Autosis is a Na+,K+-ATPase–regulated form of celldeath triggered by autophagy-inducing peptides,starvation, and hypoxia–ischemiaYang Liua,b, Sanae Shoji-Kawataa,b,1, Rhea M. Sumpter, Jr.a,b, Yongjie Weia,b,c, Vanessa Ginetd, Liying Zhange,Bruce Posnere, Khoa A. Tranf, Douglas R. Greeng, Ramnik J. Xavierh,i,j, Stanley Y. Shawf,j, Peter G. H. Clarked,Julien Puyald,2, and Beth Levinea,b,c,k,2

aCenter for Autophagy Research, Departments of bInternal Medicine, eBiochemistry, and kMicrobiology, and cHoward Hughes Medical Institute, University ofTexas Southwestern Medical Center, Dallas, TX 75390; dDepartment of Fundamental Neurosciences, University of Lausanne, CH-1005 Lausanne, Switzerland;fCenter for Systems Biology, hCenter for Computational and Integrative Biology, and iGastrointestinal Unit, Massachusetts General Hospital, HarvardMedical School, Boston, MA 02114; jBroad Institute of Harvard and MIT, Cambridge, MA 02142; and gDepartment of Immunology, St. Jude’s Children’sResearch Hospital, Memphis, TN 38105

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2013.

Contributed by Beth Levine, October 26, 2013 (sent for review September 16, 2013)

A long-standing controversy is whether autophagy is a bona fidecause of mammalian cell death. We used a cell-penetrating autoph-agy-inducing peptide, Tat-Beclin 1, derived from the autophagy pro-tein Beclin 1, to investigatewhether high levels of autophagy result incell death by autophagy. Here we show that Tat-Beclin 1 inducesdose-dependent death that is blocked by pharmacological or geneticinhibition of autophagy, but not of apoptosis or necroptosis. Thisdeath, termed “autosis,” has unique morphological features, includ-ing increased autophagosomes/autolysosomes and nuclear convolu-tion at early stages, and focal swelling of the perinuclear space at latestages. We also observed autotic death in cells during stress condi-tions, including in a subpopulation of nutrient-starved cells in vitroand in hippocampal neurons of neonatal rats subjected to cerebralhypoxia–ischemia in vivo. A chemical screen of ∼5,000 known bio-active compounds revealed that cardiac glycosides, antagonists ofNa+,K+-ATPase, inhibit autotic cell death in vitro and in vivo. Further-more, genetic knockdown of the Na+,K+-ATPase α1 subunit blockspeptide and starvation-induced autosis in vitro. Thus, we have iden-tified a unique form of autophagy-dependent cell death, a Food andDrug Administration-approved class of compounds that inhibit suchdeath, and a crucial role for Na+,K+-ATPase in its regulation. Thesefindings have implications for understanding how cells die duringcertain stress conditions and how such cell death might be prevented.

The lysosomal degradation pathway of autophagy plays a cru-cial role in enabling eukaryotic cells to adapt to environmentalstress, especially nutrient deprivation (1). The core autophagymachinery was discovered in a genetic screen in yeast for genesessential for survival during starvation, and gene knockout orknockdown studies in diverse model organisms provide strongevidence for a conserved prosurvival function of autophagy duringstarvation (1). This prosurvival function of autophagy results fromits ability to mobilize intracellular energy resources to meet thedemand for metabolic substrates when external nutrient suppliesare limited.In contrast to this well-accepted, prosurvival function of

autophagy, there has been much debate as to whether autophagy—especially at high levels—also functions as a mode of cell death(2). Historically, based on morphological criteria, three types ofprogrammed cell death have been defined: type I apoptotic celldeath; type II autophagic cell death; and type III, which includesnecrosis and cytoplasmic cell death (3). Autophagic cell deathwas originally defined as a type of cell death that occurs withoutchromatin condensation and is accompanied by large-scale auto-phagic vacuolization of the cytoplasm. This form of cell death,first described in the 1960s, has been observed ultrastructurally intissues where developmental programs (e.g., insect metamor-phosis) or homeostatic processes in adulthood (e.g., mammary

involution following lactation or prostate involution followingcastration) require massive cell elimination (4–6). Autophagiccell death has also been described in diseased tissues and in cul-tured mammalian cells treated with chemotherapeutic agents orother toxic compounds (4–6).The term “autophagic cell death” has been controversial, be-

cause it has been applied to scenarios where evidence is lackingfor a causative role of autophagy in cell death (i.e., there is celldeath with autophagy but not by autophagy). However, usingmore stringent criteria to define autophagic cell death, severalstudies in the past decade have shown that autophagy genes areessential for cell death in certain contexts. This includes cases oftissue involution in invertebrate development as well as in cul-tured mammalian cells lacking intact apoptosis pathways (6, 7).In apoptosis-competent cells, high levels of autophagy can alsolead to autophagy gene-dependent, caspase-independent celldeath (8–10). In neonatal mice, neuron-specific deletion of Atg7protects against cerebral hypoxia–ischemia-induced hippocam-pal neuron death (11), and in adult rats, shRNA targeting beclin1 decreases neuronal death in the thalamus that occurs sec-ondary to cortical infarction (12).Although such studies provide genetic support for autophagy

as a bona fide mode of cell death, the nature of autophagic cell

Significance

We show that the selective overactivation of autophagy cancause cell death with unique morphological features distinctfrom apoptosis or necrosis. This unique type of autophagic celldeath, termed “autosis,” occurs not only in vitro but also invivo in cerebral hypoxia–ischemia. Moreover, autosis is inhibi-ted both in vitro and in vivo by cardiac glycosides, which areNa+,K+-ATPase antagonists used in clinical medicine. Ourfindings contribute to the basic understanding of cell-deathmechanisms and suggest strategies for protecting cells againststresses such as hypoxia–ischemia.

Author contributions: Y.L., S.S.-K., R.M.S., B.P., D.R.G., R.J.X., S.Y.S., P.G.H.C., J.P., and B.L.designed research; Y.L., S.S.-K., R.M.S., Y.W., V.G., K.A.T., and J.P. performed research; S.S.-K.and K.A.T. contributed new reagents/analytic tools; Y.L., R.M.S., Y.W., V.G., L.Z., B.P., R.J.X.,S.Y.S., P.G.H.C., J.P., and B.L. analyzed data; and Y.L., L.Z., R.J.X., S.Y.S., P.G.H.C., J.P.,and B.L. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.1Present address: Department of Molecular Immunology and Inflammation, NationalCenter for Global Health and Medicine, Tokyo 162-8655, Japan.

