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Biomedical subjects

M Leist

Publications and source records attributed to M Leist.

At least 55 records · Page 3Linked to original sources
PubMed1999

ATP controls neuronal apoptosis triggered by microtubule breakdown or potassium deprivation.

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, 1-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.

Adenosine Triphosphate↗
PubMed1998

Intracellular ATP, a switch in the decision between apoptosis and necrosis.

Regardless of whether apoptosis or necrosis are elicited by toxicants or by pathophysiological conditions they are considered 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. The execution of the death program seems to involve a relatively limited number of pathways. In many instances, their ordered execution results in characteristic morphological and biochemical changes termed apoptosis. However, some subroutines of the degradation program may not be active in all cases of cell death. Then, the morphological appearance of dying cells and some of their biochemical alterations differ from those of classical 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-empting 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.

Adenosine Triphosphate↗
PubMed1998

Apoptosis, excitotoxicity, and neuropathology.

While a high rate of cell loss is tolerated and even required to model the developing nervous system, an increased rate of cell death in the adult nervous system underlies neurodegenerative disease. Evolutionarily conserved mechanisms involving proteases, Bcl-2-related proteins, p53, and mitochondrial factors participate in the modulation and execution of cell death. In addition, specific death mechanisms, based on specific neuronal characteristics such as excitability and the presence of specific channels or enzymes, have been unraveled in the brain. Particularly important for various human diseases are excessive nitric oxide (NO) production and excitotoxicity. These two pathological mechanisms are closely linked, since excitotoxic stimulation of neurons may trigger enhanced NO production and exposure of neurons to NO may trigger the release of excitotoxins. Depending on the experimental situation and cell type, excitotoxic neuronal death may either be apoptotic or necrotic.

Adult↗
PubMed1998

Selective proteolysis of the nuclear replication factor MCM3 in apoptosis.

Cleavage of specific protein subsets is a key event in the execution of apoptosis. Protein degradation may serve for the structural alterations that result in cell self-destruction, but it may also function as a switch in the decisions between apoptosis and necrosis or apoptosis and cell proliferation. Here, we show that MCM3, but not other members of the Mcm family of replicative proteins, is cleaved early in several models of apoptosis. Cleavage of MCM3 can be prevented by caspase inhibitors, and it does not occur when cells are forced to undergo necrosis by energy deprivation. We propose that active destruction of MCM3 inactivates the Mcm complex and serves to prevent untimely DNA replication events during the execution of the cell death program.

Apoptosis↗
PubMed1998

Hypersensitivity to seizures in beta-amyloid precursor protein deficient mice.

Secreted forms of the beta-amyloid precursor protein (beta-APP) have neuroprotective properties in vitro and may be involved in the containment of neuronal excitation. To test whether loss of secreted forms of beta-APP (sAPPs) may enhance excitotoxic responses, we injected mice homozygous for a targeted mutation of the beta-APP gene (beta-APPDelta/Delta) 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 beta-APPDelta/Delta 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 beta-APPDelta/Delta 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 beta-APP gene, this phenotype results from a loss of APP rather than from a dominant effect of APPDelta.

Amyloid beta-Protein Precursor↗
PubMed1998

1-Methyl-4-phenylpyridinium induces autocrine excitotoxicity, protease activation, and neuronal apoptosis.

The pathogenesis of several neurodegenerative diseases may involve indirect excitotoxic mechanisms, where glutamate receptor overstimulation is a secondary consequence of initial functional defects of neurons (e.g., impairment of mitochondrial energy generation). The neurotoxin 1-methyl-4-phenylpyridinium (MPP+) and other mitochondrial inhibitors (e.g., rotenone or 3-nitropropionic acid) elicited apoptosis in cerebellar granule cell cultures via stimulation of autocrine excitotoxicity. Cell death, increase in intracellular Ca2+ concentration, release of cytochrome c, and all biochemical and morphological signs of apoptosis were prevented by blockade of the N-methyl-D-aspartate receptor with noncompetitive, glycine-site or glutamate-site inhibitors. In addition, MPP+-induced apoptosis was reduced by high Mg2+ concentrations in the medium or by inhibiting exocytosis with clostridial neurotoxins. Two classes of cysteine proteases were involved in the execution of cell death: caspases and calpains. Inhibitors of either class of proteases prevented cell death, cleavage of intracellular proteins (i.e., fodrin), and the appearance of typical features of apoptosis such as phosphatidylserine translocation or DNA fragmentation. However, protease inhibitors did not interfere with the initial intracellular Ca2+ concentration increase. We suggest that MPP+ as well as other mitochondrial inhibitors trigger indirect excitotoxic processes, which lead to Ca2+ overload, protease activation, and subsequent neuronal apoptosis.

