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Chapter 17 Glutamate induced cell death: Apoptosis or necrosis?
O.P. Ottersen, I.A. Langmoen and L. Gjerstad (Eds.) Progress in Brain Research, Vol 116 0 1998 Elsevier Science BV. All rights reserved.
CHAPTER 17
Glutamate induced cell death: Apoptosis or necrosis? Maria Ankarcrona Karolinska Institutet. Department of Clinical Neuroscience and Family Medicine, Division of Geriatric Medicine, KFC, Novum, 4thJ%Or, S-141 86 Huddinge, Sweden
Introduction
Types of cell death: Necrosis and apoptosis
Glutamate toxicity is involved in the onset of cell death in several pathological conditions. For example, glutamate accumulation during ischemia and ischemic reperfusion often result in widespread cell death in the affected brain regions (Siesjo, 1992). Moreover, glutamate toxicity also seems to play a role in cell deletion in neurodegenerative disorders (Lancelot and Beal, this volume) such as Alzheimer’s disease (Copani et al., 1991; Mattson et al., 1992; Weiss et al., 1994), Parkinson’s disease (Mitchell et al., 1994), Huntingtons disease (Portera-Cailliau et al., 1995) and AIDS dementia (Lipton, 1996). Overstimulation of glutamate receptors results in sustained intracellular calcium overload (Choi, 1995) that triggers several lethal processes e.g. DNA-damage, proteolysis, mitochondrial dysfunction and disruption of cytoskeleta1 orgarkation (Reynolds, this volume). Sudden and massive intracellular calcium overload often lead to necrosis, however disturbances in calcium signalling can also trigger apoptosis (Nicotera et al., 1994). In the present review we discuss whether cell death manifests itself acutely as necrosis or slowly develops as apoptosis after glutamate exposure to a neuronal population. It appears, that to be able to determine which type or types of cell death are involved, it is necessary to use several different techniques to detect cell death and to study the full time-course of degeneration at several time-points after the insult.
Two morphologically distinct modes of cell death are in general recognized: necrosis and apoptosis. Necrosis is a passive form of cell death and a result of disorganized breakdown of the cell, often following acute violent insult. It is characterized by the concomitant disruption of several homeostatic processes, rapid energy depletion, organelle swelling and the activation of random catalytic processes. Cells are often destroyed very rapidly and the release of their content into the surroundings evokes inflammatory responses (Wyllie, 1980; Arends and Wyllie, 1991). In contrast, apoptosis is an active and organized cell deletion process, which results in removal of the cells by phagocytosis. Apoptosis occurs physiologically to eliminate unwanted, damaged or unnecessary cells and disturbances in the control of apoptosis can lead to disease (E-Lan, 1994; Thompson, 1995). Repression of apoptosis may lead to cancer or autoimmune disorders, whereas excess apoptosis may be involved in degenerative diseases. Results from in vitro and in vivo studies show that apoptosis could be the prevalent mode of cell deletion in neurodegenerative processes such as Alzheimer’s disease (Cotman and Anderson, 1999, Parkinson’s disease (Dispasquale et al., 1991; Mitchell et al., 1994) and Huntingtons chorea (PorteraCailliau et al., 1995) as well as neuronal cell death in the penumbra of a stroke (Charriaut-Marlangue et al., 1995).
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Cells dying by apoptosis undergo several morphological and biochemical changes, including: cell shrinkage, protease activation, nuclear condensation and DNA-fragmentation (Wyllie, 1980; Arends and Wyllie, 1991). Dead cells are rapidly engulfed and digested by macrophages leaving no trace in the surrounding tissue. In fact, apoptosis is often neglected because of the rapid clearance of dead cells by phagocytosis (Savill, 1993). Recent studies using cell-free systems and enucleated cells suggest that the central components of the cell death machinery are localized in the cytoplasm (Lazebnik et al., 1993; Newmeyer et al., 1994; Jacobson et al., 1994). Several lines of evidence suggest that proteases are good candidates as such cytoplasmic effectors of apoptosis. In the nematode C. elegans the exact number of cells dying by apoptosis during development is known. Several genes involved in this cell deletion are identified, among these ced-3 that must function for apoptosis to proceed (Ellis et al., 1991). Ced-3 encodes a protein with significant homology with mammalian interleukin- 1@-convertingenzyme (ICE) (Yuan et al., 1993). ICE is a cystein protease that cleaves pro-IL-l@to IL-l@ at Asp residues (Thornberry et al., 1992). A whole family of ICE-like proteases (or caspases) has now been described and these seem to be involved in the initiation as well as the execution of apoptosis. Some substrates for caspases have been identified. For example, poly (ADP-ribose) polymerase (PARP) is cleaved and inactivated by caspase-3 (Lazebnik et al., 1994; Fernandez-Alnemri et al., 1995; Tewari et al., 1995) and caspases are also involved in lamin degradation (Lazebnik et al., 1995; Zhivotovsky et al., 1995). Lamin proteolysis appears to play a critical role in nuclear degradation during apoptosis (Zhivotovsky et al., 1997) and precedes chromatin fragmentation in neurons exposed to glutamate (Ankarcrona et al., 1996a). On the other hand, PARP proteolysis is not a prerequisite for nuclear apoptosis and the ability of cells to undergo apoptosis is not affected in PARP knock-out mice (Wang et al., 1995). Still P A W cleavage is a good indicator for nuclear penetration of activated apoptotic proteases (e.g. caspase-3).
