual control group as 100%. indispensable roles of AMP-activated protein kinase (AMPK), sirtuin 1 (SIRT1), and mitochondrial activity in regulating energy restoration + during parthanatos, which also better explain the differential 2.4. Determination for 7AAD cells response in Atg5-deficient cells. Collectively, these results provide 4 a complete energy perspective for better treating parthanatos-related MEFs were seeded in a 24-well plate (5 × 10 cells per well) for 24 h disorders and reveal unexpectedly PD98059 as an effective parthanatos- followed by the treatment indicated. The supernatant was collected, alleviating compound that should guide the future direction for drug and the cells were trypsinized and then mixed with the respective development. supernatant, followed by centrifugation at 2500 rpm for 10 min at room temperature. The pelleted cells were washed with 1× PBS 2. Materials and methods once and then centrifuged in a similar manner. Then, the cells were resuspended with PBS containing 7AAD (5 μM) and then fl 2.1. Reagents and antibodies submitted to FACS analysis (FACScan ow cytometer, Becton Dickinson) using the CellQuest program. The data were obtained MNNG (O-2392) was purchased from Chem Service. 7- based on the FL3 channel. Aminoactinomycin D (7AAD, 559925) was obtained from BD Pharmingen. zVAD-fmk (219007), doxorubicin (324380), SB203580 2.5. Lactate dehydrogenase (LDH) release assay (559389), SP600125 (420119), U0126 (662005), PD98059 (513000), wortmannin (681675), and compound C (171260) were ordered from LDH release assay was performed using CytoTox 96® Non-Radioactive Calbiochem. Gallotannin (403040), rapamycin (R8781), bafilomycin Cytotoxicity Assay Kit (G1780) from Promega according to the man- A1 (B1793), rotenone (R8875), diphenyliodonium (DPI, D2926), ufacturer's protocol. In brief, cells were seeded in a 24-well plate trifluorocarbonylcyanide phenylhydrazone (FCCP, C2920), MTT (5 × 104 cells per well) for 24 h followed by the treatment as indicated. (M2128) and Dulbecco's PBS (D5652) were obtained from Sigma-Aldrich. The readout for the maximum LDH release was achieved by Lysis JC-1 (T3168), Dulbecco's Modified Eagle Medium (DMEM, 12100-046), Solution (10×) 45 min before the endpoint. The supernatant from trypsin-EDTA (25200-072), fetal bovine serum (FBS, 04001-1A), and each well was collected and centrifuged for 5 min at 4 °C, and then penicillin/streptomycin (03031-1B) were purchased from Invitrogen. transferred in 50 μl into a 96-well plate, followed by 50 μl reconstituted The FastStart Universal SYBR Green Master (14526100) was ordered substrate mix, for a desired incubation period (protected from light). from Roche Applied Science. Commercial antibodies against p-ERK1/2 A final 50 μl stop solution was added into each well, and the plate was C.-T. Huang et al. / Biochimica et Biophysica Acta 1843 (2014) 531–543 533 read at 490 nm. With background subtraction, the maximum value was (5 mg/ml) was added into the medium per well with a 5-fold dilution, represented by the complete lysis group as 100%. and the plate was incubated for 1 h at 37 °C. After that, the solution was aspirated, and the purple formazan granules metabolized by the 2.6. Immunoblot mitochondria in live cells were resolved with 300 μl DMSO. The signal was detected by LB96V MicroLumat Plus (American Laboratory

Cells were lyzed with the RIPA buffer supplemented with a cocktail Trading) at 550 and 630 nm. Data were represented by A550–630 and of proteinase inhibitors. After sonication, protein concentrations were normalized with the individual control group as 100%. determined using the Bio-Rad protein assay. Each sample with the equal protein concentration was electrophoresed on SDS-PAGE, and 2.11. Determination for mitochondrial transmembrane potential transferred to the PVDF membranes. Nonspecific binding was blocked (MTP, △Ψm) with 5% nonfat milk. After immunoblotted with the primary specific antibodies as indicated, membranes were washed and incubated JC-1 dye was used to detect △Ψm by FACS analysis. If △Ψm is main- with an HRP-conjugated secondary antibody. Protein bands were tained, JC-1 forms as a dimer and reveals red; otherwise, when △Ψm is detected with an enhanced chemiluminescence detection reagent. disrupted, JC-1 acts as a monomer and the red color declines into an The signals were quantified by ImageJ 1.48 g with suitable back- orange or yellow color. Of note, additional rotenone-treated and ground