tacrolimus and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

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.. 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.. 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.. 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.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Carrier Proteins; Caspase 3; Caspase Inhibitors; Caspases; Cerebellum; Colchicine; Cyclosporine; Cysteine Proteinase Inhibitors; Deoxyglucose; Dizocilpine Maleate; Energy Metabolism; Enzyme Activation; Enzyme Inhibitors; Glutathione; Mice; Mice, Inbred BALB C; Microfilament Proteins; Microtubules; Neurons; Neuroprotective Agents; Nitroso Compounds; Nocodazole; Oligopeptides; Paclitaxel; Potassium; Protein Biosynthesis; S-Nitrosoglutathione; Tacrolimus; Verapamil

Programmed cell death in animals is usually associated with apoptotic morphology and requires caspase activation. Necrosis and caspase-independent cell death have been reported, but mostly in experimental conditions that lead some to question their existence it in vivo. Loss of interdigital cells in the mouse embryo, a paradigm of cell death during development [1], is known to include an apoptotic [2] and caspase-dependent [3] [4] mechanism. Here, we report that, when caspase activity was inhibited using drugs or when apoptosis was prevented genetically (using Hammertoe mutant mice, or mice homozygous for a mutation in the gene encoding APAF-1, a caspase-activating adaptor protein), interdigital cell death still occurred. This cell death was negative for the terminal-deoxynucleotidyl-mediated dUTP nick end-labelling (TUNEL) assay and there was no overall cell condensation. At the electron microscopy level, peculiar 'mottled' chromatin alterations and marked mitochondrial and membrane lesions, suggestive of classical necrotic cell death, were observed with no detectable phagocytosis and no local inflammatory response. Thus, in this developmental context, although caspase activity confers cell death with an apoptotic morphotype, in the absence of caspase activity an underlying mechanism independent of known caspases can also confer cell death, but with a necrotic morphotype. This cell death can go undetected when using apoptosis-specific methodology, and cannot be blocked by agents that act on caspases.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptotic Protease-Activating Factor 1; Bone Morphogenetic Proteins; Caspase Inhibitors; Caspases; Chromatin; Cysteine Proteinase Inhibitors; Embryonic and Fetal Development; Fetal Proteins; Hindlimb; In Situ Nick-End Labeling; Mice; Mice, Knockout; Mice, Mutant Strains; Morphogenesis; Necrosis; Organelles; Proteins; Receptors, Growth Factor; Signal Transduction; Tacrolimus

Expression of the pro-apoptotic molecule BAX has been shown to induce cell death. While BAX forms both homo- and heterodimers, questions remain concerning its native conformation in vivo and which moiety is functionally active. Here we demonstrate that a physiologic death stimulus, the withdrawal of interleukin-3 (IL-3), resulted in the translocation of monomeric BAX from the cytosol to the mitochondria where it could be cross-linked as a BAX homodimer. In contrast, cells protected by BCL-2 demonstrated a block in this process in that BAX did not redistribute or homodimerize in response to a death signal. To test the functional consequence of BAX dimerization, we expressed a chimeric FKBP-BAX molecule. Enforced dimerization of FKBP-BAX by the bivalent ligand FK1012 resulted in its translocation to mitochondria and induced apoptosis. Caspases were activated yet caspase inhibitors did not block death; cytochrome c was not released detectably despite the induction of mitochondrial dysfunction. Moreover, enforced dimerization of BAX overrode the protection by BCL-XL and IL-3 to kill cells. These data support a model in which a death signal results in the activation of BAX. This conformational change in BAX manifests in its translocation, mitochondrial membrane insertion and homodimerization, and a program of mitochondrial dysfunction that results in cell death.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Caspase 3; Caspase 9; Caspases; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytosol; Dimerization; Enzyme Activation; Humans; Interleukin-3; Ligands; Membrane Potentials; Mice; Mitochondria; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Recombinant Fusion Proteins; Tacrolimus