Methods to distinguish necrotic and apoptotic cells
Apoptotic cells stained with a nuclear dye appear with condensed nuclei under a fluorescence microscope and the degraded chromatin, cleaved by proteases and endonucleases into high molecular weight and oligonucleosomal lengthened DNAfragments, form distinct patterns on agarose gels. On the other hand, necrotic cells swell, lose membrane integrity and their DNA is randomly cleaved forming a smear on agarose gels. Even though DNA-fragmentation into oligonucleosoma1 lengthed fragments is a classical feature of apoptosis, it does not occur in all situations where apoptosis is involved. Rather, it appears that DNA-cleavage in apoptosis is a multistep process beginning with the formation of high molecular weight DNA-fragments (Filipski et al., 1990; Walker et al., 1995). This type of DNA-cleavage occurs prior to, or in the absence of, internucleosoma1 fragmentation (Oberhammer et al., 1993; Brown et al., 1993; Ankarcrona et al., 1995) and better correlates with the morphological appearance of apoptotic nuclei. Another method to detect chromatin degradation during apoptosis is in situ nick-end labeling (TUNEL) of fragmentated DNA. This technique has often been used to detect apoptotic cells in tissue slices. However, the labeling is not specific for DNA-fragments formed during apoptosis and it has been shown that also necrotic cells are stained by this technique (Nishiyama et al., 1996). Therefore, the TUNEL-technique should be combined with for example morphological studies of dying cells to be able to determine the type of cell death involved. While necrotic cells lose membrane permeability, often detected as LDH-leakage or failure to exclude trypan blue, cells dying by apoptosis maintain membrane integrity. Thus necrotic cells can be distinguished from apoptotic cells by using fluorescent nuclear dyes with different properties of cell permeability and emission wavelengths. For example, a membrane permeable dye (e.g. SYTO13) and a membrane impermeable dye (e.g. propidium iodide) can be used in combination. In our
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studies, cerebellar granule cells (CGC) grown on cover-slips were loaded with SYTO- 13 and then placed under a confocal microscope. Cells were perfused with glutamate and propidium iodide for 30 minutes. During the exposure we observed that some cells turned from green (SYTO-13) to red fluorescence (propidium iodide). In these cells, apparently dying by necrosis, glutamate caused rupture of cell membranes leading to uptake of propidium iodide. In other experiments, CGC were exposed to glutamate, reincubated in the old culture medium for 6 hours, loaded with SYTO-13 and then perfused with propidium iodide under the confocal microscope. Condensed nuclei typical of apoptosis were detected, and all cells excluded propidium iodide. In this case, cells that earlier died by necrosis had been washed away and only apoptotic cells still remaining attached to the cover-slip were observed (Ankarcrona et al., 1995). Protease activity is detected by immunoblot analysis of substrate cleavage products or assayed in vitro by fluorogenic peptide substrates. In addition, peptide inhibitors of proteases can be used to block apoptosis in many systems. Activation of proteases and cleavage of substrates (e.g. lamin and PARP) often occur early during apoptosis and not in necrosis. For example, caspase-3 processing and activation seems to be specific for apoptosis and was observed only when cerebellar granule cells in culture underwent apoptosis and not necrosis (Armstrong et al., 1997). Finally, cells dying by necrosis rapidly lose mitochondrial membrane potential and cellular energy, while cells undergoing apoptosis restore mitochondrial functions and require energy to complete the death program (see below) (Ankarcrona et al., 1995; Leist et al., 1997). In summary: condensed nuclei, DNA-fragmentation, early protease activation and maintained cell membrane integrity as well as mitochondrial functions can be used as criteria for apoptosis. By using a combination of the techniques described above it is possible to distinguish apoptotic cells from necrotic cells. To determine the mode of cell death it is also important to study morphological and biochemical changes in cells
both at early and late time-points after the insult. This is especially important in the in vivo situation since apoptotic cells are rapidly phagocytised and therefore easily missed if only a few time-points are chosen. A complication in the distinction between apoptosis and necrosis comes from the observation that secondary necrosis of apoptotic cells occurs in vitro or in vivo and reflects an insufficient removal of apoptotic cells by phagocytes (Leist et al., 1995). In this case, secondary processes may cause cell disintegration, mimicking necrosis. Glutamate neurotoxicity: A succession of necrosis and apoptosis While low concentrations of glutamate exert trophic effects and promote neuronal survival and synapse formation (Zorumski and Thio, 1992), increasing doses can result in neurotoxicity. In glutamate neurotoxicity cell death is mediated by calcium signaling and free radicals and manifests as apoptosis or necrosis (Mattson and Furukawa, 1996). It seems likely that the same fundamental mechanisms of neuronal injury lie behind the two types of cell death which then develop depending on the cell type, severity and duration of the injury and on the state of the cell. Induction of both apoptosis and necrosis in a neuronal cell population exposed to excitatory amino acids has been shown in vitro (Ankarcrona et al., 1995; Bonfoco et al., 1995; Gwag et al., 1995; Cebers et al., 1997) as well as in vivo (Pollard et al., 1994; Ferrer et al., 1995; Portera-Cailliau et al., 1995; van Lookeren Campagne et al., 1995). In vivo models of ischemic brain injury show that necrosis occurs in the focus of the ischemic zone where glutamate accumulation causes severe injury. Cells in the penumbra, also triggered to die, initially survive and later undergo apoptosis (Charriaut-Marlangue et al., 1995). It is of great importance for the treatment of stroke, as well as other neurodegenerative disorders where glutamate toxicity is implicated, to find drugs that block cell death caused by overstimulation of glutamate receptors. Potential targets for such drugs are
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glutamate receptors themselves and cellular processes upstream and downstream of receptor activation. These cellular processes include release of glutamate, free radical generation, activation of proteases and nitric oxide synthase, opening of mitochondria1 transition pores and release of intracellular calcium (Lipton and Rosenberg, 1994; Lancelot and Beal, this volume). Below we discuss the role of glutamate receptors and mitochondria in the onset and progression of cell death. Glutamate receptors
The glutamate receptors are categorized into ionotropic receptors controlling ion channels and metabotropic receptors coupled to G-proteins (Wenthold and Roche, this volume; Bruno et al., this volume). Briefly, the ionotropic receptors are: i) N-methyl-D-aspartate (NMDA) receptor sensitive to NMDA and glutamate, ii) a-amino-3hydoxy-5-methyl-4-isoxazolepropionate(AMPA) receptor sensitive to AMPA, kainic acid and glutamate iii) kainic acid receptor, sensitive to kainic acid and glutamate. When activated by agonists, the NMDA-receptor channel becomes permeable to calcium and sodium. Depending on the exact subunit composition of AMPA and kainic acid receptors their channels are permeable to sodium and sometimes to calcium (Borges and Dingledine, this volume). AMPA and kainic acid receptors mediate fast responses, while NMDAreceptors produce relatively sustained depolarization (Kebabian and Neumeyer, 1994; Lipton and Rosenberg, 1994). The NMDA-receptor is positively regulated by glycine, polyamines and phosphorylation. Its activity is decreased by oxidation of sulfhydryl groups in a redox-site. Lipton and co-workers have shown that generation of nitrozonium (NO+), produced upon activation of nitric oxide synthases, may result in such downregulation of the NMDA-receptor (Lipton et al., 1993). Oxidation of the redox-site downregulates receptor activity and potentially protects from glutamate induced cell death. Nitroglycerin, a drug used to treat cardiovascular disorders, is active on the