calibration and represented as indicated the ratio of respec- FCCP-treated groups were used as positive controls. After drug tive intensity units. treatment, medium was aspirated, replaced with PBS containing JC-1 (5 μg/ml) for 30 min at 37 °C in the dark. The cells were then 2.7. Immunoprecipitation for SIRT1 activity assay trypsinized and transferred into a micro-centrifuge tube, centrifuged at 2500 rpm for 10 min, resuspended as a single-cell suspension, SIRT1 activity was measured by CycLex Sir2 Assay Kit (CY-1151) followed by FACS analysis (FACScan flow cytometer, Becton Dickinson) from MBL International Corporation according to the manufacturer's using the CellQuest program. The data were obtained based on the protocol. In brief, after SIRT1 immunoprecipitation, a mixture of SIRT1 FL1 (on the x-axis) and FL2 (on the y-axis) channels with appropriate reaction agents was added and incubated for 1 h. Finally, 50 μl superna- compensation, and expressed as the cell percentage with significant tant per sample was transferred into a 96-well dark plate, followed by loss of FL-2 signal under the user-defined quadrant analysis. 50 μl 2× Stop Solution. At this time, a blank (Cell Lysis Buffer alone) and a recombinant SIRT1 were used as background correction. Fluores- 2.12. Statistical analysis cence was read by LB96V MicroLumat Plus with excitation wavelength at 340–360 nm and emission at 440–460 nm. Data were normalized All statistical analyses were performed with unpaired two-tailed to the protein content measured by Bradford method and expressed Student's t-tests. Values were expressed as the mean ± S.E.M. from at as % compared to the WT control group (100%). least three independent experiments, each of which was done in 2–3 replicates. 2.8. Mitochondrial DNA (mtDNA) quantification 3. Results After drug treatment, the cell pellet was added with UniversALL™ Extraction Buffer (Yeastern, Cat. YGF10-1) and heated for 10 min at 3.1. Atg5−/− MEFs are vulnerable to MNNG-induced cell death 95 °C for complete extraction of total DNA, and DNA concentration was quantified by the LB96V MicroLumat Plus at 260 nm and 280 nm. The parthanatos inducer MNNG triggered the ATP depletion within A total of 1 μg DNA was used, mixed well with the FastStart SYBR 4 h treatment followed by a recovery phase in MEFs, where Atg5−/− Green Master (Roche), and then submitted to quantitative real-time MEFs showed impaired ATP restoration compared to the WT counter-

PCR analysis with automatic CT setting, with a program of 20 min at part (Fig. 1A). Similar to our previously work demonstrating that 95 °C, followed by 50 cycles for 15 s at 95 °C, 20 s at 58 °C and 20 s at MNNG-induced PARylation and necrotic cell death in MEFs could be 72 °C. Single-product amplification was verified by an integrated post- abrogated by the pharmacological inhibitors 3AB and/or DPQ [7],we run melting curve analysis. The primer sequence pairs used for quantita- also confirmed this notion in terms of ATP depletion. Our data revealed tive real-time PCR were mtDNA (16S rRNA) forward 5′-AGCTG AAGAA that MNNG-induced early-phase ATP depletion could be completely AGCGT GTTCC A-3′, mtDNA (16S rRNA) reverse 5′-AACTC TGTCC abrogated by the PARP1 inhibitor 3AB (Fig. 1B) or in the absence of AAGAT CGCTG ACT-3′, nuclear DNA (hexokinase 2, intron 9) forward PARP1 (Fig. 1C). In support of necrotic context, MNNG-elicited ATP 5′-CGGGG ACCTG ACTGA CTACC-3′ and nuclear DNA (hexokinase 2, dynamic alteration was not affected in the presence of pan-caspase intron 9) reverse 5′-AGGAA GGCTG GAAGA GTGC-3′. mtDNA content inhibitor zVAD-fmk (Fig. 1D). Consistent with the fragility in −/− was calculated from the difference in CT values between mtDNA and MNNG-treated Atg5 MEFs, LDH release was detectable at 9 h nucleus-specific DNA and expressed as fold increase based on a 2−△△CT and up to 60% at 24 h (Fig. 1E).Similarly,byuseof7AAD,which method. could intercalate double-stranded nucleic acids from dying and dead cells, we demonstrated that Atg5−/− MEFs showed more 7AAD+ 2.9. RNA extraction, reverse transcription, and quantitative real-time PCR signalsinresponsetoMNNGthantheWTcounterpart(Fig. 1F). Further- more, we confirmed this biphasic ATP alteration is specific to MNNG in RNA extraction and real time-PCR were conducted as we previously MEFs, where both WT and Atg5−/− MEFs similarly responded to the described [26]. The primer sequence pairs used for quantitative real- topoisomerase inhibitors doxorubicin (Fig. 1G) and etoposide (Fig. 1H). time PCR were PGC1α forward 5′-AGCCGTGACCACTGACAACGAG-3′, Taken together, these data suggest that Atg5−/− MEFs are vulnerable PGC1α reverse 5′-GCTGC ATGGT TCTGA GTGCT AAG-3′; β-actin forward to MNNG-induced cell death with disabled ATP recovery. 