2To whom correspondence may be addressed. E-mail: JulienPierre.Puyal@unil.ch or beth.levine@utsouthwestern.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1319661110/-/DCSupplemental.

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death that occurs in mammalian cells and tissues in response tophysiological/pathophysiological stimuli remains poorly defined.It is unclear whether cells that die by autophagy have uniquemorphological features or a unique death machinery. The onlymorphological feature that has been linked to autophagic celldeath—autophagic vacuolization—may be observed in cells un-dergoing apoptotic or necrotic cell death, and no proteins, asidefrom the core autophagy proteins, have been shown to be re-quired for autophagic cell death.Here we identify a form of autophagic cell death, autosis, which

has unique morphological features; depends on the cellular Na+,K+-ATPase; and occurs during treatment with autophagy-inducingpeptides, starvation, and cerebral hypoxia–ischemia.

ResultsAutophagy-Inducing Peptides Trigger Autophagy-Dependent Cell Death.Previously, we discovered a potent autophagy-inducing cellpermeable peptide (13), Tat-Beclin 1, composed of 11 amino acidsof the HIV Tat protein transduction domain, a diglycine linker,and 18 amino acids (267–284 aa) derived from the autophagyprotein, Beclin 1. This peptide induced autophagy without cy-totoxicity at low doses, but caused cell death at higher doses (13).This finding suggested that the Tat-Beclin 1 peptide might inducea form of autophagy-dependent cell death and serve as a modelfor defining characteristics of autophagy-dependent cell deaththat occurs in pathophysiological settings.We examined the relationship between Tat-Beclin 1-induced

autophagy and cell death. In HeLa cells, Tat-Beclin 1 led todose-dependent induction of autophagy, as measured by ratios oflight chain 3 (LC3)-II/I and degradation of the autophagy sub-strate, p62 (Fig. 1A), as well as cell death, as measured by trypanblue exclusion (Fig. 1B). Increasing durations of exposure to afixed concentration of Tat-Beclin 1 resulted in a time-dependentincrease in autophagy and cell death (Fig. S1 A and B). Noautophagy induction or cell death was observed after treatmentwith a control peptide, Tat-Scrambled (13). Thus, Tat-Beclin 1

induces cell death in parallel with its ability to induce autophagyin a dose- and time-dependent manner.We confirmed that Tat-Beclin 1 induced HeLa cell death by

detection of cells positive for Sytox Green (a nucleic dye ex-cluded by live cells) (Fig. 1C), an increase of propidium iodide(PI)-positive cells (Fig. S1 C and D), and a decline of cellularATP levels (Fig. S1E). In addition, Tat-Beclin 1, but not Tat-Scrambled, significantly reduced clonogenic survival (Fig. 1D).Tat-Beclin 1 also induced cell death in a variety of additionaltumor cell lines, in human and rat fibroblasts, and in primary andE1A/Ras-transformed murine embryonic fibroblasts (MEFs)(Fig. S1F).We investigated whether Tat-Beclin 1-induced autophagy

is mechanistically related to Tat-Beclin 1-induced cell deathby using pharmacological and genetic approaches to inhibitautophagy. Treatment with 3-methyladenine (3-MA), an inhibitorof class III PI3K activity and autophagosome formation, partiallyblocked Tat-Beclin 1-induced HeLa cell death, as measured in-creased cellular levels of ATP (Fig. S1G), a decreased percentageof trypan blue-positive cells (Fig. S1H), and increased clonogenicsurvival (Fig. 1E). siRNA knockdown of the essential autophagygene, beclin 1, decreased autophagy in HeLa cells (Fig. S1I),decreased Tat-Beclin 1-induced cell death (Fig. S1J), and in-creased clonogenic survival (Fig. 1F). Furthermore, doxycycline-inducible shRNA knockdown of ATG13 or ATG14 in U2OS cellsdecreased Tat-Beclin 1-induced autophagy (Fig. S1K), protectedagainst Tat-Beclin 1-induced cell death (Fig. S1L), and increasedclonogenic survival (Fig. 1G). Blockade of autophagosomal/lysosomal fusion by bafilomycin A1, a vacuolar proton ATPaseinhibitor, did not reduce Tat-Beclin 1-induced cell death (Fig.S1M), suggesting that this form of cell death does not require latestages of autophagy. In addition, another autophagy-inducingpeptide, Tat-vFLIP α2 (which acts by releasing ATG3 from cel-lular FLIP) (14), also induced autophagy (Fig. S1N) which wasassociated with dose- and time-dependent cell death (Fig. S1 Oand P) and reduced by ATG14 knockdown in U2OS cells (Fig.S1Q). Thus, autophagy-inducing peptides trigger cell death thatrequires the autophagy machinery.

Autophagy Peptide-Induced Death Does Not Require Apoptotic orNecropoptotic Machinery. We next asked whether the apoptosisand/or necroptosis death machinery is involved in Tat-Beclin 1-induced cell death. We found that neither z-VAD, an inhibitor ofcaspases and apoptosis, nor necrostatin-1 (Nec-1), an inhibitor ofRIPK1 kinase-mediated necroptosis, rescued Tat-Beclin 1-inducedcell death as measured by levels of cellular (Fig. S1G), trypan blueexclusion (Fig. S1H), or clonogenic survival (Fig. S2A). Adeno-virus E1A/Ras-transformed MEFs with null mutations in the twoproapoptotic genes, Bax and Bak, were susceptible to Tat-Beclin1-induced cell death (Fig. 2A and Fig. S2B) but were resistant todeath induced by the apoptosis-inducing agents, staurosporineand etoposide (Fig. S2C). Genetic deletion of two key regulatorsof necroptosis, Ripk1 and Ripk3, failed to protect primary MEFsfrom Tat-Beclin 1-induced cell death (Fig. 2B and Fig. S2D). Thus,neither the apoptotic nor necroptotic death machinery is requiredfor Tat-Beclin 1-induced cell death.Additional assays confirmed the lack of apoptosis in Tat-