NMDA-receptor redox-site and works in a manner resembling N O f . In animal models high concentrations of nitroglycerin have been found to be neuroprotective during various NMDAreceptor mediated insults, including focal ischemia (Sathi et al., 1993). NMDA-receptor activity is also inhibited by open-channel blockers e.g. dizocilpine (MK-801) and memantine. Our experiments show that the NMDA-receptor antagonist MK-801 blocks both necrosis and apoptosis developing sequentially in cerebellar granule cells exposed to glutamate (Ankarcrona et al., 1995). Moreover, NMDA and quinolinic acid cause striatal apoptosis which was blocked by MK-801 but not by NBQX (AMPA/KA antagonist) (Qin et al., 1996). In other model systems, memantine abolishes neuronal injury in focal ischemia both in vivo and in vitro (Lipton, 1996 and refs therein). The use of memantine for potential treatment of stroke and neurodegenerative disorders has several advantages: i) unlike MK-801, memantine does not remain in the channel for an excessively long time, ii) memantine is clinically tolerated and used in the treatment of Parkinson’s disease and spasticity, and iii) memantine mainly blocks effects of pathological concentrations of glutamate and normal NMDA-receptor activity, important in neuronal plasticity, is less affected. Altogether, these studies indicate a central role for the NMDA-receptor in the onset of glutamate induced cell death. However, by blocking desensitization of AMPA-receptors with cyclothiazide cerebellar granule cells exposed to glutamate were triggered to die also in the presence of NMDAantagonists (Cebers et al., 1997). These results suggest that activation of AMPA-receptors, permeable to calcium under certain conditions, also contributes to the onset of neuronal apoptosis and necrosis. Role of mitochondria in glutamate toxicity
We used primary cultures of rat cerebellar granule cells (CGC) to study the role of mitochondria in glutamate induced cell death (Ankarcrona et al.,
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1995). Differentiated CGC express glutamate receptors predominantly of the NMDA-type and may serve as a model system for studies of glutamate toxicity. CGC are cultured in the presence of cytosine arabinoside to inhibit growth of dividing cells and mature cultures contain more than 95% CGC. In vitro CGC are dependent on constant depolarization for survival and therefore the culture medium is supplemented with 25 mM potassium chloride. In fact a lower concentration of potassium chloride ( 5 mM) induces cell death by apoptosis (D’Mello et al., 1993). In our studies, CGC were exposed to glutamate for 30 minutes and subsequently reincubated in the old culture medium. Part of the cell population (30-50% depending on glutamate concentration) died rapidly by necrosis up to three hours after exposure. Necrosis was detected as impaired metabolism of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan and loss of cell membrane integrity. We also measured a loss of mitochondrial membrane potential and ATP-depletion in the whole cell population immediately after glutamate exposure. Cells initially surviving the insult recovered mitochondrial membrane potential and ATP-levels. This part of the cell population later underwent apoptosis. These experiments show that while necrotic cells undergo rapid depletion of cellular energy, cells dying by apoptosis are dependent on mitochondrial energy production. Other studies have questioned the importance of mitochondrial respiration in apoptosis (Jacobson et al., 1994; Newmeyer et al., 1994), since they observed that cells undergo apoptosis even if deficient in mitochondrial ATPproduction. However, artificial ATP-generating systems were included in both studies. We have suggested that cells lacking cellular energy undergo necrosis before the apoptotic program has a chance to develop (Nicotera et al., 1997). This hypothesis is further supported by recent observations in human T-cells triggered to die by apoptosis with staurosporin or CD95 stimulation. Cells preemptied of ATP died by necrosis, while repletion of the extramitochondrial ATP-pool with glucose prevented necrosis and restored the ability
of the cells to undergo apoptosis (Leist et al., 1997). The transient loss of mitochondria1 membrane potential and cellular energy immediately following glutamate exposure may be an important signal for apoptosis (Reynolds, this volume). Decrease in mitochondrial membrane potential precedes apoptosis in many systems (Deckwerth and Johnson, 1993; Zamzami et al., 1995; Petit et al., 1995; Ankarcrona et al., 1995; Schinder et al., 1996) and leads to opening of permeable transition pores (PT) in the mitochondrial inner membrane. It has been suggested that a molecule active in the apoptotic pathway could be released from mitochondria during PT-opening (Zamzami et al., 1996). Such an apoptosis inducing factor (AIF) has been identified as a soluble protein localized in the intermembrane space of mitochondria and possessing protease activity (Susin et al., 1996). AIF induces typical manifestations of nuclear apoptosis (e.g. nuclear condensation and DNA-fragmentation) and all in vitro activities of AIF are blocked by the cysteine protease inhibitor Z-VAD.fmk, an efficient inhibitor of apoptosis in many cell systems (Zhivotovsky et al., 1995; Pronk et al., 1996; Cain et al., 1996). AIF fails to cleave PARP and lamin, two known substrates for proteases in the caspase family. Its molecular mass and subcellular localization also differs from the proteases in this family and AIF seems to belong to another group of cysteine proteases. Overexpression of Bcl-2, a protein known to inhibit apoptosis in many cells (Hockenbery et al., 1993; Reed, 1994; Newmeyer et al., 1994; Jacobson et al., 1994), blocks PT-opening and release of AIF from mitochondria. This indicates a central role for regulation of PT-opening by Bcl-2 during the initiation of apoptosis (Susin et al., 1996). One protein released from mitochondria upon apoptotic stimuli has been identified as cytochrome c (Liu et al., 1996). Cytochrome c was required to induce apoptosis in cell-free extracts and caspase-3 activation as well as PARP cleavage were detected. Cytochrome c is encoded by nuclear genes and transported into mitochondria. This supports the notion that all mitochondrial functions critical for apoptosis are encoded by nuclear
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genes, since cells lacking mitochondrial DNA also release the “death-factor’’ and die by apoptosis (Zamzami et al., 1996). Whether AIF and cytochrome c are identical or belong to a family of “death-factors’’ released from mitochondria remains to be elucidated. We found that CGC exposed to glutamate in the presence of cyclosporin A (CsA), an inhibitor of PT-opening and calcineurin activity, were protected from both necrosis and apoptosis. In addition, the more specific calcineurin inhibitor FK-506 had the same effects (Ankarcrona et al., 1996b). Our studies suggest that both PT-opening and calcineurin activity is important for apoptosis to occur. Hypothetically, a “death-factor” is released from mitochondria into the cytoplasma during transient loss of mitochondrial membrane potential. The “death-factor’’ is subsequently dephosphorylated by calcineurin and triggers cell death presumably by protease activity. It appears that mitochondria are not only important as an energy source, maybe they also harbour molecules important for initiation of apoptosis.