5′-CGGGG ACCTG ACTGA CTACC-3′, β-actin reverse 5′-AGGAA GGCTG GAAGA GTGC-3′. 3.2. MAPKs do not regulate energy adaptive response during parthanatos in MEFs 2.10. MTT assay As previously mentioned in other contexts [13,14],wealsowondered MEFs (5 × 104 per well) were seeded in a 24-well plate for 24 h whether MAPKs have a role in energy adaptation during parthanatos in followed by the treatment as indicated. At the endpoint, MTT solution MEFs. Phosphorylation of ERK, JNK and p38 was similarly induced in 534 C.-T. Huang et al. / Biochimica et Biophysica Acta 1843 (2014) 531–543

Fig. 1. Atg5−/− MEFs are susceptible to parthanatos with disabled ATP restorability. (A, B, D) Intracellular ATP content after 150 μM MNNG administration at indicated time points was determined in Atg5+/+ and Atg5−/− paired MEFs in the absence or presence of 10 μM 3AB (B) or 20 μM zVAD-fmk (D) pretreated for 30 min. (C) Intracellular ATP content after 150 μM MNNG administration at indicated time points was determined in primary WT and PARP1−/− MEFs isolated from fresh 129S WT and PARP1−/− mice, respectively. (E) LDH release after 150 μM MNNG administration at indicated time points was measured in WT and Atg5−/− MEFs. (F) FACS analysis of cell death was performed in MEFs treated with 150 or 300 μM MNNG for 24 h, where 5 μM 7AAD was incubated 30 min prior to FACS. (G, H) Intracellular ATP content after 3 μM doxorubicin (DXR) administration (G), and intracellular ATP content and MTT activity after 100 or 200 μM etoposide administration (H) at indicated time points were determined in WT and Atg5−/− MEFs. Signiﬁcance level: *p b 0.05 compared to the WT counterpart in the same treatment and time point.

WT and Atg5−/− MEFs in response to MNNG (Fig. 2A). Moreover, (a cathepsin B inhibitor) did not interfere with MNNG-elicited ATP SB203580 (a p38 inhibitor) (Fig. 2B), SP600125 (a JNK inhibitor) dynamic responses in both WT and Atg5−/− MEFs (Fig. 2E). (Fig. 2C) and U0126 (a MEK/ERK inhibitor) (Fig. 2D) did neither affect − − nor explain the differential ATP alteration in WT and Atg5 / MEFs 3.3. PD98059 effectively facilitates ATP restoration during parthanatos and during parthanatos. Therefore, despite their participation in other saves vulnerable MEFs in a MEK-independent manner parthanatic contexts, MAPKs do not regulate the ATP adaptive response in these MEFs. Additionally, administration of either necrostatin-1 In the above MAPK screen, we discovered unexpectedly that PD98059, (a RIP1 inhibitor), BAPTA/AM (a cytosolic calcium chelator), or CA-074 a ﬂavonoid derivative considered the conventional MEK/ERK inhibitor, C.-T. Huang et al. / Biochimica et Biophysica Acta 1843 (2014) 531–543 535

Fig. 2. MAPKs do not regulate energy adaptive response during parthanatos in MEFs. (A) Immunoblot analysis of the MAPK-related proteins in WT and Atg5−/− MEFs after 150 μMMNNG challenge at indicated time points. The bands representative of Atg5 were shown as Atg12-conjugated forms. (B–E) Intracellular ATP content after 150 μM MNNG administration at indicated time points was determined in WT and Atg5−/− MEFs in the presence or absence of 3 μM SB203580 (B), 30 μM SP600125 (C), 3 μM U0126 (D), 10 μM necrostatin-1, 10 μM BAPTA/AM, or 10 μM CA-074 (E). In each row of quantitative results regarding immunoblots, the normalized reference was framed as indicated. effectively and dose-dependently facilitated ATP recovery (Fig. 3A) PD98059 at 10 μM, neither JNK nor p38 phosphorylation (Fig. 3C and dampened cell death (Fig. 3B) in response to MNNG evidently in vul- and D). Notably, inhibition of ERK phosphorylation could be readily nerable Atg5−/− MEFs at the concentration as low as 3 μM. Notably, achieved by U0126 at 3 μM(Fig. 3C), while only partially achieved MNNG-induced ERK1/2 phosphorylation was not attenuated by by PD98059 at 30 μM(Fig. 3D). Furthermore, post-treatment of 536 C.-T. Huang et al. / Biochimica et Biophysica Acta 1843 (2014) 531–543

