David Chauvier

Specific caspase inhibitor Q-VD-OPh prevents neonatal stroke in P7 rat: a role for gender.

Hypoxia–ischaemia in the developing brain results in brain injury with prominent features of apoptosis. In the present study, a third generation dipeptidyl broad‐spectrum caspase inhibitor, quinoline‐Val‐Asp(Ome)‐CH2‐O‐phenoxy (Q‐VD‐OPh), was tested in a model of unilateral focal ischaemia with reperfusion in 7‐day‐old rats. Q‐VD‐OPh (1 mg/kg, i.p.) reduced cell death, resulting in significant neuroprotection at 48 h of recovery (infarct volume of 12.6 ± 2.8 vs. 24.3 ± 2.2%, p = 0.006). The neuroprotective effects observed at 48 h post‐ischaemia hold up at 21 days of survival time and attenuate neurological dysfunction. Analysis by gender revealed that females were strongly protected (6.7 ± 3.3%, p = 0.006), in contrast to males in which there was no significant effect, when Q‐VD‐OPh was given after clip removal on the left common carotid artery. Immunoblot analysis demonstrated that Q‐VD‐OPh inhibits caspase 3 cleavage into its p17 active form and caspase 1 up‐regulation and cleavage in vivo. Following ischaemia in P7 rats, males and females displayed different time course and pattern of cytochrome c release and active p17 caspase 3 during the first 24 h of recovery. In contrast, no significant difference was observed for caspase 1 expression between genders. These results indicate that ischaemia activates caspases shortly after reperfusion and that the sex of the animal may strongly influences apoptotic pathways in the pathogenesis of neonatal brain injury. The specificity, effectiveness, and reduced toxicity of Q‐VD‐OPh may determine the potential use of peptide‐derived irreversible caspase inhibitors as promising therapeutics.

Ryanodine receptor leak mediated by caspase-8 activation leads to left ventricular injury after myocardial ischemia-reperfusion

Myocardial ischemic disease is the major cause of death worldwide. After myocardial infarction, reperfusion of infracted heart has been an important objective of strategies to improve outcomes. However, cardiac ischemia/reperfusion (I/R) is characterized by inflammation, arrhythmias, cardiomyocyte damage, and, at the cellular level, disturbance in Ca2+ and redox homeostasis. In this study, we sought to determine how acute inflammatory response contributes to reperfusion injury and Ca2+ homeostasis disturbance after acute ischemia. Using a rat model of I/R, we show that circulating levels of TNF-α and cardiac caspase-8 activity were increased within 6 h of reperfusion, leading to myocardial nitric oxide and mitochondrial ROS production. At 1 and 15 d after reperfusion, caspase-8 activation resulted in S-nitrosylation of the RyR2 and depletion of calstabin2 from the RyR2 complex, resulting in diastolic sarcoplasmic reticulum (SR) Ca2+ leak. Pharmacological inhibition of caspase-8 before reperfusion with Q-LETD-OPh or prevention of calstabin2 depletion from the RyR2 complex with the Ca2+ channel stabilizer S107 (“rycal”) inhibited the SR Ca2+ leak, reduced ventricular arrhythmias, infarct size, and left ventricular remodeling after 15 d of reperfusion. TNF-α–induced caspase-8 activation leads to leaky RyR2 channels that contribute to myocardial remodeling after I/R. Thus, early prevention of SR Ca2+ leak trough normalization of RyR2 function is cardioprotective.

Targeting neonatal ischemic brain injury with a pentapeptide-based irreversible caspase inhibitor

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.

A critical role for Fas/CD‐95 dependent signaling pathways in the pathogenesis of hyperoxia‐induced brain injury

Prematurely born infants are at risk for development of neurocognitive impairment in later life. Oxygen treatment has been recently identified as a trigger of neuronal and oligodendrocyte apoptosis in the developing rodent brain. We investigated the role of the Fas death receptor pathway in oxygen‐triggered developmental brain injury.

Predicting sensorimotor and memory deficits after neonatal ischemic stroke with reperfusion in the rat

Among experimental models of perinatal ischemic stroke, Renolleaus model mimics selected types of stroke at birth, including ischemia and reperfusion. However, its behavioural consequences on development have been poorly described. Here, ischemia-reperfusion was performed in 7-day-old Wistar rats. Between the ages of 9 and 40 days, sensorimotor and memory functions were assessed. The infarcted area was analysed by immunohistochemistry at 40 days of age. The remaining lesion was in the parietal cortex, in the form of a cone-shaped area. This area contained glial cells but neither neurons nor macrophages. Transient focal neonatal ischemia led to sensorimotor alterations in early adulthood, such as postural asymmetry, motor coordination and somatosensory deficits, and hyperactivity, as well as cognitive impairments, such as spatial reference memory deficits. Based on these results, we propose here a selection of behavioural tests that should constitute meaningful tools for assessing sensory and cognitive functions after experimental neonatal ischemic stroke.

A flavivirus protein M-derived peptide directly permeabilizes mitochondrial membranes, triggers cell death and reduces human tumor growth in nude mice

Dengue viruses belong to the Flavivirus family and are responsible for hemorrhagic fever in Human. Dengue virus infection triggers apoptosis especially through the expression of the small membrane (M) protein. Using isolated mitochondria, we found that synthetic peptides containing the C-terminus part of the M ectodomain caused apoptosis-related mitochondrial membrane permeabilization (MMP) events. These events include matrix swelling and the dissipation of the mitochondrial transmembrane potential (ΔΨm). Protein M Flavivirus sequence alignments and helical wheel projections reveal a conserved distribution of charged residues. Moreover, when combined to the cell penetrating HIV-1 Tat peptide transduction domain (Tat-PTD), this sequence triggers a caspase-dependent cell death associated with ΔΨm loss and cytochrome c release. Mutational approaches coupled to functional screening on isolated mitochondria resulted in the selection of a protein M derived sequence containing nine residues with potent MMP-inducing properties on isolated mitochondria. A chimeric peptide composed of a Tat-PTD linked to the 9-mer entity triggers MMP and cell death. Finally, local administration of this chimeric peptide induces growth inhibition of xenograft prostate PC3 tumors in immuno-compromised mice, and significantly enhances animal survival. Together, these findings support the notion of using viral genomes as valuable sources to discover mitochondria-targeted sequences that may lead to the development of new anticancer compounds.