Conclusions Accumulating evidence from in vivo and in vitro studies shows that both necrosis and apoptosis occur in neuronal cell death after glutamate exposure. It is not known why some cells in a neuronal population die by necrosis while others, triggered to die by the same insult, later undergo apoptosis. Parameters that could influence which type of cell death will occur are: type of glutamate receptor activated, cell type, maturity of the cell, effects on mitochondria and cellular energy. Our studies show that glutamate exposure causes a succession of necrosis and apoptosis in cerebellar granule cells depending on mitochondrial functions. Necrosis associated with extreme energy loss in mitochondria may reflect the failure of neurons to carry out the “default” apoptotic program. The maintenance of mitochondrial function may therefore be a decisive factor in determining the degree and progression of neuronal injury caused by excitotoxins. The recent detection
of “death-factors’’ released from mitochondria upon loss of membrane potential and PT-opening further emphasizes the importance of mitochondria for the onset and progresssion of apoptosis (Susin et al., 1996; Zamzami et al., 1996; Liu et al., 1996; Ankarcrona et al., 1996b). Strong evidence shows a central role for proteases in the apoptotic pathway, and PT-opening might well be an equally important switch in the death program (Marchetti et al., 1996).
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272 Nicotera, P., Zhirotorsky, B., Bellozo, G., and Orrenius, S. (1994) Ion signalling in apoptosis. In: Apoptosis (Schimke, R.T. and Mihich, E. eds) pp 97-115, Plenum Press, New York. Nicotera, P., Ankarcrona, M., Bonfoco, E., Orrenius, S. and Lipton, S.A. (1997) Neuronal necrosis and apoptosis: Two distinct events induced by exposure to glutamate or oxidative stress. In: Neuronal Regeneration, Reorganization, and Repair, (Seil FJ ed), pp 95-101. Philadelphia: Lippincott-Raven Publishers. Nishiyama, K., Kwak, S., Takekoshi, S., Watanabe, K. and Kanazawa, I. (1996) In situ nick end-labeling detects necrosis of hippocampal pyramidal cells induced by kainic acid. Neurosci. Lett., 212: 139-142. Oberhammer, F., Wilson, J.W., Dive, C., Morris, I.D., Hickman, J.A., Wakeling, A.E., Walker, P.R. and Sikorska, M. (1993) Apoptotic death in epithelial cells: Cleavage of DNA to 300 and/or 50 kb fragments prior to in the absence of internucleosomal fragmentation. EMBO J., 1 2 367S3684. Petit, P.X., LeCoeur, H., Zorn, E., Dauguet, C., Mignotte, B. and Gougeon, M.L. (1995) Alterations of mitochondrial structure and function are early events of dexamethasoneinduced thymocyte apoptosis. f. Cell Biol., 130: 157-167. Pollard, H., Charriaut-Marlangue, C., Cantagrel, S., Represa, A,, Robain, O., Moreau, J. and Ben-Ari, Y. (1994) Kainateinduced apoptotic cell death in hippocampal neurons. Neurosci., 63: 7-18. Portera-Cailliau, C., Hedreen, J.C., Price, D.L. and Koliatsos, V.E. (1995) Evidence for apoptotic cell death in Huntington disease and excitotoxic animal models. J. Neurosci, 15: 37753787. Pronk, G.J. Ramer, K., Amiri, P. and Williams, L.T. (1996) Requirement of an ICE-like protease for induction of apoptosis and ceramide generation by REAPER. Science (Wash. D C ) , 271: 808-810. Qin, Z., Wang, Y. and Chase, T.N. (1996) Stimulation of Nmethyl-D-aspartate receptors induce apoptosis in rat brain. Brain Res., 725: 166-176. Reed, J.C. (1994) Bcl-2 and the regulation of programmed cell death. J. Cell Biol., 124: 1 4 . Sahti, S., Edgecomb, P., Warach, S., Manchester, K., Donaghey, T. Stieg, P.E., Jensen, F.E. and Lipton, S.A. (1993) Chronic transdermal nitroglycerin (NTG) is neuroprotective in experimental rodent stroke models. SOC.Neurosci. Abstr., 19: 849. Savill, J.S., Fadok, V., Henson, P. and Haslett, C. (1993) Phagocyte recognition of cells undergoing apoptosis. fmmunol. Today, 1 4 131-136. Schinder, A.F., Olson, E.C., Spitzer, N.C. and Montal, M. (1996) Mitochondria1 dysfunction is a primary event in glutamate neurotoxicity. J . Neurosci., 16: 61254133, Siesjo, B.K. (1992) Pathophysiology and treatment of focal cerebral ischemia. J. Neurosurg., 77: 169-184.