Fig. 3. PD98059 effectively facilitates ATP restoration during parthanatos and saves vulnerable MEFs independently of MEK inhibition. Intracellular ATP content (A) and LDH release (B) after 150 μM MNNG administration at indicated time points were determined in WT and Atg5−/− MEFs in the presence or absence of PD98059 at 3, 10, or 30 μM pretreated for 30 min. (C) Immunoblot analysis of ERK phosphorylation was conducted in WT and Atg5−/− MEFs after 150 μM MNNG challenge at indicated time points in the presence or absence of 10 μM PD98059 (top panel) or 3 μM U0126 (bottom panel), pretreated for 30 min. (D) Immunoblot analysis of the indicated proteins was conducted in WT MEFs after 150 μM MNNG challenge at indicated time points in the presence or absence of PD98059 at 10 or 30 μM pretreated for 30 min. (E) Intracellular ATP content was determined in WT and Atg5−/− MEFs in the presence or absence of 10 μM PD98059 pre-treated for 30 min or post-treated at 1 or 4 h after 150 μM MNNG administration at indicated time points. In each row of quantitative results regarding immunoblots, the normalized reference was framed as indicated. C.-T. Huang et al. / Biochimica et Biophysica Acta 1843 (2014) 531–543 537

PD98059 (10 μM) at 1 and 4 h after MNNG administration rendered 3.4. PD98059 protects MNNG-treated MEFs without modulation on DNA the similar protectiveness as pre-treatment (Fig. 3E). Collectively, damage responses these data implied PD98059 as an effective parthanatos-inhibiting compound whose protectiveness is through a MEK-independent We questioned whether protection by PD98059 at effective dosage mechanism. against parthanatos comes from modulation of MNNG-elicited PARP1

Fig. 4. PD98059 protects MNNG-treated MEFs without modulation on DNA damage responses. (A–D) Immunoblot analyses of PARylated PARP1 (A, B), p53 (C), and γH2AX (D) were performed in WT (A–D) and Atg5−/− MEFs (B, C) after 150 μM MNNG challenge at indicated time points in the presence or absence of 10 μM (B, C) or 30 μM (A) PD98059, or 3 μM U0126 (C), pretreated for 30 min. (E) Intracellular ATP content after 150 μM MNNG administration at indicated time points was determined in WT and Atg5−/− MEFs in the presence or absence of 10 μM PD98059 and/or 30 μM gallotannin (GLTN), pretreated for 30 min. In each row of quantitative results regarding immunoblots, the normalized reference was framed as indicated. PAR-PARP1 indicates PARylated PARP1. 538 C.-T. Huang et al. / Biochimica et Biophysica Acta 1843 (2014) 531–543 activation and DNA damages in MEFs. To address these possibilities, we activity was rather impaired in Atg5−/− MEFs but not significantly af- first determined PARP1-dependent PARylation as an index of PARP1 fected in WT counterpart (Fig. 6A). We also noted here that Atg5−/− activity. Administration of MNNG rapidly induced PARylation of several MEFs harbored an inherently compromised SIRT1 activity at basal proteins, especially with the apparent effect on protein(s) at the same level (about 80% compared to WT; Fig. 6A). However, with addition molecular weight as PARP1 (113 kD) (Fig. 4A and B). As PARP1 itself of PD98059, SIRT1 activity was significantly elevated in both MNNG- can undergo PARylation [27], this obvious PARylation signal might be challenged WT and Atg5−/− MEFs (Fig. 6A). In line with the necessity representative of PARP1 and its activity. Importantly, we found that of SIRT1, pharmacologic inhibition of SIRT1 by sirtinol completely PARP1 PARylation following MNNG treatment was unaffected in the amplified MNNG cytotoxicity in both WT and Atg5−/− MEFs (Fig. 6B). presence of PD98059 in both WT and Atg5−/− MEFs, albeit a higher With a check in mitochondrial DNA (mtDNA) content, however, we PAR signal was noted in Atg5−/− MEFs (Fig. 4B). This PARP1 PARylation found that MNNG was able to increase mtDNA content at 1 h in both after MNNG treatment was neither significantly affected by PD98059 at WT and Atg5−/− MEFs, but the intensity was attenuated thereafter higher concentration (30 μM) when ERK inhibition was achieved (Fig. 6C). Besides, a higher mtDNA induction was noted in Atg5−/− (Fig. 4A). Next, to further evaluate if PD98059 might affect MNNG- MEFs than in the WT counterpart in response to MNNG (Fig. 6C). We induced DNA damage responses, we determined p53 and γH2AX reasoned that this compensatory higher burst was reflective of intrinsic expression. We found that MNNG-induced p53 protein expression in deficiencies in mitochondrial quality control in Atg5−/− MEFs, even both WT and Atg5–/− MEFs was neither disturbed by PD98059 nor though it was still not comparable for providing sufficient energy U0126 (Fig. 4C) and ditto for γH2AX expression in WT MEFs (Fig. 4D). resources from these