Beclin 1-induced death. In contrast to staurosporine, Tat-Beclin1 did not activate caspase 3, as shown biochemically by the lackof cleavage of caspase 3 or its substrate PARP (Fig. 2C) and bythe lack of immunofluorescence staining for active caspase 3(Fig. 2D). In addition, minimal pancaspase activity was detectedin Tat-Beclin 1-treated cells by flow cytometry (Fig. S2 E and F).Consistent with nonapoptotic cell death, no TUNEL staining(Fig. S2G) or DNA ladder formation (Fig. S2H) was detected inTat-Beclin 1-treated cells. We also confirmed that Tat-Beclin 1[derived from a structurally flexible region in the Beclin 1 evo-lutionarily conserved domain (15)] did not exhibit a pore-form-ing ability to release cytochrome c from mitochondria (Fig. S2I),as do certain other amphipathic α-helical peptides (16). Fur-thermore, antioxidants that block reactive oxygen species-mediated

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Fig. 1. Tat-Beclin 1 induces autophagy-dependent cell death. (A) Westernblot of LC3 and p62 in HeLa cells treated with Tat-Scrambled (T-S) or Tat-Beclin 1 (T-B) peptides for 5 h. (B) Cell death of HeLa cells treated T-S or T-Bfor 5 h. (C) Representative images of Sytox Green staining of HeLa cellstreated with T-S or T-B (20 μM, 5 h). (Scale bar, 50 μm.) (D) Clonogenic cellsurvival of HeLa cells treated with T-S or T-B (20 μM, 5 h). (E) Clonogenicsurvival of HeLa cells treated with T-B (20 μM, 4 h) ± 10 mM 3-MA. (F) Clo-nogenic survival of siRNA-transfected HeLa cells treated with T-B (20 μM,3h). (G) Clonogenic survival of doxycycline (Dox)-inducible U2OS/TR cellsstably transfected with empty vector, shATG13, or shATG14 ± Dox (1 μg/mL)for 5 d before treatment with T-B (25 μM, 5 h). For B and D–G, error barsrepresent mean ± SEM and similar results were observed in three in-dependent experiments. For D–G, the number of colonies in untreatedcontrols was standardized as 100%. NS, not significant; **P < 0.01; ***P <0.001; t test. See also Fig. S1.

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cell death failed to rescue Tat-Beclin 1-induced cell death(Fig. S2J). Similar to Tat-Beclin 1, Tat-vFLIP α2 also failed toinduce caspase 3 or PARP cleavage (Fig. S2K). Thus, takentogether, our data indicate that autophagy-inducing peptide-triggered cell death is genetically and biochemically distinctfrom apoptosis or necroptosis.

Autophagy Peptide-Induced Cell Death Has Unique MorphologicalFeatures. To characterize the nature of this autophagy-dependent,nonapoptotic, and nonnecrotic cell death, we performed live-cellimaging of Tat-Beclin 1-treated cells (Fig. 3A and Movie S1).During the initial phase, cells exhibit relatively normal morphol-ogy with increased vacuolar dynamics and perinuclear accumu-lation of numerous vacuoles. After a few hours, cells undergo anabrupt demise (lasting ∼15–20 min) characterized by the rapidshrinkage of the nucleus with a portion of its surface developinga concave appearance corresponding to a round, vacuole-likeentity, reflecting (based on our EM data; see below) a local sep-aration of the inner and outer nuclear membranes. This is followedby focal plasma membrane rupture and extracellular extrusionof cytoplasmic contents. Cells treated with Tat-Beclin 1 displayincreased substrate adherence that persists until their final demise(unlike apoptotic or necrotic cells, which generally float).The concave nuclear appearance observed in Tat-Beclin 1-

induced cell death is associated with abnormalities in nuclearlamin-A/C staining (lack of a uniform circular appearance andfocal regions of dense staining) (Fig. 3B and Fig. S3A). Tat-Beclin 1-treated dying cells also exhibit an abnormal fragmentedpattern of Tom20 (mitochondrial marker) and PDI [endoplasmicreticulum (ER) marker] staining, and a striking increase in ex-pression of LAMP1, a marker of late endosomes/autolyosomes(which would be expected in the setting of a robust autophagyresponse) (Fig. 3B). Tat-vFLIP α2-treated dying cells have asimilar concave nuclear appearance and similar abnormalities inlamin-A/C, Tom20, PDI, and LAMP1 staining (Fig. S3B).We performed ultrastructural analyses to further characterize

the morphology of Tat-Beclin 1-induced death (Fig. 3C and Fig.S3C). As apparent from live-cell imaging, there were two phasesof the death process; phase 1 is characterized by a slow phase ofgradual change and phase 2 is characterized by an abrupt phaseof final collapse and cell death. Morphologically, phase 1 can bedivided into two stages. In phase 1a, the nucleus becomes con-voluted (but the perinuclear space is normal); chromatin ismoderately condensed, forming darker regions in the nucleuswith borders that are fuzzy (in contrast to clumps of chromatinthat typically have sharp borders in apoptosis); many of the mi-tochondria are electron dense and some have an abnormal

internal structure (clumps instead of bands); the ER is dilatedand fragmented; and numerous autophagosomes, autolysosomes,and empty vacuoles are present. In phase 1b, the perinuclearspace becomes swollen at discrete regions surrounding the innernuclear membrane, and these swollen areas contain membrane-bound regions with a density and granularity resembling thecytosol. In some cases, the perinuclear space extends throughsubstantial distances in the cytoplasm. In phase 2, there is focalballooning of the perinuclear space (which appears empty), of-ten associated with a concavity of the nuclear surface. At this latestage, the morphology appears necrotic; mitochondria and otherorganelles are swollen; and autophagosomes, autolysosomes, andER are rare. There appears to be lysis of the plasma membrane(which is difficult to discern from EM analyses but is sub-stantiated by PI staining, Sytox Green staining, and live-cellimaging of Tat-Beclin 1-treated cells).These ultrastructural changes are distinct from previous clas-

sifications of cell death (3), including type-3B cytoplasmic death(also called “paraptosis”), which also has perinuclear swelling.In type-3B cell death, perinuclear swelling is moderate and uni-form around the entire nuclear perimeter, whereas in death ofautophagy-inducing peptide-treated cells, there is a pronouncedballooning in a focal region of the perinuclear space. To avoidconfusion with terms such as “autophagic cell death” (which issometimes applied to states in which it is not clear that autophagyis required for cell death and/or in which autophagic featurescoexist with apoptosis or necrosis), we coined the term “autosis”to define cell death mediated by autophagy genes and charac-terized by focal perinuclear swelling. Although several studieshave described cell death that is blocked by genetic inhibition of