Susin, S.A., Zamzami, N., Castedo, M., Hirsch, T., Marchetti, P., Macho, A., Daugas, E., Geuskens, M. and Kroemer, G. (1996) Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J. Exp. Med., 184 1331-1341. Tewari, M., Quan, L.T., ORourke, K., Desnoyers, S., Zeng, Z., Beidler, D.R., Poirier, G.G., Salvesen, G.S. and Dixit, V.M (1995) Yama/CPP32b, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymearse. CeZl, 8 1: 801-809. Thompson, C.B. (1995) Apoptosis in the pathogenesis and treatment of disease. Science, 267: 1456-1462. Thornberry, N.A., Bull, H.G., Calaycay, J.R., Chapman, K.T. and Howard, A.D. (1992) A novel heterodimeric cysteine protease is required for interleukin-18, processing in monocytes. Nature, 356: 76&774. van Lookeren Campagne, M., Lucassen, P.J., Vermeulen, J.P. and Balazs, R. (1995) NMDA and kainate induce internucleosomal DNA cleavage associated with both apoptotic and necrotic cell death in the neonatal brain. Eur. f. Neurosci., 7: 1627- 1640. Walker, P.R., Pandrey, S. and Sikorska, M. (1995) Degradation 2: 97-104. of chromatin in apoptotic cells. Cell Death Drff., Wang, Z.-Q., Auer, B., Stingl, L., Berghammer, H., Haidacher, D., Schweiger, M. and Wagner, E.F. (1995) Mice lacking ADPRT and poly(ADP-ribosy1)ation develop normally but are susceptible to skin disease. Genes Dev., 9: 509-520. Weiss, J.H., Pike, C.J. and Cotman, C.W. (1994) Ca2+ channel blockers attenuate 8-amyloid peptide toxicity to cortical neurons in culture. f. Neurochem., 62: 372-315. Wyllie, A.H., Kerr, J.F.R. and Currie, A.R. (1980) Cell death: The significance of apoptosis. In?. Rev. Cytol., 68: 251-306. Yuan, J., Shaham, S., Ledoux, S., Ellis, H.M. and Horvitz, H.R. (1993) The c. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-18-converting enzyme. Cell, 75: 641452. Zamzami, N., Marchetti, P., Castedo, M., Zanin, C., Vayssiere, J.-L., Petit, P.X. and Kroemer, G. (1995) Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. f.Exp. Med., 181: 1661-1 672. Zamzami, N., Susin, S.A., Marchetti, P., Hirsch, T., GomezMonterrey, I., Castedo, M. and Kroemer, G. (1996) Mitochondrial control of nuclear apoptosis. J. Exp. Med., 183: 1533-1544. Zhivotovsky, B., Gahm, A,, Ankarcrona, M., Nicotera, P. and Orrenius, S. (1995) Multiple proteases are involved in thymocyte apoptosis. Exp. Cell Res., 221: 404412. Zhivotovsky, B., Gahm, A. and Orrenius, S. (1997) Two different proteases are involved in the proteolysis of lamin during apoptosis. Biochem. Biophys. Res. Com., 233: 96101. Zorumski, C.F. and Thio, L.L. (1992) Properties of vertebrate glutamate receptors: Calcium mobilization and desensitization. Prog. Neurobiol., 39: 295-336.
CHAPTER 17
Glutamate induced cell death: Apoptosis or necrosis? Maria Ankarcrona Karolinska Institutet. Department of Clinical Neuroscience and Family Medicine, Division of Geriatric Medicine, KFC, Novum, 4thJ%Or, S-141 86 Huddinge, Sweden
Introduction
Types of cell death: Necrosis and apoptosis
Glutamate toxicity is involved in the onset of cell death in several pathological conditions. For example, glutamate accumulation during ischemia and ischemic reperfusion often result in widespread cell death in the affected brain regions (Siesjo, 1992). Moreover, glutamate toxicity also seems to play a role in cell deletion in neurodegenerative disorders (Lancelot and Beal, this volume) such as Alzheimer’s disease (Copani et al., 1991; Mattson et al., 1992; Weiss et al., 1994), Parkinson’s disease (Mitchell et al., 1994), Huntingtons disease (Portera-Cailliau et al., 1995) and AIDS dementia (Lipton, 1996). Overstimulation of glutamate receptors results in sustained intracellular calcium overload (Choi, 1995) that triggers several lethal processes e.g. DNA-damage, proteolysis, mitochondrial dysfunction and disruption of cytoskeleta1 orgarkation (Reynolds, this volume). Sudden and massive intracellular calcium overload often lead to necrosis, however disturbances in calcium signalling can also trigger apoptosis (Nicotera et al., 1994). In the present review we discuss whether cell death manifests itself acutely as necrosis or slowly develops as apoptosis after glutamate exposure to a neuronal population. It appears, that to be able to determine which type or types of cell death are involved, it is necessary to use several different techniques to detect cell death and to study the full time-course of degeneration at several time-points after the insult.
Two morphologically distinct modes of cell death are in general recognized: necrosis and apoptosis. Necrosis is a passive form of cell death and a result of disorganized breakdown of the cell, often following acute violent insult. It is characterized by the concomitant disruption of several homeostatic processes, rapid energy depletion, organelle swelling and the activation of random catalytic processes. Cells are often destroyed very rapidly and the release of their content into the surroundings evokes inflammatory responses (Wyllie, 1980; Arends and Wyllie, 1991). In contrast, apoptosis is an active and organized cell deletion process, which results in removal of the cells by phagocytosis. Apoptosis occurs physiologically to eliminate unwanted, damaged or unnecessary cells and disturbances in the control of apoptosis can lead to disease (E-Lan, 1994; Thompson, 1995). Repression of apoptosis may lead to cancer or autoimmune disorders, whereas excess apoptosis may be involved in degenerative diseases. Results from in vitro and in vivo studies show that apoptosis could be the prevalent mode of cell deletion in neurodegenerative processes such as Alzheimer’s disease (Cotman and Anderson, 1999, Parkinson’s disease (Dispasquale et al., 1991; Mitchell et al., 1994) and Huntingtons chorea (PorteraCailliau et al., 1995) as well as neuronal cell death in the penumbra of a stroke (Charriaut-Marlangue et al., 1995).