defective mitochondria during parthanatos. In To test whether PD98059 could still be protective under enhanced the presence of PD98059, we observed that a higher induction fold death signal, we utilized the poly(ADP-ribose) glycohydrolase (PARG) and a longer retention intensity of mtDNA content were achieved in inhibitor gallotannin (GLTN) for PAR retention. Within 8 h screening, both WT and Atg5−/− MEFs after MNNG challenge (Fig. 6C). Further- ATP restoration following MNNG treatment was significantly impaired more, we also noticed that, in both WT and Atg5−/− MEFs, PD98059 in the presence of GLTN in both MEFs, while addition of PD98059 in con- alone was capable of stimulating mtDNA synthesis (Fig. 6C) and raising text with GLTN still facilitated ATP recovery in both WT and Atg5−/− the transcription of peroxisome proliferator-activated receptor (PPAR) MEFs (Fig. 4E). Therefore, these data argue that PD98059 protects gamma coactivator 1 alpha (PGC1α)(Fig. 6D), whose product is a mas- MEFs from parthanatos without modulation on DNA damage responses. ter regulator of mitochondrial gene expression and energy metabolism [29]. 3.5. AMPK is indispensable to energy adaptation during parthanatos To further confirm the mitochondrial setting for energy restoration after MNNG challenge, we used the mitochondrial pharmacologic inhib- Since the energy stress is a clear signature of parthanatos, we turned itors rotenone (a complex I/II inhibitor), DPI (a flavoprotein inhibitor), to examine other master stress kinases about their contribution to and FCCP (a mitochondrial uncoupler). In the presence of rotenone, parthanatic energy responses in MEFs. Western blot analysis demon- DPI or FCCP, MNNG cytotoxicity was extremely exasperated in both strated that phosphorylation of AMPKα1 at Thr172 was induced as WT and Atg5−/− MEFs (Fig. 6E). Of note, all of these mitochondrial early as 30 min after MNNG administration in MEFs, but faded down inhibitors alone did not lead to differential energy level between WT thereafter (Fig. 5A). Here, we noted a higher basal AMPKα1phosphor- and Atg5−/− MEFs at 24 h treatment (Fig. 6E). For a close view about ylation in Atg5−/− MEFs. Within 30 min challenge, the levels of phos- the ATP-depleting process, we found that the ATP level was severely phorylation on Akt at Ser473 and GSK3β at Ser9 decreased whenever exhausted in the presence of rotenone or DPI in both WT and Atg5−/− AMPK was active (Fig. 5A), suggesting a reciprocal activation relation- MEFs as early as 15 min after MNNG challenge and without subsequent ship between AMPK and Akt/GSK3β signal pathways immediately rebound (Fig. 6F). On the other hand, we also utilized the MTT, a tetra- after MNNG administration. After this early inhibitory response, the zolium dye which could be converted into a purple formazan by the Akt and GSK3β phosphorylation statuses concordantly rebounded mitochondrial succinate dehydrogenase in viable cells as an indicator (Fig. 5A). However, in the presence of PD98059, the phosphorylation of mitochondrial activity. Concordantly, MNNG challenge decreased dynamics of these kinases inducible by MNNG was not significantly MTT activity to higher extent in Atg5−/− MEFs than in the WT counter- affected in both MEFs (Fig. 5A). To understand the essential role of part (Fig. 6G). In 1 h MNNG treatment, the MTT activities were equally AMPK in energy regulation upon parthanatos, we used the pharmaco- suppressed to half their basal levels, but thereafter, this compromised logic AMPK inhibitor compound C. Indeed, in the presence of compound mitochondrial activity was relieved in WT MEFs while continuously C, the ATP restorability during parthanatos was both impaired in WT suppressed in Atg5−/− MEFs (Fig. 6G). Moreover, addition of PD98059 and Atg5−/− MEFs (Fig. 5B). Remarkably, addition of PD98059 still better recovered mitochondrial activity in Atg5−/− MEFs 10 h after facilitated ATP recovery in MNNG-challenged WT and Atg5−/− MEFs in MNNG administration, but not significantly improved further in WT the presence of compound C (Fig. 5B). On the other hand, pharmacologic MEFs (Fig. 6G). To determine whether necrotic events preceded the inhibition of PI3K/Akt by wortmannin or mTORC1 by rapamycin only early suppression of ATP level and mitochondrial activity, we used the slightly worsened the energy restoration in MNNG-treated Atg5−/− JC-1 dye to evaluate the mitochondrial transmembrane potential