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MEFs treated with peptide (5 h). (B) Clonogenic survival of wild-type, Ripk1+/+;Ripk3−/−, and Ripk1−/−;Ripk3−/− MEFs treated with peptide (20 μM, 5 h). In Aand B, the number of colonies of Tat-Scrambled-treated cells was standardizedas 100%. (C) Western blot of cleaved caspase 3 and cleaved PARP in HeLacells treated with 20 μM Tat-Scrambled (T-S), 20 μM Tat-Beclin 1 (T-B), 1 μMstaurosporine (STS) ± 100 μM Z-VAD-FMK (z-VAD), or 32 mM H2O2 for 5 h.The asterisk denotes a cross-reacting band. (D) Representative images ofcleaved caspase 3 staining in HeLa cells treated with 20 μM Tat-Beclin 1 or1 μM staurosporine for 5 h. Scale bar, 50 μm. For A and B, error bars rep-resent mean ± SEM of triplicate samples and similar results were observed inthree independent experiments. NS, not significant; t test. See also Fig. S2.

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Fig. 3. Morphological features of Tat-Beclin 1-induced autosis. (A) Repre-sentative live-cell imaging of HeLa cells treated with 25 μM Tat-Beclin 1 for5 h (Movie S1; times shown as hh:mm). The black arrow denotes releasedintracellular components from a ruptured cell membrane and the whitearrow denotes perinuclear space between the inner nuclear membrane andcytoplasm at a region of nuclear concavity. (Scale bar, 10 μm.) (B) Repre-sentative images of mitochondrial (Tom20), ER (PDI), late endosome/lyso-some (LAMP1), and nuclear lamin-A/C staining in HeLa cells treated with Tat-Scrambled (T-S) or Tat-Beclin 1 (T-B) (20 μM, 5 h). (Scale bar, 20 μm.) (C) EManalysis of HeLa cells treated with peptide (20 μM, 5 h). White arrows showdilated and fragmented ER; black arrows show regions where the peri-nuclear space has swollen and contains clumps of cytoplasmic material.(Scale bars, 1 μm.) See also Fig. S3.

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autophagy (6), such studies have not described increased sub-strate adherence and focal perinuclear swelling of dying cells.Thus, autosis represents a previously undescribed form of celldeath by autophagy.

Starvation Induces Autosis. We next investigated whether autosisoccurs during physiological stress conditions associated with highlevels of autophagy. Nutrient starvation is a potent physiologicalinducer of autophagy in eukaryotic cells, and previous studieshave shown that autophagy delays apoptosis in cells subjected tostarvation, including HeLa cells (1). However, upon subjectingHeLa cells to amino-acid and serum starvation, we found that,unlike the vast majority of cells which detach from their substrateand undergo apoptosis (as evidenced by active caspase 3 stain-ing), a small subpopulation (∼1%) of cells become more sub-strate-adherent and lack evidence of caspase 3 activation (Fig.4A). This population of substrate-adherent, caspase 3-negativecells undergoes plasma-membrane rupture and cell death, as

identified by Sytox-Green staining (Fig. S4A), and also displaysa marked increase (approximately threefold) in numbers ofautophagosomes (GFP-LC3 puncta) compared with numbers inthe majority population of starved cells that float and undergoapoptosis (Fig. 4 B and C). These substrate-adherent cells havenuclei with concave regions and focal swelling of the perinuclearspace (Fig. 4 A, B, and D and Fig. S4A) and display similar ab-normalities in lamin-A/C staining as observed in Tat-Beclin 1and Tat-vFLIP α2 peptide-treated cells (Fig. S4A). Similar toTat-Beclin 1 peptide treatment, we observed phase-1 starvedcells with increased autophagosomes/autolysosomes, and regionsof perinuclear swelling containing organelles and phase-2 cellswith rare autophagosomes/autolysosomes and dilated emptyregions in the perinuclear space (Fig. 4D). We also observedfeatures of autosis in starved adherent murine bone marrow-derived macrophages (BMDMs) and primary MEFs (Fig. 4E),suggesting that starvation-induced autosis occurs in primary cellsand is not merely a consequence of mutations that confer re-sistance to apoptosis in continuous cell lines. Moreover, thefrequency of autotic cell death was higher (∼5%) in primary cellsthan in HeLa cells.To confirm that autophagy is required for starvation-induced

death in cells with autotic morphology, we assessed the effects ofautophagy gene knockdown on the clonogenic survival of sub-strate-adherent starved cells. In this assay, floating (apoptoticand necrotic) cells were washed away after 48 h (HeLa cells) or72 h (U2OS cells and BMDMs) starvation, and the clonogenicpotential of the remaining adherent cells was assessed (Fig. S4B).Both ATG7 and beclin 1 siRNA treatment (Fig. S4C) increasedcolony numbers formed by starved adherent HeLa cells (Fig.4F), and ATG14 shRNA expression (Fig. S4D) increased colonynumbers formed by starved adherent U2OS cells (Fig. 4G). Ly-sozyme:Cre-mediated deletion of Atg5 (Fig. S4E) also increasedcolony numbers formed by starved adherent Atg5flox/flox BMDMs(Fig. 4H). [ATG7 siRNA, beclin 1 siRNA, ATG14 shRNA andAtg5 deletion had minimal effect on the clonogenic survival ofcells cultured in normal media (Fig. S4 C–E)]. Thus, autophagygenes are required for starvation-induced autosis.

