2002-07, Simon, Bernadett, Malisan, Florence, Testi, Roberto, Nicotera, Pierluigi, Leist, Marcel

Increased brain ganglioside levels are a hallmark of various neuroinflammatory pathologies. Here, we provide evidence that murine microglia can secrete disialoganglioside GD3 upon exposure to inflammatory stimuli. Comparison of different neural cell types revealed a particular and specific sensitivity of oligodendrocytes towards exogenous GD3. Oligodendrocyte death triggered by GD3 was preceded by degeneration of cellular processes, and associated with typical features of apoptosis, such as chromatin condensation, exposure of phosphatidylserine, release of cytochrome c from mitochondria, and loss of mitochondrial membrane potential, followed by the loss of plasma membrane integrity and detachment of disintegrated oligodendrocytes. Overexpression of bcl-2 partially protected oligodendrocytes from death. In contrast, treatment with the pan-caspase inhibitor zVAD-fmk did not prevent phosphatidylserine exposure, chromatin margination at the nuclear periphery, and death, although caspase-3 was blocked. Thus, GD3 produced by microglia under neuroinflammatory conditions may function as a novel mediator triggering mitochondria-mediated, but caspase-independent, apoptosis-like death of oligodendrocytes.

1998, Steinbach, Joachim P., Müller, Ulrike, Leist, Marcel, Li, Zhi-Wei, Nicotera, Pierluigi, Aguzzi, Adriano

Secreted forms of the β-amyloid precursor protein (β-APP) have neuroprotective properties in vitro and may be involved in the containment of neuronal excitation. To test whether loss of secreted forms of β-APP (sAPPs) may enhance excitotoxic responses, we injected mice homozygous for a targeted mutation of the β-APP gene (β-APP∆/∆) intraperitoneally with kainic acid. We found that in these mice, kainic acid induced seizures initiated earlier, and acute mortality was enhanced compared to isogenic wild-type mice independently from the callosal agenesis phenotype observed to occur at increased frequency in APP mutant mice. Expression of c-fos in cortex and cingulate gyrus was enhanced in β-APP∆/∆ mice, although the amount of structural damage and apoptosis in the hippocampal pyramidal cell layer and cortex was similar to that of controls. When cerebellar granule cell cultures and cortical neuronal cultures were challenged with glutamate receptor agonists, the rates of cell death and apoptosis of β-APP∆/∆ mice were indistinguishable from those of controls. Therefore, deficiency of sAPPs causes facilitation of seizure activity in the absence of enhanced cell death. Since enhanced seizures were observed also in mice homozygous for a deletion of the entire β-APP gene, this phenotype results from a loss of APP rather than from a dominant effect of APP∆.

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.

1997, Nicotera, Pierluigi, Leist, Marcel

Apoptosis and necrosis are considered as conceptually distinct forms of cell death. Nevertheless, there is increasing evidence that classical apoptosis and necrosis represent only the extreme ends of a wide range of possible morphological and biochemical deaths. The two classical types of demise can occur simultaneously in tissues or cell cultures exposed to the same stimulus, and often the intensity of the same initial insult decides the prevalence of either apoptosis or necrosis. This suggests that, while some early events may be common to both types of cell death, a downstream controller may be required to direct cells towards the organised execution of apoptosis. We have recently shown that intracellular energy levels and mitochondrial function are rapidly compromised in necrosis, but not in apoptosis of neuronal cells. Then, we went on to show that pre-emptying human T cells of ATP switches the type of demise caused by two classic apoptotic triggers (staurosporin and CD95 stimulation) from apoptosis to necrosis. Conditions of controlled intracellular ATP depletion, which was obtained by blocking mitochondrial and/or glycolytic ATP generation, were used in combination with repletion of the cytosolic ATP pool with glucose to redirect the death program towards apoptosis or necrosis. At least two distinct steps, the typical nuclear degradation, and the expression of annexin V-recognisable determinants on the cell surface require sufficient ATP generation. This suggests that some upstream regulators of cell death maybe common to both types of cell demise, whereas yet unknown downstream processes decide its shape and the implications for the neighbouring tissue.

1998, Leist, Marcel, Kühnle, Simone, Single, Barbara, Nicotera, Pierluigi

Apoptosis and necrosis are two forms of cell death that have been defined on the basis of distinguishable morphological criteria. However, these different types of cell death may involve several common signalling and execution mechanisms. Since various stimuli induce both apoptotic and necrotic death, the mode of cell demise seems to be dependent on intracellular factors. One of these factors is the concentration of ATP. By modulating ATP levels, apoptosis or necrosis can be triggered selectively under otherwise identical conditions. By controlling ATP levels in staurosporine treated Jurkat cells, apoptotic (but not necrotic) cell death was detected selectively, and was quantitated with high sensitivity by the BM Cell Death Detection ELISA or by staining with annexin V.

1997, Leist, Marcel, Fava, Eugenio, Montecucco, Cesare, Nicotera, Pierluigi

Endogenous generation of nitric oxide and its congeners, including peroxynitrite (ONOO-), has been implicated in the mechanism of neuron loss in neurodegenerative diseases. Accordingly, nitric oxide donors and ONOO-can elicit both apoptosis and necrosis in neuron cultures. Here we show that nitric oxide donors and ONOO- are each able to trigger apoptosis of mouse cerebellar granule cells by an excitotoxic mechanism requiring exocytosis and NMDA receptor-mediated intracellular Ca2+ overload. This conclusion is supported by the following findings. Apoptosis was induced by various nitric oxide donors or by direct addition of ONOO- to differentiated cerebellar granule cell cultures that were sensitive to NMDA toxicity, but not in cerebellar granule cells that did not display NMDA-induced cell death (i.e. early days in culture) or in various glial cell populations. Donors of ONOO- or nitric oxide stimulated a sustained increase in intracellular Ca2+, which was prevented by inhibitors of NMDA receptors, such as MK-801 and 5-phospho-aminovaleric acid, or by dampening neuronal electrical activity with high concentrations of extracellular Mg2+. Moreover, these treatments and the exposure of cerebellar granule cells in nominally Ca2+-free media prevented apoptotic cell death. Both the intracellular Ca2+ increase and apoptosis elicited by ONOO- or the nitric oxide donors were prevented by blocking exocytosis with tetanus toxin or botulinum neurotoxin C.