MEFs, not comparable to that caused by compound C (Fig. 5C). Consis- (△Ψm), whose dissipation is a common signature of mitochondrial tently, LDH release was aggravated in both MEFs by compound C, where- depolarization in necrotic cell death. Intriguingly, △Ψm dissipation as addition of PD98059 alleviated this aggravation (Fig. 5D). Conjointly, was undetectable at 1, 4, and 8 h after MNNG challenge in both WT these data indicate that AMPK is a vital kinase for cells against energy and Atg5−/− MEFs (Fig. 6H), yet the validity of experiment was deprivation and cytotoxicity during parthanatos. confirmed by the positive controls using FCCP and rotenone with detectable dissipation occurring as early as in 4 h treatment in Atg5−/− MEFs 3.6. Diminished mitochondrial activity in Atg5−/− MEFs in response (Fig. 6H). Collectively, these results suggest that mitochondrial activity to parthanatos predetermines the tolerability of cells to MNNG and unveil its diminished activity in Atg5−/− MEFs during parthanatos. Considering the versatility of AMPK, we first intuitively tested whether mitochondrial signaling and activity are an inevitable part of 3.7. Autophagic flux is not obligatory for energy supply and protection AMPK-mediated cytoprotective effects against parthanatos via SIRT1 during parthanatos and mitochondrial biogenesis [28]. At 8 h after MNNG administration, when the ATP level in WT MEFs was ready for a significant rebound With observations on a requirement of AMPK in energy adaptation yet not in Atg5−/− MEFs (Fig. 1A), we consistently observed that SIRT1 and the susceptibility of Atg5−/− MEFs, together with the notion for C.-T. Huang et al. / Biochimica et Biophysica Acta 1843 (2014) 531–543 539

Fig. 5. AMPK is indispensable to energy adaptation during parthanatos. (A) Immunoblot analysis of major stress response-related proteins was conducted in WT and Atg5−/− MEFs after 150 μM MNNG challenge at indicated time points in the presence or absence of 10 μM PD98059 pretreated for 30 min. (B, C) Intracellular ATP content after 150 μM MNNG administration at indicated time points was determined in WT and Atg5−/− MEFs in the presence or absence of 10 μM compound C (CC) and/or 10 μM PD98059 (B), 100 nM wortmannin (C, top panel), or 100 nM rapamycin (C, bottom panel), respectively, pretreated for 30 min. (D) LDH release after 150 μM MNNG administration at indicated time points was measured in WT and Atg5−/− MEFsinthepresenceorabsenceof10μM compound C and/or 10 μM PD98059, pretreated for 30 min. In each row of quantitative results regarding immunoblots, the normalized reference was framed as indicated. Signiﬁcance level: *p b 0.05. 540 C.-T. Huang et al. / Biochimica et Biophysica Acta 1843 (2014) 531–543

Fig. 6. Atg5−/− MEFs reveal diminished mitochondrial activity essential to surviving parthanatos. (A) SIRT1 activity was determined in WT and Atg5−/− MEFs 8 h after 150 μM MNNG and/or 10 μM PD98059 administration, pretreated for 30 min if necessary. (B) Intracellular ATP content after 150 or 300 μM MNNG administration at 24 h was determined in WT and Atg5−/− MEFs inthepresenceorabsenceof50μM sirtinol pretreated for 30 min. (C) Mitochondrial DNA (mtDNA) content was determined after 150 μMMNNGand/or10μM PD98059 administration at indicated time points in WT and Atg5−/− MEFs, pretreated for 30 min if necessary. (D) PGC1α transcripts following 10 μM PD98059 at indicated time points were measured in WT and Atg5−/− MEFs. (E) Intracellular ATP content 24 h after 150 μM MNNG administration was determined in WT and Atg5−/− MEFs in the presence or absence of 500 nM rotenone (Ro), 10 μMDPI,or30μM FCCP, pretreated for 30 min if necessary. (F) Intracellular ATP content after 150 μM MNNG administration at indicated time points was determined in WT and Atg5−/− MEFs in the presence or absence of 500 nM rotenone (top panel) or 30 μM DPI (bottom panel) pretreated for 30 min. (G) MTT activity after 150 μM MNNG administration at indicated time points was determined in WT and Atg5−/− MEFsinthepresenceorabsenceof10μM PD98059 pretreated for 30 min. (H) Integrity of mitochondrial transmembrane potential −/− (△Ψm) after 150 μM MNNG administration at indicated time points was determined in WT and Atg5 MEFs. The validity of experiment was conﬁrmed by 30 μM FCCP and 500 nM rotenone as positive controls in Atg5−/− MEFs. ND, not determined. Signiﬁcance level: *p b 0.05 compared to the individual DMSO control groups. #p b 0.05 compared to the individual MNNG challenge groups at the same time point.