Autosis Occurs During Rat Cerebral Hypoxic–Ischemic Injury. Afterestablishing ultrastructural criteria for autosis in cultured cells,we evaluated whether autosis occurs in vivo. We performed EManalysis of neuronal death following cerebral hypoxia–ischemiain the brains of neonatal rats (Fig. 5); we focused on dying neuronsin the hippocampus CA3 region because we had previously shownthat most of these neurons degenerate with autophagic features(from 6 h after hypoxia–ischemia) without signs of apoptosis ornecrosis (17). At 24 h after cerebral hypoxia–ischemia, most ofthe dying neurons displayed prominent autophagic features, suchas numerous autophagosomes and autolysosomes, and emptyvacuoles (phase 1a). Strikingly, some dying neurons displayedcharacteristics of phase-1b or the full phase-2 features of autosis—focal ballooning of the perinuclear space associated with nuclearconcavity. Thus, autotic cell death occurs in certain pathophysi-ological settings in vivo.

A High-Throughput Chemical Screen Identifies Cardiac Glycosides asPotent Inhibitors of Autosis. To gain insight into the regulation ofautosis, we performed high-throughput compound screening toidentify inhibitors of Tat-Beclin 1-induced cell death, focusing oncompound libraries consisting of bioactive agents with knowntargets. We measured levels of cellular ATP (as a proxy of cel-lular viability) 5 h after Tat-Beclin 1 treatment of HeLa cells inthe presence of ∼5,000 Food and Drug Administration (FDA)-approved drugs and bioactive compounds with characterizedmechanisms of action (Fig. S5A). We chose for further analysisthe 36 top hits that had z scores ≥3.0 in the primary screen (Fig.6A and Dataset S1). These 36 hits were classified into nine fam-ilies based on their chemical structures and/or biological functions(Dataset S2). Of these 36 hits, eight compounds demonstrated>40% rescue of autosis in a repeat ATP assay and were chosen

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for further analysis (Dataset S3). Of these eight compounds, onlyfive, including three cardiac glycosides (digoxin, digitoxigenin,and strophanthidin) and two purinergic receptor antagonists(suramin and NF 023) demonstrated more than 80% rescue ofTat-Beclin 1 peptide-induced cell death as measured by SytoxGreen staining (Fig. S5B). The purinergic receptor antagonists,but not the cardiac glycosides, blocked cellular peptide entry(Fig. S5 C and D) and were not studied further. Thus, our chemicalscreen identified cardiac glycosides as the only class of agents thatinhibited the Tat-Beclin 1-induced cell death without blockingcellular peptide entry.To assess the significance of the effects of cardiac glycosides

on autosis in an unbiased manner, we tested whether the ninecardiac glycosides present in our library were statistically enrichedamong top-scoring compounds. We calculated a weighted Kol-mogorov–Smirnov-like statistic, the normalized enrichment score(NES), using compound set enrichment analysis (CSEA). CSEAdemonstrated strong, highly significant enrichment for cardiac gly-cosides (P < 10−4) (Fig. 6B). In addition to the three cardiac gly-cosides with z scores ≥3.0 in the primary screen, we confirmedthat other cardiac glycosides in the compound libraries alsoexhibited a significant rescue effect (Dataset S4), as did a cardiacglycoside, neriifolin, which was not in the compound libraries andis known to exert neuroprotective actions (18) (Fig. S5E). We alsoperformed CSEA using other previously identified compound setsfor autophagy inducers, specific necrosis inducers, and specificapoptosis inducers (19); none of these sets were enriched amongour top-scoring compounds (Fig. 6B). Although there are previousreports that cardiac glycosides may increase basal autophagy (20,21), an extensive set of autophagy inducers drawn from the lit-erature were overrepresented among compounds that enhanced,rather than rescued, Tat-Beclin 1-induced cell death. The lack ofconcordance between compounds that inhibited autophagy, apo-ptosis, or necrosis with the rescue of Tat-Beclin 1-induced autosisis consistent with the latter representing a distinct death process.Consistent with these bioinformatics analyses, the cardiac gly-

coside, digoxin, had no effect on apoptotic death induced bystaurosporine or necrotic death induced by H2O2 (Fig. S5 Fand G), whereas digoxin rescued Tat-Beclin 1–induced celldeath with IC50 values below 0.1 μM (Fig. S5H). Digoxin alsorescued Tat-vFLIP α2-induced death in HeLa cells (Fig. S5H)and Tat-Beclin 1-induced cell death in U2OS cells (Fig. S5I). Wealso found that clonogenic survival was rescued in Tat-Beclin 1-treated HeLa cells by digoxin, digitoxigenin, and strophanthidin(Fig. 6C); in Tat-vFLIP α2-treated HeLa cells by digoxin (Fig.S6J); and in the adherent subpopulation of HeLa cells subjectedto prolonged starvation by digoxin (Fig. 6D) and neriifolin (Fig.

6E). Thus, cardiac glycosides rescue cell death triggered by mul-tiple inducers of autosis.Digoxin partially reversed the majority of morphological ab-

normalities in cells undergoing Tat-Beclin 1-induced autosis.Upon light microscopy analysis, cells treated with Tat-Beclin 1and digoxin displayed minimal nuclear abnormalities and lackedabnormal patterns of mitochondrial (Tom20), ER (PDI), lateendososome/lysosome (LAMP1), and nuclear lamin-A/C stain-ing (Fig. S6A). Ultrastructurally, the majority of digoxin-rescuedcells had a normal-shaped nuclear membrane without any focalswelling of the perinuclear space, intact ER structure, and theabsence of increased numbers of autophagosomes and autoly-sosomes (Fig. 6F). The only morphological abnormality of autosisnot reversed by digoxin in Tat-Beclin 1-treated cells was the pres-ence of electron-dense mitochondria; however, digoxin alone (in theabsence of Tat-Beclin 1) resulted in electron-dense mitochondria(Fig. S6B). Together, these data indicate that digoxin reversesthe morphological changes of autosis except for mitochondrialabnormalities, but these do not appear to be related to cell death.We performed Western blot analyses of LC3 and p62 and

quantitation of GFP-LC3 puncta to further evaluate the effectsof digoxin on autophagy. Under basal conditions, consistent withprior reports (20, 21), we observed a dose-dependent increase inLC3-II conversion and mild reduction in p62 levels (although wedid not detect an increase in GFP-LC3 puncta) (Fig. 6G and Fig.