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Cells dying by apoptosis undergo several morphological and biochemical changes, including: cell shrinkage, protease activation, nuclear condensation and DNA-fragmentation (Wyllie, 1980; Arends and Wyllie, 1991). Dead cells are rapidly engulfed and digested by macrophages leaving no trace in the surrounding tissue. In fact, apoptosis is often neglected because of the rapid clearance of dead cells by phagocytosis (Savill, 1993). Recent studies using cell-free systems and enucleated cells suggest that the central components of the cell death machinery are localized in the cytoplasm (Lazebnik et al., 1993; Newmeyer et al., 1994; Jacobson et al., 1994). Several lines of evidence suggest that proteases are good candidates as such cytoplasmic effectors of apoptosis. In the nematode C. elegans the exact number of cells dying by apoptosis during development is known. Several genes involved in this cell deletion are identified, among these ced-3 that must function for apoptosis to proceed (Ellis et al., 1991). Ced-3 encodes a protein with significant homology with mammalian interleukin- 1@-convertingenzyme (ICE) (Yuan et al., 1993). ICE is a cystein protease that cleaves pro-IL-l@to IL-l@ at Asp residues (Thornberry et al., 1992). A whole family of ICE-like proteases (or caspases) has now been described and these seem to be involved in the initiation as well as the execution of apoptosis. Some substrates for caspases have been identified. For example, poly (ADP-ribose) polymerase (PARP) is cleaved and inactivated by caspase-3 (Lazebnik et al., 1994; Fernandez-Alnemri et al., 1995; Tewari et al., 1995) and caspases are also involved in lamin degradation (Lazebnik et al., 1995; Zhivotovsky et al., 1995). Lamin proteolysis appears to play a critical role in nuclear degradation during apoptosis (Zhivotovsky et al., 1997) and precedes chromatin fragmentation in neurons exposed to glutamate (Ankarcrona et al., 1996a). On the other hand, PARP proteolysis is not a prerequisite for nuclear apoptosis and the ability of cells to undergo apoptosis is not affected in PARP knock-out mice (Wang et al., 1995). Still P A W cleavage is a good indicator for nuclear penetration of activated apoptotic proteases (e.g. caspase-3).
Methods to distinguish necrotic and apoptotic cells
Apoptotic cells stained with a nuclear dye appear with condensed nuclei under a fluorescence microscope and the degraded chromatin, cleaved by proteases and endonucleases into high molecular weight and oligonucleosomal lengthened DNAfragments, form distinct patterns on agarose gels. On the other hand, necrotic cells swell, lose membrane integrity and their DNA is randomly cleaved forming a smear on agarose gels. Even though DNA-fragmentation into oligonucleosoma1 lengthed fragments is a classical feature of apoptosis, it does not occur in all situations where apoptosis is involved. Rather, it appears that DNA-cleavage in apoptosis is a multistep process beginning with the formation of high molecular weight DNA-fragments (Filipski et al., 1990; Walker et al., 1995). This type of DNA-cleavage occurs prior to, or in the absence of, internucleosoma1 fragmentation (Oberhammer et al., 1993; Brown et al., 1993; Ankarcrona et al., 1995) and better correlates with the morphological appearance of apoptotic nuclei. Another method to detect chromatin degradation during apoptosis is in situ nick-end labeling (TUNEL) of fragmentated DNA. This technique has often been used to detect apoptotic cells in tissue slices. However, the labeling is not specific for DNA-fragments formed during apoptosis and it has been shown that also necrotic cells are stained by this technique (Nishiyama et al., 1996). Therefore, the TUNEL-technique should be combined with for example morphological studies of dying cells to be able to determine the type of cell death involved. While necrotic cells lose membrane permeability, often detected as LDH-leakage or failure to exclude trypan blue, cells dying by apoptosis maintain membrane integrity. Thus necrotic cells can be distinguished from apoptotic cells by using fluorescent nuclear dyes with different properties of cell permeability and emission wavelengths. For example, a membrane permeable dye (e.g. SYTO13) and a membrane impermeable dye (e.g. propidium iodide) can be used in combination. In our
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studies, cerebellar granule cells (CGC) grown on cover-slips were loaded with SYTO- 13 and then placed under a confocal microscope. Cells were perfused with glutamate and propidium iodide for 30 minutes. During the exposure we observed that some cells turned from green (SYTO-13) to red fluorescence (propidium iodide). In these cells, apparently dying by necrosis, glutamate caused rupture of cell membranes leading to uptake of propidium iodide. In other experiments, CGC were exposed to glutamate, reincubated in the old culture medium for 6 hours, loaded with SYTO-13 and then perfused with propidium iodide under the confocal microscope. Condensed nuclei typical of apoptosis were detected, and all cells excluded propidium iodide. In this case, cells that earlier died by necrosis had been washed away and only apoptotic cells still remaining attached to the cover-slip were observed (Ankarcrona et al., 1995). Protease activity is detected by immunoblot analysis of substrate cleavage products or assayed in vitro by fluorogenic peptide substrates. In addition, peptide inhibitors of proteases can be used to block apoptosis in many systems. Activation of proteases and cleavage of substrates (e.g. lamin and PARP) often occur early during apoptosis and not in necrosis. For example, caspase-3 processing and activation seems to be specific for apoptosis and was observed only when cerebellar granule cells in culture underwent apoptosis and not necrosis (Armstrong et al., 1997). Finally, cells dying by necrosis rapidly lose mitochondrial membrane potential and cellular energy, while cells undergoing apoptosis restore mitochondrial functions and require energy to complete the death program (see below) (Ankarcrona et al., 1995; Leist et al., 1997). In summary: condensed nuclei, DNA-fragmentation, early protease activation and maintained cell membrane integrity as well as mitochondrial functions can be used as criteria for apoptosis. By using a combination of the techniques described above it is possible to distinguish apoptotic cells from necrotic cells. To determine the mode of cell death it is also important to study morphological and biochemical changes in cells