the involvement of AMPK in autophagy induction [30],wereasonably we did neither observe LC3 lipidation in HeLa cells after MNNG challenge speculated a role of autophagy during parthanatos. In the presence (Fig. 7B), while a gradual decline in expression of LC3-II was noted of bafilomycin A1, a vacuolar H+ ATPase inhibitor that prevents (Fig. 7B). Much strikingly, MNNG-mediated ATP response curves and cy- autolysosomal fusion, we found, however, that MNNG did not promote totoxicity were not exacerbated in the presence of bafilomycin A1 in LC3 lipidation, a molecular signature of autophagosome maturation. both WT and Atg5−/− MEFs (Figs. 7C and D). Compatibly with above, Consistently MNNG did not induce p62 degradation, a consequence of the addition of PD98059 still facilitated ATP recovery in WT and Atg5 cargo elimination within autolysosomal compartment, in WT MEFs −/− MEFs in the presence of bafilomycin A1 after MNNG administration without bafilomycin A1 treatment. Noticeably, the defective autophagy (Fig. 7E). Therefore, these results together with Fig. 6 indicate that induction machinery in Atg5−/− MEFs was confirmed by the absence parthanatos does not induce autophagic flux but is aggravated by the of lipidated (LC3-II) and the p62 accumulation (Fig. 7A). Consistently, deficits in mitochondrial quality control underlying Atg5 deficiency. C.-T. Huang et al. / Biochimica et Biophysica Acta 1843 (2014) 531–543 541

Fig. 7. Autophagic flux is not obligatory for energy supply and protection during parthanatos. (A, B) Immunoblot analyses of autophagic flux-related proteins were conducted in WT and Atg5−/− MEFs (A) and in HeLa cells (B) after 150 μM MNNG and/or 100 nM bafilomycin A1 (baf A1) administration at indicated time points, pretreated for 30 min if necessary. (C–E) Intracellular ATP content (C, E) and LDH release (D) after 150 μM MNNG, 100 nM bafilomycin A1 (baf A1), and/or 10 μM PD98059 administration at indicated time points in WT and Atg5−/− MEFs, pretreated for 30 min if necessary. In some rows of quantitative results regarding immunoblots, the normalized reference was framed as indicated if necessary.

4. Discussion Our findings indicate that the MAPK signaling pathways are not involved in the regulation of parthanatic energy responses, albeit their Massive NAD+ and ATP depletion is a general mark of PARP1- reported modalities elsewhere to DNA repair, cell cycle arrest, and mediated necrotic cell death. Despite the current progress that exogenous defense response to stress [13,14,35,36]. Furthermore, both WT and supplementation of metabolic intermediates could save neurons or glia Atg5−/− MEFs showed comparable MAPK activation during parthanatos, cells from PARP1-mediated cell death [15,16,31,32],lessisknownabout possibly excluding a link underlying Atg5 deficiency to those activation. the regulation of energy turnover during the parthanatos. Here, we char- A MEK-independent PD98059 protectiveness from parthanatos is acterized the depleting and recovering phases of ATP level during exceptionally highlighted throughout this study. The differential actions parthanatos and underscored the recovery capability as a determinant between the conventional MEK kinase inhibitors PD98059 and U0126 of severity of parthanatic cell death. By using macroautophagy-defective have been reported somewhere in literature, where, for example, Atg5−/− MEFs, we highlighted their extraordinary vulnerability to the PD98059 could potently stimulate adipogenesis but U0126 did not [37]. alkylating agent MNNG, but not to the DNA-intercalating topoisomerase In line with their findings, here we also showed that only PD98059 II inhibitors doxorubicin and etoposide, which could potently initiate administration effectively saved MEFs from parthanatos. Indeed, several apoptotic cell death [33]. Regarding a potential dual role of Atg5 in MEK-independent actions for PD98059 have been reported and might autophagy and apoptosis [34], our observed fragility and impaired be correlated to its property as a flavonoid derivative, including weak ATP restorability of Atg5−/− MEFs to MNNG were unaffected by the estrogenic activity [37,38], elevation of glutathione [39–41], and behaving pan-caspase inhibitor zVAD-fmk, together with the unequivocally similar as an aryl hydrocarbon receptor (AHR) antagonist [42]. Notably, in this responses of WT and Atg5−/− MEFs to doxorubicin suggesting that Atg5 study we implied that PD98059 protectiveness from parthanatos comes deficiency confers a specificinfirmity to parthanatos. from an enhancement of mitochondrial activity, without modulation on 542 C.