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S6C). In contrast, doses as low as 100 nM resulted in a milddecrease in starvation-induced autophagy and a more dramaticdecrease in Tat-Beclin 1-induced autophagy (Fig. 6G and Fig.S6C). The increased p62 accumulation was not due to changes inp62 mRNA expression (Fig. S6D). Although cardiac glycosideshave been reported to induce apoptosis (22, 23), we did notobserve caspase activation in Tat-Beclin 1-treated cells in thepresence of digoxin (Fig. S6E).

Na+,K+-ATPase Regulates Autosis. Cardiac glycosides are inhibitorsof Na+,K+-ATPase, a plasma membrane pump that generatesNa+ and K+ gradients across the membrane and acts as a versa-tile signal transducer (22). We therefore examined whether Na+,K+-ATPase regulates autosis using siRNA knockdown of the α1subunit of Na+,K+-ATPase. Similar to digoxin treatment, Na,K-α1–subunit knockdown (Fig. S7 A and B) resulted in a milddecrease in starvation-induced autophagy and a more dramaticdecrease in Tat-Beclin 1-induced autophagy (Fig. 7A and Fig.S7A). The observed increase in p62 accumulation was not due tochanges in p62 mRNA expression (Fig. S7C) or to a block inpeptide delivery into cells (Fig. S7D). In parallel with inhibitionof autophagy, three different siRNAs against Na,K-α1 inhibitedTat-Beclin 1- and Tat-vFLIP α2-induced death (Fig. S7E). Theyalso increased clonogenic survival of Tat-Beclin 1-treated cells(Fig. 7B) and adherent cells subjected to starvation (Fig. 7C).Na,K-α1 siRNA also exerted a protective effect against autosis inhuman U2OS (Fig. S7 F and G) and in mouse NIH 3T3 (Fig. S7H and I) cells. Digoxin did not enhance Na,K-α1 siRNA-medi-ated protection against autosis triggered by autophagy-inducingpeptides (Fig. S7J), suggesting that digoxin and Na+,K+-ATPaseinhibition block autosis through the same mechanism.

Cardiac Glycoside-Mediated Protection Against Neuronal Autosis inRat Cerebral Hypoxia–Ischemia. A previous chemical screen toidentify compounds that provide neuroprotection in a mousebrain slice-based model for ischemic stroke revealed neriifolin asa strong hit, and whole-animal studies have shown that neriifolinand other cardiac glycosides provide neuroprotection in neonatalmodels of cerebral hypoxia–ischemia (18, 24, 25). Given theseobservations, coupled with our findings described above that rathippocampal CA3 region neurons die by autosis following hyp-oxia–ischemia, we evaluated whether neriifolin could protectneonatal rats against cerebral hypoxia–ischemia and reduceautosis in the hippocampal region CA3.In agreement with results in mice (18), we found that neriifolin

was highly neuroprotective in rats; it dramatically increased thevolume of intact tissue in the ipsilateral hemisphere of neonatal

animals 1 wk after cerebral hypoxia–ischemia (Fig. 8 A and B).This effect was particularly notable in the hippocampus (Fig. 8Band Fig. S8A), where significant neuronal pathology, especially inthe CA3 region, was detected as early as 24 h after hypoxia–ischemia injury (Fig. S8B). The CA3 region of the hippocampuswas protected at 24 h and 7 d after neonatal hypoxia–ischemia byneriifolin treatment compared with vehicle-treated pups (Fig. S8 Aand B). In parallel with this neuroprotection, neriifolin preventedthe increase in autophagy in the CA3 region of the hippocampusthat occurred after hypoxia–ischemia injury, as measured by de-tection of decreased numbers of endogenous LC3 puncta andLAMP1 puncta by immunofluorescence and immunoperoxidasestaining (Fig. 8C and Fig. S8C) and decreased levels of LC3-II(Fig. 8 D and E). Strikingly, in contrast to the characteristicfeatures of autosis (numerous autophagosomes, autolysosomes,and empty vacuoles; abnormal mitochondria and ER; and focalseparation of the inner and outer nuclear membrane) observedin the CA3 region of vehicle-treated pups 24 h after cerebralhypoxia–ischemia, the CA3-region neurons of neriifolin-treatedanimals displayed no ultrastructural features associated withautosis (Fig. 8F). Thus, cardiac glycosides block the increase inautophagy and protect hippocampal neurons against cerebralhypoxia–ischemia-induced autosis in vivo.

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Fig. 7. Na+,K+-ATPase regulates autosis. (A) Quantitation of GFP-LC3 dots(>100 cells analyzed per sample) in HeLa/GFP-LC3 cells 72 h after transfectionwith indicated siRNA and treatment with Tat-Beclin 1 (20 μM, 2 h) or star-vation (HBSS, 2 h) ± 20 nM bafilomycin A1. (B) Clonogenic survival of HeLacells transfected with indicated siRNA for 72 h and then treated with Tat-Scrambled or Tat-Beclin 1 (20 μM, 5 h). Shown are the percentage of coloniesin Tat-Beclin 1– vs. Tat-Scrambled–treated cells for each siRNA. (C) Clono-genic survival of adherent HeLa cells transfected with indicated siRNA for72 h, and then starved for 48h. Clonogenic survival of control siRNA trans-fected cells in starvation conditions relative to normal medium standardizedas 100%. For A–C, error bars represent mean ± SEM of triplicate samples andsimilar results were observed in three independent experiments. NS, notsignificant; *P < 0.05; **P < 0.01; ***P < 0.001; t test. See also Fig. S7.

