Prevention of endotoxin-induced lethality, but not of liver apoptosis in Poly(ADP-ribose) polymerase-deficient mice
1999-09-24, Kühnle, Simone, Nicotera, Pierluigi, Wendel, Albrecht, Leist, Marcel
Activation of poly-(ADP-ribose) polymerase (PARP) is often associated with cytotoxicity, but its precise role in shock-induced lethality and in different modes of tissue injury is still unknown. We took advantage of the existence of mice with a targeted deletion of the PARP gene (PARP−/−) to examine the differential sensitivity of wild-type (wt) and PARP−/− mice toward endotoxin (LPS)-induced lethality and different forms of liver damage. All PARP−/− animals survived high-dose (20 mg/kg) LPS-mediated shock, which killed 60% of wt animals. Moreover, LPS-induced necrotic liver damage was significantly reduced. In contrast, when apoptotic liver damage was induced via injection of low concentrations of LPS (30 μg/kg) into D-galactosamine-sensitized mice, or via activation of hepatic cell death receptors, PARP−/− animals were not protected. We conclude that PARP is involved in systemic LPS toxicity, while it plays a minor role in apoptotic liver damage mediated by TNF or CD95.
Inhibition of Mitochondrial ATP Generation by Nitric Oxide Switches Apoptosis to Necrosis
1999-06-15, Leist, Marcel, Single, Barbara, Naumann, Heike, Fava, Eugenio, Simon, Bernadett, Kühnle, Simone, Nicotera, Pierluigi
Under pathological conditions, the mode of cell death, apoptosis or necrosis, is relevant for the subsequent fate of the tissue. Cell demise may be shaped by endogenous mediators such as nitric oxide (NO) which interfere with subroutines of the death program. Here we show that apoptosis of Jurkat cells elicited by either staurosporine (STS) or anti-CD95 antibodies in glucose-free medium is converted to necrosis by NO donors. In the presence of NO, release of mitochondrial cytochrome c was delayed and activation of execution caspases was prevented. Stimulated cells died nonetheless. The switch in the mode of cell death was due to NO-dependent failure of mitochondrial energy production. Restoration of intracellular ATP by glucose supplementation recovered the cells' ability to activate caspases and undergo apoptosis. In this system, the apoptosis/necrosis conversion promoted by NO was not mediated by cyclic guanosine monophosphate-dependent mechanisms, poly-(ADP-ribose)-polymerase (PARP) activation, or inhibition of caspases due to S-nitrosylation and glutathione depletion. In contrast, depleting intracellular ATP with rotenone, an inhibitor of mitochondrial complex I mimicked the effect of NO. The findings presented here suggest that NO can decide the shape of cell death by lowering intracellular ATP below the level required to allow the coordinated execution of apoptosis.
Tributyltin-Induced Apoptosis Requires Glycolytic Adenosine Trisphosphate Production
1999, Stridh, Hélène, Fava, Eugenio, Single, Barbara, Nicotera, Pierluigi, Orrenius, Sten, Leist, Marcel
The toxicity of tributyltin chloride (TBT) involves Ca2+ overload, cytoskeletal damage, and mitochondrial failure leading to cell death by apoptosis or necrosis. Here, we examined whether the intracellular ATP level modulates the mode of cell death after exposure to TBT. When Jurkat cells were energized by the mitochondrial substrate, pyruvate, low concentrations of TBT (1-2 íM) triggered an immediate depletion of intracellular ATP followed by necrotic death. When ATP levels were maintained by the addition of glucose, the mode of cell death was typically apoptotic. Glycolytic ATP production was required for apoptosis at two distinct steps. First, maintenance of adequate ATP levels accelerated the decrease of mitochondrial membrane potential, and the release of the intermembrane proteins adenylate kinase and cytochrome c from mitochondria. A possible role of the adenine nucleotide exchanger in this first ATPdependent step is suggested by experiments performed with the specific inhibitor, bongkrekic acid. This substance delayed cytochrome c release in a manner similar to that caused by ATP depletion. Second, caspase activation following cytochrome c release was only observed in ATPcontaining cells. Bcl-2 had only a minor effect on TBT-triggered caspase activation or cell death. We conclude that intracellular ATP concentrations control the mode of cell death in TBTtreated Jurkat cells at both the mitochondrial and caspase activation levels.