-T. Huang et al. / Biochimica et Biophysica Acta 1843 (2014) 531–543 the DNA damage responses. Therefore, from our results in combination SIRT1 activation, rendering protection against several metabolic diseases with previous findings [37,38] and a body of knowledge that links as well as cancer [58]. However, considering the versatility and broad estrogenic activities to mitochondrial respiration [43–45], we believe participation of SIRT1 and PARP1 under several physiologic and patholog- that PD98059 is able to boost up mitochondrial functionality which ic conditions [17,59,60], the background metabolic status underlying seems extremely essential to energy restoration during parthanatos. SIRT1/PARP1 imbalance in Atg5−/− MEFs is worthy of investigation to a AMPK is a multifunctional protein kinase that participates in energy greater extent, which may help clarify the interrelationship among expenditure and autophagy induction in response to diverse stimuli SIRT1, PARP1, and macroautophagy, and benefitthetreatmentofmany [30,46]. In this study we uncovered an absolute requirement of AMPK disorders linking to dysregulation in these aspects. to protect cells from parthanatos and dissected its potential down- Our results provide a complete energy perspective dealing with its stream cellular processes [28,47,48], i.e., underlining the importance of regulation on the parthanatic response and explicitly point out the ener- mitochondrial metabolism but, unexpectedly, not of autophagy induc- gy status as a key determinant in the death outcome. PARP1-mediated tion as an adaptive response to parthanatos. The unnecessariness of cell death has been frequently described in type I diabetes [10],in autophagic flux machinery for energy restoration during parthanatos neuropathology such as stroke [61], excitotoxicity [61,62],andneurode- was also exemplified in our study by the absence of LC3 lipidation and generation [12,21], as well as at necrotic loci from ischemia–reperfusion p62 elimination after MNNG challenge, the parthanatos irresponsive injury like myocardial infarcts [63]. Concomitantly, autophagy dysregu- to bafilomycin A1, as well as parthanatos insensitive to enforced lation has also been implicated in these neurological and cardiovascular autophagy induction by mTORC1 inhibition, which is supposed to be diseases [25,64,65]. In contrast, we clarify in this study a link of responsible for autophagy induction downstream of AMPK activation mitochondrial quality maintained by autophagy to the severity of [49]. Furthermore, the PD98059 protectiveness was also rechecked to parthanatos. Moreover, concerning the urgent need of efficacious be independent of autophagy induction as illustrated with its effective- PARP inhibitors for better therapeutic benefits within a wide range of ness in the presence of bafilomycin A1. In this study, we also observed inflammatory disorders [17], we also identify unexpectedly PD98059 the higher basal AMPK signaling in Atg5−/− MEFs. We reasoned that as a cytoprotective compound against parthanatos via enhancing the increased AMPK activity might be the compensatory adaptation in mitochondrial metabolism and facilitating energy restoration following Atg5−/− MEFs. However, an alternative macroautophagy has also the ATP depletion. Taken together, our findings establish a fundamental been reported in Atg5−/− MEFs in response to starvation or etoposide basis for treating these parthanatos-related disorders and should guide [50], but here we precisely excluded the need of autophagy flux in the future direction for drug development. parthanatos by use of the autophagy inhibitor bafilomycin A1, which shall block all the autophagic clearance events. Acknowledgement After ruling out the necessity of autophagic flux in restoring mitochondrial activity and subsequent energy level during parthanatos, we rea- This work was supported by grants from the National Science soned that it is deficits in mitochondrial quality control associated with Council (NSC 100-2320-B-002-088) and Taipei Medical University– Atg5 knockout [51,52] that contribute to impaired mitochondrial energy Shuang Ho Hospital (102TMU-SHH-08). adaptability in response to this stress. 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