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Fig. 8. Neonatal hypoxic–ischemic brain damage and hippocampal CA3region autophagy and autosis are reduced by treatment with the cardiacglycoside, neriifolin. (A) Representative Nissl-stained coronal sectionsthrough the brain showing the neuroprotective effect of neriifolin (Lower)compared with vehicle (Upper) 1 wk after hypoxia–ischemia (HI). (Scale bar,1 mm.) (B) Volumes of intact tissue ipsilaterally compared with con-tralaterally 1 wk after neonatal cerebral hypoxia–ischemia and indicatedtreatment. Values are mean ± SD (n = 6 for neriifolin and n = 9 for vehicle).***P < 0.001; Welch’s ANOVA test. (C) Representative confocal microscopyimages of LC3 dots (red) and LAMP1 dots (green) in CA3 hippocampalneurons after 24h hypoxia–ischemia and indicated treatment or sham op-eration. NeuN (green) and MAP2 (red) are neuronal markers. Hoechststaining (blue) shows cell nuclei. (Scale bars, 20 μm.) (D and E) RepresentativeLC3 immunoblots (D) and quantification of LC3-II/tubulin levels (E) fromimmunoblots of hippocampi of rats subjected to hypoxia–ischemia. Valuesare mean ± SD (n = 6 for neriifolin and n = 9 for vehicle). NS, not significant;*P < 0.05; **P < 0.001; Kruskal–Wallis test. (F) EM analysis of neriifolineffects in hippocampal region CA3 of 7-d-old rats 24 h after hypoxia–is-chemia. INM, inner nuclear membrane; M, mitochondrion; N, nucleus; ONM,outer nuclear membrane; PNS, perinuclear space. (Scale bars, 1 μm.)

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DiscussionOur findings identify a unique form of autophagic cell death—autosis—that meets two essential criteria put forth by the No-menclature Committee on Cell Death (2): suppression by in-hibition of the autophagic pathway, and lack of features ofapoptosis and necrosis. The form of death that we observed inadherent cells subjected to starvation and in cells treated withautophagy-inducing peptides not only meets these criteria forautophagic cell death, but also has a distinctive morphologicaland chemical inhibition signature. In addition to the classicalmorphological criteria of autophagic cell death (increased auto-lysosomes in dying cells lacking features of apoptosis and necrosis),death induced in starved adherent cells and by autophagy-inducingpeptides is accompanied by ER dilation and stereotypic nuclearchanges, involving an early convoluted appearance, the forma-tion of focal concave regions of the nucleus with surroundingfocal swelling of the perinuclear space, and the accumulation ofstructures within this space at early stages of the process. Thisform of cell death, but not apoptosis or necrosis, is also selec-tively blocked by pharmacological and genetic inhibition of Na+,K+-ATPase.Although the underlying mechanisms of the morphological

changes of autosis and the pathway by which Na+,K+-ATPasemediates autosis remain to be determined, the discovery of thisunique morphological and chemical inhibition signature hasimportant biological implications. Our findings pave the road tothe discovery of physiological and pathophysiological conditionsin which autophagy functions as a death mechanism, and mayprovide a candidate treatment for diseases in which such deathcontributes to pathogenesis. For example, using the morpho-logical criteria we established for autosis in cells subjected tostarvation or treatment with autophagy-inducing peptides, weidentified the presence of autotic death in rat hippocampalneurons subjected to hypoxic–ischemic injury. Moreover, weshowed that a class of FDA-approved chemical compounds—cardiac glycosides—that inhibited autosis in an in vitro chemicalcompound screen also reduced hippocampal neuronal autosisand conferred neuroprotection in vivo in neonatal rats subjectedto cerebral hypoxia–ischemia. Thus, by defining a unique form ofautophagic cell death and by performing an in vitro chemicalscreen that identified a specific class of inhibitors of this form ofcell death (e.g., cardiac glycosides), we have been able to es-tablish a scientific rationale for the use of cardiac glycosides inthe treatment of a clinically important disease, neonatal cerebralhypoxia–ischemia. Based on our identification of specific mor-phological criteria for autosis, it should be possible to determineadditional pathophysiological settings in which autosis plays arole and which may be ameliorated by cardiac glycosides. Con-versely, mediators in the regulatory network of autosis may serveas candidate targets in cancer chemotherapy or other settingswhere pharmacological induction of cell death may be beneficial.We found that autosis occurs in at least two distinct physio-

logical/pathophysiological conditions: starvation and cerebralhypoxia–ischemia. At present, it is not yet known which, if any,previously reported instances of autophagic cell death involveautosis (except for hypoxia–ischemia-induced hippocampal-region-CA3 death evaluated in this study). It is possible that theunique morphological changes we describe for autosis are presentbut have been missed in observations of autophagic cell death inother settings, especially those that lack concurrent features ofapoptosis or necrosis and/or in tissues (e.g., heart and kidneys)where high levels of autophagy are postulated to play a role inischemia–reperfusion injury (26, 27). While the future identifi-cation of specific biochemical markers of autosis will facilitatesuch investigations, it should be possible to determine whethercell death occurs via autosis using the morphological criteria wedescribe, as well as studies examining the inhibitory effect ofcardiac glycosides. One cautionary note is that certain isoformsof the Na+,K+-ATPase in the rodent (but not human) are resistantto cardiac glycosides; for example, the rodent-α3 subunit expressed

predominantly in brain is sensitive, whereas the rodent-α1 subunitexpressed in many peripheral tissues is resistant (28).Although we are not aware of previous reports of similar nu-

clear morphological abnormalities in autophagic cell death, theexpression of sterol reductases that are localized to the ER andouter nuclear membrane, TM7SF2 and DHCR1, results in mas-sive ER and perinuclear space expansion resembling that observedin autotic cells (29). These observations suggest that disruptionof ER/outer nuclear membrane cholesterol metabolism mayproduce the phenotype of ER and focal perinuclear space ex-pansion. This phenotype is possibly caused by alterations of ER-membrane properties, including transport or channel conductance,which would result in osmotic changes and disruption of sig-naling through the nuclear envelope. Given the crucial role ofthe ER in autophagosomal biogenesis (30), we speculate thatstimulation of very high levels of autophagy may perturb normalER membrane biogenesis/homeostatic mechanisms, leading tosimilar expansions of the ER lumen and perinuclear space.Further studies are needed to investigate the underlying mech-anisms of the morphological abnormalities observed in autosis.Cardiac glycosides, a large family of naturally derived steroidal

