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  • Proc. Natl. Acad. Sci. USAVol. 92, pp. 7162-7166, August 1995Neurobiology

    Apoptosis and necrosis: Two distinct events induced, respectively,by mild and intense insults with N-methyl-D-aspartate or nitricoxide/superoxide in cortical cell cultures

    (excitotoxicity/cortical neurons/peroxynitrite)

    EMANUELA BONFOCO*t, DIMITRI KRAINC*, MARLA ANKARCRONAt, PIERLUIGI NICOTERAt, AND STUART A. LIPTON*t*Laboratory of Cellular and Molecular Neuroscience, Children's Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA 02115; andtDepartment of Environmental Medicine, Division of Toxicology, Karolinska Institute, S-17177 Stockholm, Sweden

    Communicated by Richard L. Sidman, New England Regional Primate Research Center, Southborough, MA, March 27, 1995

    ABSTRACT N-Methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity may depend, in part, on the generationof nitric oxide (NO') and superoxide anion (Oi-), which reactto form peroxynitrite (OONOl). This form of neurotoxicity isthought to contribute to a final common pathway of injury ina wide variety of acute and chronic neurologic disorders,including focal ischemia, trauma, epilepsy, Huntington dis-ease, Alzheimer disease, amyotrophic lateral sclerosis, AIDSdementia, and other neurodegenerative diseases. Here, wereport that exposure of cortical neurons to relatively shortdurations or low concentrations ofNMDA, S-nitrosocysteine,or 3-morpholinosydnonimine, which generate low levels ofperoxynitrite, induces a delayed form of neurotoxicity pre-dominated by apoptotic features. Pretreatment with superox-ide dismutase and catalase to scavenge Oi- partially preventsthe apoptotic process triggered by S-nitrosocysteine or 3-mor-pholinosydnonimine. In contrast, intense exposure to highconcentrations of NMDA or peroxynitrite induces necroticcell damage characterized by acute swelling and lysis, whichcannot be ameliorated by superoxide dismutase and catalase.Thus, depending on the intensity of the initial insult, NMDAor nitric oxide/superoxide can result in either apoptotic ornecrotic neuronal cell damage.

    Oxidative stress, leading to the formation of free radicals, hasbeen implicated in a final common pathway for neurotoxicityin a wide variety of acute and chronic neurologic diseases (1).Excessive stimulation of excitatory amino acid receptors inthese disorders may trigger the production of free radicals. Inparticular, neurotoxicity associated with overstimulation ofN-methyl-D-aspartate (NMDA) receptors is thought to bemediated by an excessive Ca2+ influx, leading to a series ofpotentially neurotoxic events (1). One of these events is theactivation of nitric oxide synthase and the subsequent produc-tion of nitric oxide (NO') (2). Another event is the stimulationof phospholipase A2 or Ca2+ overload of mitochondria, leadingto the generation of superoxide anion (O-) (3). NO can reactwith 02- to form peroxynitrite (OONO-) (4), which results indose-dependent neuronal damage (5).The mode of neuronal cell death after damage initiated by

    excitotoxins or free radicals, however, has remained contro-versial, with some groups finding features of necrosis butothers reporting characteristics of apoptosis (6-9). Althoughsimilar factors might trigger either phenomenon, this does notmean that the nature of the two forms of cell death is the same.

    Necrosis and apoptosis are distinct mechanisms of cell deathwith very different characteristics. Necrosis is caused by cat-astrophic toxic or traumatic events with passive cell swelling,injury to cytoplasmic organelles including mitochondria, and

    rapid collapse of internal homeostasis. Necrosis leads tomembrane lysis, release of cellular contents, and resultinginflammation (10, 11). In contrast, apoptosis is an activeprocess of neuronal cell destruction with specific definingmorphologic and molecular features, the description of whichis still evolving. Apoptosis is characterized by cell shrinkage,membrane blebbing, and, if a nucleus is present, nuclearpyknosis, chromatin condensation, and genomic fragmenta-tion (12, 13). A major difference between the two types of celldeath is the generalized involvement of neighboring cellswithin necrotic tissue. To prevent leakage of excitatory aminoacids, proteolytic enzymes, DNA, and oxidized lipids with aproinflammatory response, apoptotic cells condense theirchromatin, shrink, and in some cases shield their intracellularmilieu by cross-linking membrane proteins. In a sense, apo-ptosis represents, at a cellular level, "death with dignity"-theinternal and external membranes are preserved so that thecellular contents are safely sealed within the dying cells untilphagocytosis intervenes.