both at early and late time-points after the insult. This is especially important in the in vivo situation since apoptotic cells are rapidly phagocytised and therefore easily missed if only a few time-points are chosen. A complication in the distinction between apoptosis and necrosis comes from the observation that secondary necrosis of apoptotic cells occurs in vitro or in vivo and reflects an insufficient removal of apoptotic cells by phagocytes (Leist et al., 1995). In this case, secondary processes may cause cell disintegration, mimicking necrosis. Glutamate neurotoxicity: A succession of necrosis and apoptosis While low concentrations of glutamate exert trophic effects and promote neuronal survival and synapse formation (Zorumski and Thio, 1992), increasing doses can result in neurotoxicity. In glutamate neurotoxicity cell death is mediated by calcium signaling and free radicals and manifests as apoptosis or necrosis (Mattson and Furukawa, 1996). It seems likely that the same fundamental mechanisms of neuronal injury lie behind the two types of cell death which then develop depending on the cell type, severity and duration of the injury and on the state of the cell. Induction of both apoptosis and necrosis in a neuronal cell population exposed to excitatory amino acids has been shown in vitro (Ankarcrona et al., 1995; Bonfoco et al., 1995; Gwag et al., 1995; Cebers et al., 1997) as well as in vivo (Pollard et al., 1994; Ferrer et al., 1995; Portera-Cailliau et al., 1995; van Lookeren Campagne et al., 1995). In vivo models of ischemic brain injury show that necrosis occurs in the focus of the ischemic zone where glutamate accumulation causes severe injury. Cells in the penumbra, also triggered to die, initially survive and later undergo apoptosis (Charriaut-Marlangue et al., 1995). It is of great importance for the treatment of stroke, as well as other neurodegenerative disorders where glutamate toxicity is implicated, to find drugs that block cell death caused by overstimulation of glutamate receptors. Potential targets for such drugs are
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glutamate receptors themselves and cellular processes upstream and downstream of receptor activation. These cellular processes include release of glutamate, free radical generation, activation of proteases and nitric oxide synthase, opening of mitochondria1 transition pores and release of intracellular calcium (Lipton and Rosenberg, 1994; Lancelot and Beal, this volume). Below we discuss the role of glutamate receptors and mitochondria in the onset and progression of cell death. Glutamate receptors
The glutamate receptors are categorized into ionotropic receptors controlling ion channels and metabotropic receptors coupled to G-proteins (Wenthold and Roche, this volume; Bruno et al., this volume). Briefly, the ionotropic receptors are: i) N-methyl-D-aspartate (NMDA) receptor sensitive to NMDA and glutamate, ii) a-amino-3hydoxy-5-methyl-4-isoxazolepropionate(AMPA) receptor sensitive to AMPA, kainic acid and glutamate iii) kainic acid receptor, sensitive to kainic acid and glutamate. When activated by agonists, the NMDA-receptor channel becomes permeable to calcium and sodium. Depending on the exact subunit composition of AMPA and kainic acid receptors their channels are permeable to sodium and sometimes to calcium (Borges and Dingledine, this volume). AMPA and kainic acid receptors mediate fast responses, while NMDAreceptors produce relatively sustained depolarization (Kebabian and Neumeyer, 1994; Lipton and Rosenberg, 1994). The NMDA-receptor is positively regulated by glycine, polyamines and phosphorylation. Its activity is decreased by oxidation of sulfhydryl groups in a redox-site. Lipton and co-workers have shown that generation of nitrozonium (NO+), produced upon activation of nitric oxide synthases, may result in such downregulation of the NMDA-receptor (Lipton et al., 1993). Oxidation of the redox-site downregulates receptor activity and potentially protects from glutamate induced cell death. Nitroglycerin, a drug used to treat cardiovascular disorders, is active on the
NMDA-receptor redox-site and works in a manner resembling N O f . In animal models high concentrations of nitroglycerin have been found to be neuroprotective during various NMDAreceptor mediated insults, including focal ischemia (Sathi et al., 1993). NMDA-receptor activity is also inhibited by open-channel blockers e.g. dizocilpine (MK-801) and memantine. Our experiments show that the NMDA-receptor antagonist MK-801 blocks both necrosis and apoptosis developing sequentially in cerebellar granule cells exposed to glutamate (Ankarcrona et al., 1995). Moreover, NMDA and quinolinic acid cause striatal apoptosis which was blocked by MK-801 but not by NBQX (AMPA/KA antagonist) (Qin et al., 1996). In other model systems, memantine abolishes neuronal injury in focal ischemia both in vivo and in vitro (Lipton, 1996 and refs therein). The use of memantine for potential treatment of stroke and neurodegenerative disorders has several advantages: i) unlike MK-801, memantine does not remain in the channel for an excessively long time, ii) memantine is clinically tolerated and used in the treatment of Parkinson’s disease and spasticity, and iii) memantine mainly blocks effects of pathological concentrations of glutamate and normal NMDA-receptor activity, important in neuronal plasticity, is less affected. Altogether, these studies indicate a central role for the NMDA-receptor in the onset of glutamate induced cell death. However, by blocking desensitization of AMPA-receptors with cyclothiazide cerebellar granule cells exposed to glutamate were triggered to die also in the presence of NMDAantagonists (Cebers et al., 1997). These results suggest that activation of AMPA-receptors, permeable to calcium under certain conditions, also contributes to the onset of neuronal apoptosis and necrosis. Role of mitochondria in glutamate toxicity
We used primary cultures of rat cerebellar granule cells (CGC) to study the role of mitochondria in glutamate induced cell death (Ankarcrona et al.,