Nitric Oxide Inhibits Execution of Apoptosis at Two Distinct ATP-Dependent Steps Upstream and Downstream of Mitochondrial Cytochrome c Release
1999, Leist, Marcel, Single, Barbara, Naumann, Heike, Fava, Eugenio, Simon, Bernadett, Kühnle, Simone, Nicotera, Pierluigi
The endogenous mediator nitric oxide (NO) blocked apoptosis of Jurkat cells elicited by staurosporine, anti-CD95 or chemotherapeutics, and switched death to necrosis. The switch in the mode of cell death was dependent on the ATP loss elicited by NO. This affected two distinct steps of the apop· totic cascade. First, the release of cytochrome c from mitochondria was delayed by NO. Second, processing of procaspases-317 to the active proteases was prevented even after cytochrome c had been released. Thus, NO interferes with execution steps of apoptosis both upstream and downstream of cytochrome c release.
Execution of Apoptosis : Converging or Diverging Pathways?
1999-09, Nicotera, Pierluigi, Leist, Marcel, Single, Barbara, Volbracht, Christiane
There is increasing evidence that apoptosis and necrosis represent only two of several possible ways for cells to die. These two types of demise can occur simultaneously in tissues or cell cultures exposed to the same stimulus, and often local metabolic conditions and the intensity of the same initial insult decide the prevalence of either apoptosis or necrosis. Recent work has shown that execution of the apoptotic programme involves a relatively limited number of pathways. According to a general view, these would converge to activate the caspase family of proteases. However, there is increasing evidence that apoptotic-like features can be observed also in cells where caspases are inhibited by cell-permeable tripeptides, such as z-VaD-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), or analogous compounds. This has posed the question as to whether apoptosis may or may not occur in a caspase independent way, and whether caspase inhibitors may be effective in the treatment of disease. Also relevant is the understanding that low intracellular energy levels during apoptosis can preclude caspase activation, and consequently decide the occurrence and mode of demise in damaged cells. In vivo, incomplete execution of damaged cells by apoptosis may have profound implications, as their persistence within a tissue, followed by delayed lysis, may elicit delayed pro-inflammatory reactions. In this minireview, we discuss some recent findings suggesting that cells may use diverging execution pathways, with different implications in pathology and therapy.
Neuronal cell death : a demise with different shapes
1999, Nicotera, Pierluigi, Leist, Marcel, Manzo, Luigi
Severe neuronal loss is common to many neurodegenerative diseases. Although necrotic features are often prevalent in neuropathological conditions, there is now increasing evidence to show that apoptosis can significantly contribute to neuronal demise in neurodegenerative diseases, including Huntington's and Alzheimer's diseases and HIV-associated dementia. Furthermore, a role in other disorders such as stroke, trauma, Parkinson's disease, multiple sclerosis and amyotrophic lateral sclerosis has been suggested from animal studies1. Nevertheless, it is unclear which of the two types of demise, apoptosis or necrosis, prevail in acute and slowly developing neurogenerative disorders, and whether the mode of cell death is relevant for the ultimate progression of the disease.
The debate on the occurrence and prevalence of either type of death in pathological conditions such as stroke or neurotoxic injury could be resolved in part by considering that different types of cell death within a tissue reflect either the complete or the partial execution of a common death programme. Some endogenous mediators might modulate the shape (morphological appearance) of cells in the death process; such modulation could have implications for the neighbouring tissue by interfering with the execution of apoptosis-specific subroutines, thereby changing apoptosis to necrosis.
ATP Controls Neuronal Apoptosis Triggered by Microtubule Breakdown or Potassium
1999, Volbracht, Christiane, Leist, Marcel, Nicotera, Pierluigi
Background: Early loss of neurites followed by delayed damage of neuronal somata is a feature of several neurodegenerative diseases. Death by apoptosis would ensure the rapid removal of injured neurons, whereas conditions that prevent apoptosis may facilitate the persistence of damaged cells and favor inflammation and disease progression. Materials and Methods: Cultures of cerebellar granule cells (CGC) were treated with microtubule disrupting agents. These compounds induced an early degeneration of neurites followed by apoptotic destruction of neuronal somata. The fate of injured neurons was followed after co-exposure to caspase inhibitors or agents that decrease intracellular ATP (deoxyglucose, S-nitrosoglutathione, l-methyl 4-phenylpyridinium). We examined the implications of energy loss for caspase activation, exposure of phagocytosis markers, and long-term persistence of damaged cells. Results: In CGC exposed to colchicine or nocodazole, axodendritic degeneration preceded caspase activation and apoptosis. ATP-depleting agents or protein synthesis inhibition prevented caspase activation, translocation of the phagocytosis marker, phosphatidylserine, and apoptotic death. However, they did not affect the primary neurite loss. Repletion of ATP by enhanced glycolysis restored all apoptotic features. Peptide inhibitors of caspases also prevented the apoptotic changes in the cell bodies, although the axodendritic net was lost. Under this condition cell demise still occurred 48 hr later in a caspase-independent manner and involved plasma membrane lysis at the latest stage. Conclusions: Inhibition of the apoptotic machinery by drugs, energy deprivation, or endogenous mediators may result in the persistence and subsequent lysis of injured neurons. In vivo, this may favor the onset of inflammatory processes and perpetuate neurodegeneration.