    In attempting to define the mechanism and events leadingto these different forms of neuronal cell death, it is extremelyimportant to be aware of an artifact that can occur in tissueculture models-the distinction between apoptosis and necro-sis can be confused because of the lack of scavenging cells, andthus the phagocytic step after apoptosis may not occur.Instead, an apoptotic cell can eventually undergo secondarynecrosis and rupture its contents into the surrounding medium(14). Thus, it is mandatory to look for features of apoptosis atvarious time points after the insult and not to unduly delaythese observations, as secondary necrosis may intervene andtherefore obfuscate the true nature of the injury to thecompromised cell. In vivo, a similar situation may ensue whenthe number of apoptotic cells overwhelms the ability of thetissue to remove them. In this case, a late necrosis may developin the tissue. It is true that not all forms of apoptosis aresimilar, and probably further classifications will be required.However, most authorities believe that apoptosis is an activeform of cell death, entirely distinct from necrosis. In the latter,a general failure of cellular homeostasis results in energy lossand often uncontrolled Ca2+ overload, rapidly resulting in cellblebbing and lysis.We noted for neurons that apoptosis may take some time to

    develop after the inciting event, whereas massive necrosiscould sometimes be observed within minutes or an hour.Therefore, we wondered whether the intensity of the originalinsult could be related, at least in some cases, to the pathway

    Abbreviations: LDH, lactate dehydrogenase; NMDA, N-methyl-D-aspartate; SNOC, S-nitrosocysteine; SOD, superoxide dismutase;TUNEL technique, terminal deoxynucleotidyltransferase-mediateddUTP-biotin nick end-labeling; SIN-1, 3-morpholinosydnonimine.*To whom reprint requests should be addressed at: Children's Hos-pital, 300 Longwood Avenue, Enders Building, Suite 361, Boston,MA 02115.

    7162

    The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. 1734 solely to indicate this fact.

  • Proc. Natl. Acad. Sci. USA 92 (1995) 7163

    to neuronal cell death? In this study we show that mildexcitotoxic or free radical insults result most often in apoptoticneuronal cell death, whereas necrosis predominates afterintense, fulminate insults.

    MATERIALS AND METHODS

    Generation of NO, Oi-, and OONO-. S-Nitrosocysteine(SNOC) and peroxynitrite (OONO-) were prepared as we havedescribed (5, 15). 3-Morpholinosydnonimine (SIN-1; 1 mM) hasbeen shown to produce NO and 02-, resulting in OONO-formation (16). With the short half-life of OONO-, its steady-state concentration generated by SIN-1 should only approach lowmicromolar levels in our culture system. SNOC (200 ,uM) de-composes homolytically to yield NO0 at a relatively rapid rate (tlh

  • 7164 Neurobiology: Bonfoco et al.

    10-

    10-

    0-

    o-

    0

    B 10

    -o-.-I. 610

    .4cIB 44C

    z

    NMDA 300 gM

    )0 * LDH D-100 Apoptotic nuclei

    10 -C 8

    0- ~~~~~~~~~~~~~0 61O 4 _

    3h 18h

    SIN-1 1 mM

    >0 * LDH that time (Fig. 1B). During the necrotic process, neuronal cell0 Apoptotic nuclei bodies and nuclei swelled in contradistinction to the nuclear

    condensation evident during apoptosis. NMDA receptor an-;o tagonists D-2-amino-5-phosphonopentanoate (AP5, 200

    ,M-2 mM) and dizocilpine (MK-801, 1-10 tkM) preventedo - - both NMDA-induced necrosis and apoptosis in our system buto t were ineffective after exposure to free radicals or peroxynitrite

    (ref. 4 and data not shown).0 3h 18h To generate low levels of peroxynitrite (at most a few

    NMDA 2 mM micromolar), we used 1 mM SIN-1 or 200 ILM SNOC. Alter-natively, we synthesized peroxynitrite and directly exposed the* LDH cortical cultures to low micromolar levels. Under our condi-)O0 Apoptotic nuclei tions, exposure to these relatively low concentrations of per-

    30- t oxynitrite resulted in delayed injury that was apoptotic inio

    t7 nature. For example, an increased number of apoptotic nuclei0o _I was observed 18 h after insult compared with 3 h (Figs. 1 C-Eo0. and 2A b-d). In these experiments, LDH levels eventually rose

    but only after many hours. This rise in LDH reflects the!o _; -previously reported observation that apoptotic cells, under0 tissue culture conditions, will eventually leak their cytoplasmic

    3h 18h contents because they do not undergo phagocytosis as in vivo.SNOC 200 I1M However, in the initial phases of apoptosis the integrity of the

    E 100- LDH F ioo * LDH0 Apoptotic nuclei t Apoptotic nuclei

    O 10 80

    ND60(A, 60B

    ,uM)40r OONO (1 u) ncnrs,LHeesrsatrwt

    C

    20- 20

    z 04

    3h 18h 3h 18h

    Nio10M cMONON- (100 ,uM

    FIG. 1. Time course of neurotoxicity induced by NMDA, SIN-i,

    SNOC, or peroxynitrite (00N0-). Dose and time dependence ofNMDA (A, B) and 00N0 (E, F) on neuronal death, as determinedby LDH leakage and number of apoptotic nuclei. Apoptotic nucleiincreased most dramatically

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