1-Methyl-4-phenylpyridinium↗
PubMed1998

Attempt to differentiate between individual glutathione peroxidases in biological samples.

Routine determinations of glutathione peroxidases (GPx) still suffer from poor standardization and usually from lack of specification. The different types of glutathione peroxidases present in cellular homogenates may be differentially estimated by their distinct substrate specificities. Complex lipid hydroperoxides required for this approach, however, are not generally available nor easily standardized due to their molecular heterogeneity and tendency to form micelles. We therefore developed a simple procedure for the differential estimation of the major cellular types of GPx, the cytosolic GPx (cGPx) and the phospholipid hydroperoxide glutathione peroxidase (PHGPx) taking advantage of the peculiar susceptibility of PHGPx to deoxycholate. It proved to reliably determine the activities of both purified cGPx and PHGPx, in mixtures thereof, and in homogenates of tissue samples (e.g., testes), and some (e.g. ECV 304) but not all (e.g. THP-1) cultured cell lines. The method allows the differential estimation of cGPx and PHGPx, if the samples do not contain further types of GPx.

Animals↗
PubMed1997

The novel SAR-binding domain of scaffold attachment factor A (SAF-A) is a target in apoptotic nuclear breakdown.

The scaffold attachment factor A (SAF-A) is an abundant component of the nuclear scaffold and of chromatin, and also occurs in heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. Evidence from previous experiments had suggested that SAF-A most likely has at least two different functions, being involved both in nuclear architecture and RNA metabolism. We now show that the protein has a novel scaffold-associated region (SAR)-specific bipartite DNA-binding domain which is independent from the previously identified RNA-binding domain, the RGG box. During apoptosis, but not during necrosis, SAF-A is cleaved in a caspase-dependent way. Cleavage occurs within the bipartite DNA-binding domain, resulting in a loss of DNA-binding activity and a concomitant detachment of SAF-A from nuclear structural sites. On the other hand, cleavage does not compromise the association of SAF-A with hnRNP complexes, indicating that the function of SAF-A in RNA metabolism is not affected in apoptosis. Our results suggest that detachment of SAF-A from SARs, caused by apoptotic proteolysis of its DNA-binding domain, is linked to the formation of oligonucleosomal-sized DNA fragments and could therefore contribute to nuclear breakdown in apoptotic cells.

Amino Acid Sequence↗
PubMed1997

The shape of cell death.

Cell death, a scheduled event during development and tissue turnover, or the ultimate consequence of toxic or pathologic insults seems to involve a relatively limited number of execution pathways. This reflects the evolution of an organized sequence of events perhaps converging onto final common pathways that are used to dispose of unwanted or injured cells. In many cases, the ordered execution of this internal death program leads to typical morphological and biochemical changes that have been termed apoptosis. Apoptosis, often equated with developmental or programmed cell death, has been opposed to unscheduled or accidental cell lysis/necrosis. However, increasing evidence suggests that the two forms of cell demise share similar characteristics, at least in the signaling and early progression phase. Recent studies have shown that, when the intensity of the insult is very high and/or when ATP generation is deficient, cells fail to execute the ordered changes ensuing in apoptosis. Then cell lysis/necrosis supervenes before the processes leading to nuclear condensation and exposure of surface molecules can be completed. Thus, apoptosis and necrosis seem to represent only different shapes of cell demise, resulting from a more or less complete execution of the internal death program.