compounds, were first described for the treatment of heart dis-eases in 1785 (22). Approximately 50 y ago, Na+,K+-ATPase wasidentified as the cellular target of cardiac glycosides. This mem-brane protein uses energy from ATP hydrolysis to facilitate thetransport of potassium ions into cells and sodium ions out ofcells; inhibition of Na+,K+-ATPase results in an increase in in-tracellular sodium and calcium ions. Cardiac glycosides also havediverse effects on cellular signaling, proliferation, metabolism,survival, gene expression, attachment, and protein trafficking.Our chemical screen revealed cardiac glycosides as the mostpotent inhibitors of autotic cell death, and we found that theyinhibited both autophagy and autotic cell death in the setting ofstarvation, autophagy-inducing peptide treatment, and neonatalhippocampal hypoxic–ischemic injury. The mechanism of actionappears to be inhibition of the target of cardiac glycosides, Na+,K+-ATPase, as we observed similar effects with Na+,K+-ATPase α1subunit siRNA knockdown in human and mouse cells. We specu-late that the effects of the Na+,K+-ATPase on increasing cell at-tachment (31) may contribute to the increased substrate adherenceof cells undergoing autotic death. In addition, it is possible thatnuclear envelope-associated Na+,K+-ATPase activity (32) may altermembrane ionic transport and osmolarity and thereby con-tribute to the ER and perinuclear space expansion observed inautotic cells.Previous studies have shown that neriifolin and other cardiac

glycosides reduce cerebral infarct size in rodent cerebral hyp-oxia–ischemia models (18, 24, 25); however, their mechanism ofneuroprotection has been unknown. Our observations suggestthat inhibition of autophagy and autophagy-dependent deathpathways may be a central mechanism of cardiac glycoside-mediated neuroprotection. Several cell-death morphologies havebeen identified in different regions of the brain after neonatalhypoxic–ischemic injury, but neuron-specific deletion of Atg7 orintracerebroventricular treatment with the autophagy inhibitor,3-MA, is sufficient to reduce infarct lesion volume, indicatingthat autophagy may be upstream of multiple death pathways.This supports our previous recommendation that postischemictreatment of neonatal cerebral hypoxia–ischemia should targetautophagy (17, 33). In the present study, we observed inhibitionof autophagy and autotic cell death in hippocampal-CA3-regionneurons of rats treated with neriifolin, but the inhibition of autoticcell death in this region of the hippocampus is not sufficient toexplain the dramatic reduction in overall ipsilateral infarct sizefollowing hypoxia–ischemia. Taken together with previous studieson autophagy, cell death, and neonatal cerebral hypoxia–ischemia,the most likely explanation for the overall neuroprotectionin neriifolin-treated rats is the blockade of both autophagy-dependent autotic death, as well as other death pathways triggeredby high levels of autophagy. Thus, cardiac glycosides and/or otheragents targeting Na+,K+-ATPase may not only ameliorate diseases

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associated with autotic cell death, but also diseases in whichautophagy is upstream of other death execution pathways. Wecannot definitively rule out indirect effects of neriifolin on neu-roprotection, but these seem unlikely in view of our in vitroobservations that cardiac glycosides inhibit stress-inducedautophagy and autosis in a cell-autonomous manner.It is noteworthy that—during cerebral hypoxia or ischemia—

the brain releases an endogenous form of cardiac glycoside(ouabain or endobain) that inhibits Na+,K+-ATPase (34). Thus,by releasing its own inhibitor of Na+,K+-ATPase in response tohypoxia–ischemia, the neonatal brain may have developed animportant mechanism to reduce autophagy and cell death byautosis. A broader question is whether basal levels of endogenouscardiac glycosides may serve as a naturally occurring “brake” whichfunctions in multiple mammalian tissues to maintain autophagyat physiological levels that promote cell survival, rather than atpathological levels that promote cell death.

Materials and MethodsCell Culture.HeLa cells were obtained fromAmerican Type Culture Collection.Information on the source of wild-type, Ripk3−/−, Ripk1−/−;Ripk3−/−, and Bax−/−;Bak−/− MEFs, Atg5flox/flox and Atg5flox/flox-LysM-Cre BMDMs, and U2OSTetR,U2OSTetR/shATG14, and U2OSTetR/shATG13, and other cells used in this studyand culture conditions is provided in SI Materials and Methods.

Autophagy-Inducing Peptides. Tat-Scrambled, Tat-Beclin 1, and Tat-vFLIP α2(14) were synthesized and administered to cells as described (13).

Antibodies and siRNAs. See SI Materials and Methods for details of antibodiesand siRNAs used in this study.

Cell Death Assays. See SI Materials and Methods for details of trypan-bluestaining, Sytox Green staining, CellTiter-Glo assays, PI staining, active cas-pase-3 detection, TUNEL staining, DNA fragmentation assays, and clono-genic survival assays.

Microscopy Studies. See SI Materials and Methods for details.

High-Throughput Chemical Screening. Chemical screening and CSEA wereperformed as previously described (35). Details of screening methodologyand analyses are provided in SI Materials and Methods.

Autophagy Analyses. See SI Materials and Methods for details.

Rat Model of Neonatal Cerebral Hypoxia–Ischemia. Neonatal rat cerebralhypoxia–ischemia experiments were performed as described (17). Immedi-ately after carotid artery occlusion, rat pups were injected intraperitoneallywith either neriifolin (0.25 mg/kg diluted in 0.5% ethanol/PBS) (Sigma,S961825) or vehicle (0.5%ethanol/PBS). See SI Materials and Methods fordetails. All experiments were performed in accordance with Swiss laws forthe protection of animals and were approved by the Vaud Cantonal Vet-erinary Office (authorization no. 1745.2).

ACKNOWLEDGMENTS. We thank Noboru Mizushima, Qiong Shi, HerbertVirgin, Xiaodong Wang, Qing Zhong, and Sandra Zinkel for providing criticalreagents; Shuguang Wei for assistance with high-throughput screening;Beatriz Fontoura for helpful discussions; Zhongju Zou for technical support;Haley Harrington for assistance with manuscript preparation; the Universityof Texas (UT) Southwestern Live Cell Imaging Facility; and the Electron Mi-croscopy Facility of the University of Lausanne. This work was supported byNational Institutes of Health Grants U54 AI057156 and RO1 CA109618 (toB.L.), RO1 A140646 (to D.R.G.); Contract HHSN268201000044C (to S.Y.S.),PO1 CA95471 (to Dr. Steven McKnight, UT Southwestern Medical Center),and P30 CA142543 (to the UT Southwestern Simmons Cancer Center); and bythe Swiss National Science Foundation Grant 310030-130769 (to J.P.).

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