ABSTRACT
Brain protection of the newborn remains a challenging priority and represents a totally unmet medical need. Pharmacological inhibition of caspases appears as a promising strategy for neuroprotection. In a translational perspective, we have developed a pentapeptide-based group II caspase inhibitor, TRP601/ORPHA133563, which reaches the brain, and inhibits caspases activation, mitochondrial release of cytochrome c, and apoptosis in vivo. Single administration of TRP601 protects newborn rodent brain against excitotoxicity, hypoxia-ischemia, and perinatal arterial stroke with a 6-h therapeutic time window, and has no adverse effects on physiological parameters. Safety pharmacology investigations, and toxicology studies in rodent and canine neonates, suggest that TRP601 is a lead compound for further drug development to treat ischemic brain damage in human newborns.
Subject(s)
Caspase Inhibitors , Cysteine Proteinase Inhibitors/therapeutic use , Hypoxia-Ischemia, Brain/drug therapy , Ischemia/drug therapy , Neuroprotective Agents/therapeutic use , Oligopeptides/therapeutic use , Quinolines/pharmacology , Animals , Animals, Newborn , Apoptosis/drug effects , Binding Sites , Caspases/metabolism , Cysteine Proteinase Inhibitors/chemistry , Cytochromes c/metabolism , Disease Models, Animal , Hypoxia-Ischemia, Brain/pathology , Ischemia/pathology , Mice , Neuroprotective Agents/chemistry , Oligopeptides/chemistry , Oligopeptides/pharmacology , Quinolines/chemistry , RatsSubject(s)
Caspase Inhibitors , Enzyme Inhibitors/pharmacology , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Benzyl Compounds/pharmacology , Cathepsins/antagonists & inhibitors , Humans , Hydrocarbons, Fluorinated/pharmacology , Mice , Neurons/drug effects , Neurons/enzymology , Quinolines/pharmacologyABSTRACT
The HIV-1 encoded apoptogenic protein Vpr induces mitochondrial membrane permeabilization (MMP) via interactions with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocator (ANT). We have designed a peptide, TEAM-VP, composed of two functional domains, one a tumor blood vessel RGD-like 'homing' motif and the other an MMP-inducing sequence derived from Vpr. When added to isolated mitochondria, TEAM-VP interacts with ANT and VDAC, reduces oxygen consumption and overcomes Bcl-2 protection to cause inner and outer MMP. TEAM-VP specifically recognizes cell-surface expressed alpha(V)beta(3) integrins, internalizes, temporarily localizes to lysosomes and progressively co-distributes with the mitochondrial compartment with no sign of lysosomal membrane permeabilization. Finally TEAM-VP reaches mitochondria of angiogenic endothelial cells to induce mitochondrial fission, dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), cytochrome c release and apoptosis hallmarks. Hence, this chimeric peptide constitutes the first example of a virus-derived mitochondriotoxic compound as a candidate to kill selectively tumor neo-endothelia.
Subject(s)
Endothelial Cells/physiology , Gene Products, vpr/pharmacokinetics , Integrin alphaVbeta3/metabolism , Mitochondria/metabolism , Peptides/pharmacokinetics , Amino Acid Sequence , Animals , Apoptosis , Cell Survival , Dose-Response Relationship, Drug , Endothelial Cells/metabolism , Gene Products, vpr/pharmacology , Humans , Lysosomes/metabolism , Mice , Mice, Inbred BALB C , Mitochondrial Membranes/metabolism , Molecular Sequence Data , Peptides/pharmacology , PermeabilityABSTRACT
During development as well as in pathological situations, neurons that fail to find appropriate targets or neurotrophic factors undergo cell death. Using primary cortical neurons subjected to acute serum-deprivation (SD), we have examined caspases activation, mitochondrial dysfunction and cell death parameters. Among a panel of metabolic, signaling and caspases inhibitors only those able to interfere with caspase-2 like activity protect primary neurons against SD-induced cell death. In situ detection and subcellular fractionation demonstrate a very early activation of cytosolic caspase-2, which controls Bax cleavage, relocalization and mitochondrial membrane permeabilization (MMP). Both z-VDVAD-fmk and a siRNA specific for caspase-2 abolish Bax changes, mitochondrial membranes permeabilization, as well as cytochrome c release-dependent activation of caspase-9/caspase-3, nuclear alterations, phosphatidylserine exposure, neurites dismantling and neuronal death. Hence, caspase-2 is an early checkpoint for apoptosis initiation in primary neurons subjected to serum deprivation.
Subject(s)
Apoptosis , Caspase 2/metabolism , Neurons/cytology , Neurons/enzymology , Serum , Animals , Apoptosis/drug effects , Caspase 2/deficiency , Cell Survival/drug effects , Cells, Cultured , Cytochromes c/metabolism , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Humans , Mice , Mitochondria/drug effects , Mitochondria/enzymology , Mitochondria/metabolism , Models, Biological , Neurons/drug effects , Peptides/pharmacology , Protein Transport/drug effects , RNA Interference , bcl-2-Associated X Protein/metabolismABSTRACT
We describe here a cytofluorometric technology for the characterization of decision, execution, and degradation steps of neuronal apoptosis. Multiparametric flow cytometry was developed and combined to detailed fluorescence microscopy observations to establish the chronology and hierarchy of death-related events: neuron morphological changes, mitochondrial transmembrane potential (DeltaPsi(m)) collapse, caspase-3 and -9 activation, phosphatidyl-serine exposure, nuclear dismantling and final plasma membrane permeabilization. Moreover, we developed a reliable real-time flow cytometric monitoring of DeltaPsi(m) and plasma membrane integrity in response to neurotoxic insults including MPTP treatment. Taking advantage of recently developed specific fluorescent probes and a third generation pan-caspase inhibitor, this integrated approach will be pertinent to study the cell biology of neuronal apoptosis and to characterize new neuro-toxic/protective molecules.