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1995). Differentiated CGC express glutamate receptors predominantly of the NMDA-type and may serve as a model system for studies of glutamate toxicity. CGC are cultured in the presence of cytosine arabinoside to inhibit growth of dividing cells and mature cultures contain more than 95% CGC. In vitro CGC are dependent on constant depolarization for survival and therefore the culture medium is supplemented with 25 mM potassium chloride. In fact a lower concentration of potassium chloride ( 5 mM) induces cell death by apoptosis (D’Mello et al., 1993). In our studies, CGC were exposed to glutamate for 30 minutes and subsequently reincubated in the old culture medium. Part of the cell population (30-50% depending on glutamate concentration) died rapidly by necrosis up to three hours after exposure. Necrosis was detected as impaired metabolism of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan and loss of cell membrane integrity. We also measured a loss of mitochondrial membrane potential and ATP-depletion in the whole cell population immediately after glutamate exposure. Cells initially surviving the insult recovered mitochondrial membrane potential and ATP-levels. This part of the cell population later underwent apoptosis. These experiments show that while necrotic cells undergo rapid depletion of cellular energy, cells dying by apoptosis are dependent on mitochondrial energy production. Other studies have questioned the importance of mitochondrial respiration in apoptosis (Jacobson et al., 1994; Newmeyer et al., 1994), since they observed that cells undergo apoptosis even if deficient in mitochondrial ATPproduction. However, artificial ATP-generating systems were included in both studies. We have suggested that cells lacking cellular energy undergo necrosis before the apoptotic program has a chance to develop (Nicotera et al., 1997). This hypothesis is further supported by recent observations in human T-cells triggered to die by apoptosis with staurosporin or CD95 stimulation. Cells preemptied of ATP died by necrosis, while repletion of the extramitochondrial ATP-pool with glucose prevented necrosis and restored the ability
of the cells to undergo apoptosis (Leist et al., 1997). The transient loss of mitochondria1 membrane potential and cellular energy immediately following glutamate exposure may be an important signal for apoptosis (Reynolds, this volume). Decrease in mitochondrial membrane potential precedes apoptosis in many systems (Deckwerth and Johnson, 1993; Zamzami et al., 1995; Petit et al., 1995; Ankarcrona et al., 1995; Schinder et al., 1996) and leads to opening of permeable transition pores (PT) in the mitochondrial inner membrane. It has been suggested that a molecule active in the apoptotic pathway could be released from mitochondria during PT-opening (Zamzami et al., 1996). Such an apoptosis inducing factor (AIF) has been identified as a soluble protein localized in the intermembrane space of mitochondria and possessing protease activity (Susin et al., 1996). AIF induces typical manifestations of nuclear apoptosis (e.g. nuclear condensation and DNA-fragmentation) and all in vitro activities of AIF are blocked by the cysteine protease inhibitor Z-VAD.fmk, an efficient inhibitor of apoptosis in many cell systems (Zhivotovsky et al., 1995; Pronk et al., 1996; Cain et al., 1996). AIF fails to cleave PARP and lamin, two known substrates for proteases in the caspase family. Its molecular mass and subcellular localization also differs from the proteases in this family and AIF seems to belong to another group of cysteine proteases. Overexpression of Bcl-2, a protein known to inhibit apoptosis in many cells (Hockenbery et al., 1993; Reed, 1994; Newmeyer et al., 1994; Jacobson et al., 1994), blocks PT-opening and release of AIF from mitochondria. This indicates a central role for regulation of PT-opening by Bcl-2 during the initiation of apoptosis (Susin et al., 1996). One protein released from mitochondria upon apoptotic stimuli has been identified as cytochrome c (Liu et al., 1996). Cytochrome c was required to induce apoptosis in cell-free extracts and caspase-3 activation as well as PARP cleavage were detected. Cytochrome c is encoded by nuclear genes and transported into mitochondria. This supports the notion that all mitochondrial functions critical for apoptosis are encoded by nuclear
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genes, since cells lacking mitochondrial DNA also release the “death-factor’’ and die by apoptosis (Zamzami et al., 1996). Whether AIF and cytochrome c are identical or belong to a family of “death-factors’’ released from mitochondria remains to be elucidated. We found that CGC exposed to glutamate in the presence of cyclosporin A (CsA), an inhibitor of PT-opening and calcineurin activity, were protected from both necrosis and apoptosis. In addition, the more specific calcineurin inhibitor FK-506 had the same effects (Ankarcrona et al., 1996b). Our studies suggest that both PT-opening and calcineurin activity is important for apoptosis to occur. Hypothetically, a “death-factor” is released from mitochondria into the cytoplasma during transient loss of mitochondrial membrane potential. The “death-factor’’ is subsequently dephosphorylated by calcineurin and triggers cell death presumably by protease activity. It appears that mitochondria are not only important as an energy source, maybe they also harbour molecules important for initiation of apoptosis.
Conclusions Accumulating evidence from in vivo and in vitro studies shows that both necrosis and apoptosis occur in neuronal cell death after glutamate exposure. It is not known why some cells in a neuronal population die by necrosis while others, triggered to die by the same insult, later undergo apoptosis. Parameters that could influence which type of cell death will occur are: type of glutamate receptor activated, cell type, maturity of the cell, effects on mitochondria and cellular energy. Our studies show that glutamate exposure causes a succession of necrosis and apoptosis in cerebellar granule cells depending on mitochondrial functions. Necrosis associated with extreme energy loss in mitochondria may reflect the failure of neurons to carry out the “default” apoptotic program. The maintenance of mitochondrial function may therefore be a decisive factor in determining the degree and progression of neuronal injury caused by excitotoxins. The recent detection
of “death-factors’’ released from mitochondria upon loss of membrane potential and PT-opening further emphasizes the importance of mitochondria for the onset and progresssion of apoptosis (Susin et al., 1996; Zamzami et al., 1996; Liu et al., 1996; Ankarcrona et al., 1996b). Strong evidence shows a central role for proteases in the apoptotic pathway, and PT-opening might well be an equally important switch in the death program (Marchetti et al., 1996).
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