Differential effects of Bcl‐2 overexpression on fibre outgrowth and survival of embryonic dopaminergic neurons in intracerebral transplants
1999-09, Schierle, Gabriele S., Leist, Marcel, Martinou, Jean-Claude, Widner, H, Nicotera, Pierluigi, Brundin, Patrik
The causes of death of transplanted neurons are not known in detail, but apoptotic mechanisms involving caspase activation are likely to play a role. We examined whether overexpression of the anti‐apoptotic protein Bcl‐2 may enhance the survival of dopaminergic [tyrosine hydroxylase (TH)‐immunoreactive] grafted neurons. For this purpose, we prepared cells from embryonic day 13 ventral mesencephalon (VM) of mice overexpressing human Bcl‐2, or from their wild‐type littermates. The bcl‐2 transgene was strongly expressed in these cells, and resulted in protection of neuronal cultures from death triggered by serum deprivation or exposure to staurosporine. To model pretransplantation stress more closely in vitro, we stored dissociated embryonic mesencephalic cells for 8 h in the same type of medium used for intracerebral transplantation. This resulted in massive cell death as quantified by lactate dehydrogenase (LDH) release, and increased DNA fragmentation. Although this cell loss was strongly reduced by a caspase inhibitor, Bcl‐2 had no significant protective effect. Finally, mesencephalic cell suspensions were xenografted into the striatum of immunosuppressed hemiparkinsonian rats. Neither the survival of TH‐immunopositive transplanted neurons nor the functional recovery of the rats was improved by Bcl‐2, although the Bcl‐2 protein was strongly expressed in transgenic grafts 5 weeks after implantation, and dopaminergic fibre outgrowth from the grafts was significantly improved. These data suggest that cell death in neuronal transplants involves apoptotic mechanisms that can bypass negative regulation by Bcl‐2.
Neuronal death in nigral grafts in the absence of poly (ADP-ribose) polymerase activation
1999, Schierle, Gabriele S. Kaminski, Hansson, Oskar, Ferrando-May, Elisa, Nicotera, Pierluigi, Brundin, Patrik, Leist, Marcel
The exact causes of the extensive cell death in nigral transplants are still unknown. Since poly-(ADP-ribose) polymerase (PARP) overactivation has been implicated in neuronal death, we examined the effects of PARP on the survival of nigral grafts by using donor tissue from PARP knock-out or wild-type mice. Eight hours after preparation of the nigral cell suspension, cell damage was quantified by measurement of lactate dehydrogenase release, DNA fragmentation and caspase activation. At this stage, PARP deletion had no protective effect. Moreover, neither the survival of transplanted dopaminergic neurons, nor the functional recovery of hemiparkinsonian graft recipients were improved by the absence of PARP. We conclude that cell death in embryonic nigral grafts is not affected by the absence of PARP activation.
Caspase inhibition reduces apoptosis and increases survival of nigral transplants
1999-01, Schierle, Gabriele S., Hansson, Oskar, Leist, Marcel, Nicotera, Pierluigi, Widner, Håkan, Brundin, Patrik
Transplantation of embryonic nigral tissue ameliorates functional deficiencies in Parkinson disease. The main practical constraints of neural grafting are the shortage of human donor tissue and the poor survival of dopaminergic neurons grafted into patients, which is estimated at 5−10% (refs.). The required amount of human tissue could be considerably reduced if the neuronal survival was augmented. Studies in rats indicate that most implanted embryonic neurons die within 1 week of transplantation, and that most of this cell death is apoptotic6. Modified peptides, such as acetyl−tyrosinyl−valyl−alanyl−aspartyl−chloro−
methylketone (Ac−YVAD−cmk), that specifically inhibit proteases of the caspase family effectively block apoptosis in a plethora of experimental paradigms, such as growth factor withdrawal, excitotoxicity, axotomy, cerebral ischemia and brain trauma. Here we examined the effects of caspase inhibition by Ac−YVAD−cmk on cell death immediately after donor tissue preparation and on long−term graft survival. Treatment of the embryonic nigral cell suspension with Ac−YVAD−cmk mitigated DNA fragmentation and reduced apoptosis in transplants. It also increased survival of dopaminergic neurons grafted to hemiparkinsonian rats, and thereby substantially improved functional recovery.