Etienne Jacotot

Characterization of phenotype markers and neuronotoxic potential of polarised primary microglia in vitro

Highlight ► A unique catalogue of phenotype markers and neuronotoxic effects of polarised primary microglia, as a comparative tool to screen neurotherapies.

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.

Chemosensitization by knockdown of adenine nucleotide translocase-2.

Mitochondrial membrane permeabilization (MMP) is a rate-limiting step of apoptosis, including in anticancer chemotherapy. Adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP on the inner mitochondrial membrane in healthy cells. In addition, ANT can cooperate with Bax to form a lethal pore during apoptosis. Humans possess four distinct ANT isoforms, encoded by four genes, whose transcription depends on the cell type, developmental stage, cell proliferation, and hormone status. Here, we show that the ANT2 gene is up-regulated in several hormone-dependent cancers. Knockdown of ANT2 by RNA interference induced no major changes in the aspect of the mitochondrial network or cell cycle but provoked minor increase in mitochondrial transmembrane potential and reactive oxygen species level and reduced intracellular ATP concentration without affecting glycolysis. At expression and functional levels, ANT2 depletion was not compensated by other ANT isoforms. Most importantly, ANT2, but not ANT1, silencing facilitated MMP induction by lonidamine, a mitochondrion-targeted antitumor compound already used in clinical studies for breast, ovarian, glioma, and lung cancer as well as prostate adenoma. The combination of ANT2 knockdown with lonidamine induced apoptosis irrespective of the Bcl-2 status. These data identify ANT2 as an endogenous inhibitor of MMP and suggest that its selective inhibition could constitute a promising strategy of chemosensitization.

Genetic inhibition of caspase-2 reduces hypoxic-ischemic and excitotoxic neonatal brain injury

Perinatal brain injury is a major cause of neurodevelopmental handicaps. Multiple pathways of oxidant stress, inflammation, and excitotoxicity lead to cell damage and death, including caspase‐dependent apoptosis. Caspase‐2 (Casp2; Nedd‐2, Ich‐1) is a developmentally regulated initiator caspase, which poorly cleaves other caspases but can initiate mitochondrial outer membrane permeabilization. We have investigated if Casp2 could mediate perinatal ischemic brain damage.

Use of Human Cancer Cell Lines Mitochondria to Explore the Mechanisms of BH3 Peptides and ABT-737-Induced Mitochondrial Membrane Permeabilization

Current limitations of chemotherapy include toxicity on healthy tissues and multidrug resistance of malignant cells. A number of recent anti-cancer strategies aim at targeting the mitochondrial apoptotic machinery to induce tumor cell death. In this study, we set up protocols to purify functional mitochondria from various human cell lines to analyze the effect of peptidic and xenobiotic compounds described to harbour either Bcl-2 inhibition properties or toxic effects related to mitochondria. Mitochondrial inner and outer membrane permeabilization were systematically investigated in cancer cell mitochondria versus non-cancerous mitochondria. The truncated (t-) Bid protein, synthetic BH3 peptides from Bim and Bak, and the small molecule ABT-737 induced a tumor-specific and OMP-restricted mitochondrio-toxicity, while compounds like HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37 or EM20-25 did not. We found that ABT-737 can induce the Bax-dependent release of apoptotic proteins (cytochrome c, Smac/Diablo and Omi/HtrA2 but not AIF) from various but not all cancer cell mitochondria. Furthermore, ABT-737 addition to isolated cancer cell mitochondria induced oligomerization of Bax and/or Bak monomers already inserted in the mitochondrial membrane. Finally immunoprecipatations indicated that ABT-737 induces Bax, Bak and Bim desequestration from Bcl-2 and Bcl-xL but not from Mcl-1L. This study investigates for the first time the mechanism of action of ABT-737 as a single agent on isolated cancer cell mitochondria. Hence, this method based on MOMP (mitochondrial outer membrane permeabilization) is an interesting screening tool, tailored for identifying Bcl-2 antagonists with selective toxicity profile against cancer cell mitochondria but devoid of toxicity against healthy mitochondria.

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.

The fourth isoform of the adenine nucleotide translocator inhibits mitochondrial apoptosis in cancer cells