Animals↗
PubMed1997

Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis.

Apoptosis and necrosis are considered conceptually and morphologically distinct forms of cell death. Here, we report that demise of human T cells caused by two classic apoptotic triggers (staurosporin and CD95 stimulation) changed from apoptosis to necrosis, when cells were preemptied of adenosine triphosphate (ATP). Nuclear condensation and DNA fragmentation did not occur in cells predepleted of ATP and treated with either of the two inducers, although the kinetics of cell death were unchanged. Selective and graded repletion of the extramitochondrial ATP/pool with glucose prevented necrosis and restored the ability of the cells to undergo apoptosis. Pulsed ATP/depletion/repletion experiments also showed that ATP generation either by glycolysis or by mitochondria was required for the active execution of the final phase of apoptosis, which involves nuclear condensation and DNA degradation.

Adenosine Triphosphate↗
PubMed1997

Apoptosis in the absence of poly-(ADP-ribose) polymerase.

Cleavage of poly-(ADP-ribose) polymerase is a process occurring early during the execution phase of apoptosis. Although in many experimental systems PARP cleavage indicates a point of no return, the significance of this proteolytic step for apoptosis remains unclear. Here we compare the susceptibility of cells from wild-type mice and PARP-/- mice to several inducers of apoptosis. Neither the susceptibility of hepatocytes towards CD95 or TNF-mediated apoptosis nor the activation of PARP-cleaving caspases was modified in PARP-/- liver cells. Thymocytes with either genotype exhibited similar sensitivity to treatments with ceramide, dexamethasone, or etoposide. The sensitivity of primary neurons towards apoptosis induced by staurosporine, colchicine, potassium withdrawal, peroxynitrite, or the neurotoxin MPP+ was also unaltered. These data suggest that neither activation nor cleavage of PARP has a causal role in apoptotic cell death of primary, non-transformed cells.

Animals↗
PubMed1997

ICE-protease inhibitors block murine liver injury and apoptosis caused by CD95 or by TNF-alpha.

The two apoptosis receptors of mammalian cells, i.e. the 55 kDa TNF receptor (TNF-R1) and CD95 (Fas/APO1) are activated independently of each other, however, their signaling involves a variety of ICE-related proteases [I]. We used a cell-permeable inhibitor of ICE-like protease activity to examine in vivo whether post-receptor signaling of TNF and CD95 are fully independent processes. Mice pretreated with the inhibitor, Z-VAD-fluoromethylketone (FMK) were dose-dependently protected from liver injury caused by CD95 activation as determined by plasma alanine aminotransferase and also from hepatocyte apoptosis assessed by DNA fragmentation (ID50 = 0.1 mg/kg). A dose of 10 mg/kg protected mice also from liver injury induced by TNF-alpha. Similar results were found when apoptosis was initiated via TNF-alpha or via CD95 in primary murine hepatocytes (IC50 = 1.5 nM) or in various human cell lines. In addition to prevention, an arrest of cell death by Z-VAD-FMK was demonstrated in vivo and in vitro after stimulation of apoptosis receptors. These findings show in vitro and in vivo in mammals that CD95 and the TNF-alpha receptor share a distal proteolytic apoptosis signal.

Alanine Transaminase↗
PubMed1997

Detectable concentrations of Fas ligand in cerebrospinal fluid after severe head injury.

When the cell surface molecule Fas is triggered by its agonist Fas ligand the result is apoptosis of these cells and tissue destruction. To elucidate the pathophysiological relevance of Fas ligand in patients with cerebral oedema caused by trauma, we examined its concentrations in cerebrospinal fluid in 18 patients using specific ELISA. Serum and cerebrospinal fluid from healthy people and injured patients without head trauma did not contain detectable Fas ligand. In contrast, cerebrospinal fluid from patients with severe brain injury contained high concentrations of Fas ligand without detectable concentrations in serum. Soluble Fas ligand concentrations in cerebrospinal fluid correlated significantly with severity of brain injury. The Fas-Fas ligand-system may have a pivotal role in causing oedema and local tissue destruction in the brain after severe head injury.

Adult↗