The adenine nucleotide translocator (ANT) is a mitochondrial bi-functional protein, which catalyzes the exchange of ADP and ATP between cytosol and mitochondria and participates in many models of mitochondrial apoptosis. The human adenine nucleotide translocator sub-family is composed of four isoforms, namely ANT1-4, encoded by four nuclear genes, whose expression is highly regulated. Previous studies have revealed that ANT1 and 3 induce mitochondrial apoptosis, whereas ANT2 is anti-apoptotic. However, the role of the recently identified isoform ANT4 in the apoptotic pathway has not yet been elucidated. Here, we investigated the effects of stable heterologous expression of the ANT4 on proliferation, mitochondrial respiration and cell death in human cancer cells, using ANT3 as a control of pro-apoptotic isoform. As expected, ANT3 enhanced mitochondria-mediated apoptosis in response to lonidamine, a mitochondriotoxic chemotherapeutic drug, and staurosporine, a protein kinase inhibitor. Our results also indicate that the pro-apoptotic effect of ANT3 was accompanied by decreased rate of cell proliferation, alteration in the mitochondrial network topology, and decreased reactive oxygen species production. Of note, we demonstrate for the first time that ANT4 enhanced cell growth without impacting mitochondrial network or respiration. Moreover, ANT4 differentially regulated the intracellular levels of hydrogen peroxide without affecting superoxide anion levels. Finally, stable ANT4 overexpression protected cancer cells from lonidamine and staurosporine apoptosis in a manner independent of Bcl-2 expression. These data highlight a hitherto undefined cytoprotective activity of ANT4, and provide a novel dichotomy in the human ANT isoform sub-family with ANT1 and 3 isoforms functioning as pro-apoptotic while ANT2 and 4 isoforms render cells resistant to death inducing stimuli.

β-amyloid induces a dying-back process and remote trans-synaptic alterations in a microfluidic-based reconstructed neuronal network

IntroductionRecent histopathological studies have shown that neurodegenerative processes in Alzheimers and Parkinsons Disease develop along neuronal networks and that hallmarks could propagate trans-synaptically through neuronal pathways. The underlying molecular mechanisms are still unknown, and investigations have been impeded by the complexity of brain connectivity and the need for experimental models allowing a fine manipulation of the local microenvironment at the subcellular level.ResultsIn this study, we have grown primary cortical mouse neurons in microfluidic (μFD) devices to separate soma from axonal projections in fluidically isolated microenvironments, and applied β-amyloid (Aβ) peptides locally to the different cellular compartments. We observed that Aβ application to the somato-dendritic compartment triggers a “dying-back” process, involving caspase and NAD+ signalling pathways, whereas exposure of the axonal/distal compartment to Aβ deposits did not induce axonal degeneration. In contrast, co-treatment with somatic sub-toxic glutamate and axonal Aβ peptide triggered axonal degeneration. To study the consequences of such subcellular/local Aβ stress at the network level we developed new μFD multi-chamber devices containing funnel-shaped micro-channels which force unidirectional axon growth and used them to recreate in vitro an oriented cortico-hippocampal pathway. Aβ application to the cortical somato-dendritic chamber leads to a rapid cortical pre-synaptic loss. This happens concomitantly with a post-synaptic hippocampal tau-phosphorylation which could be prevented by the NMDA-receptor antagonist, MK-801, before any sign of axonal and somato-dendritic cortical alteration.ConclusionThanks to μFD-based reconstructed neuronal networks we evaluated the distant effects of local Aβ stress on neuronal subcompartments and networks. Our data indicates that distant neurotransmission modifications actively take part in the early steps of the abnormal mechanisms leading to pathology progression independently of local Aβ production. This offers new tools to decipher mechanisms underlying Braaks staging. Our data suggests that local Aβ can play a role in remote tauopathy by distant disturbance of neurotransmission, providing a putative mechanism underlying the spatiotemporal appearance of pretangles.

Combined effect of hypothermia and caspase-2 gene deficiency on neonatal hypoxic-ischemic brain injury

Intoduction:Hypoxia–ischemia (HI) injury in term infants develops with a delay during the recovery phase, opening up a therapeutic window after the insult. Hypothermia is currently an established neuroprotective treatment in newborns with neonatal encephalopathy (NE), saving one in nine infants from developing neurological deficits. Caspase-2 is an initiator caspase, a key enzyme in the route to destruction and, therefore, theoretically a potential target for a pharmaceutical strategy to prevent HI brain damage.Methods:The aim of this study was to explore the neuroprotective efficacy of hypothermia in combination with caspase-2 gene deficiency using the neonatal Rice–Vannucci model of HI injury in mice.Results:HI brain injury was moderately reduced in caspase-2−/− mice as compared with wild-type (WT) mice. Five hours of hypothermia (33 °C ) vs. normothermia (36 °C) directly after HI provided additive protection overall (temperature P = 0.0004, caspase-2 genotype P = 0.0029), in the hippocampus and thalamus, but not in other gray matter regions or white matter. Delayed hypothermia initiated 2 h after HI in combination with caspase-2 gene deficiency reduced injury in the hippocampus, but not in other brain areas.Discussion:In conclusion, caspase-2 gene deficiency combined with hypothermia provided enhanced neuroprotection as compared with hypothermia alone.

Isolation of brain mitochondria from neonatal mice.

J. Neurochem. (2011) 10.1111/j.1471‐4159.2011.07525.x