Biomedical subjects

Christophe Bonny

Publications and source records attributed to Christophe Bonny.

At least 19 recordsLinked to original sources

PubMed2006

A peptide inhibitor of c-Jun NH2-terminal kinase reduces myocardial ischemia-reperfusion injury and infarct size in vivo.

The c-Jun NH(2)-terminal kinase (JNK) pathway of the mitogen-activated protein kinase (MAPK) signaling cascade regulates cell function and survival after stress stimulation. Equally robust studies reported dichotomous results suggesting both protective and detrimental effects of JNK during myocardial ischemia-reperfusion (I/R). The lack of a highly specific JNK inhibitor contributed to this controversy. We recently developed a cell-penetrating, protease-resistant peptide inhibitor of JNK, d-JNKI-1. Here we report on the effects of d-JNKI-1 in myocardial I/R. d-JNKI-1 was tested in isolated-perfused adult rat hearts. Increased activation of JNK, p38-MAPK, and extracellular signal-regulated kinase-1/2 (ERK1/2), as assessed by kinase assays and Western blotting, occurred during I/R. d-JNKI-1 delivered before onset of ischemia prevented the increase in JNK activity while not affecting ERK1/2 and p38-MAPK activation. JNK inhibition reduced ischemic injury, as manifested by increased time to contracture (P < 0.05) and decreased left ventricular end-diastolic pressure during ischemia (P < 0.01), and enhanced posthypoxic recovery of systolic and diastolic function (P < 0.01). d-JNKI-1 reduced mitochondrial cytochrome-c release, caspase-3 activation, and the number of apoptotic cells determined by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (P < 0.05), indicating suppression of the mitochondrial machinery of apoptosis. d-JNKI-1 delivered at the time of reperfusion did not improve functional recovery but still prevented apoptosis. In vivo, d-JNKI-1 reduced infarct size after coronary artery occlusion and reperfusion by approximately 50% (P < 0.01). In conclusion, d-JNKI-1 is an important compound that can be used in preclinical models to investigate the role of JNK signaling in vivo. Inhibition of JNK during I/R is cardioprotective in anesthetized rats in vivo.

Animals↗

PubMed2006

Inhibition of the c-Jun N-terminal kinase-mediated mitochondrial cell death pathway restores auditory function in sound-exposed animals.

We tested and characterized the therapeutic value of round window membrane-delivered (RWM) d-JNKI-1 peptide (Bonny et al., 2001) against sound trauma-induced hearing loss. Morphological characteristics of sound-damaged hair cell nuclei labeled by Hoechst staining show that apoptosis is the predominant mode of cell death after sound trauma. Analysis of the events occurring after sound trauma demonstrates that c-Jun N-terminal kinase (JNK)/stress-activated protein kinase activates a mitochondrial cell death pathway (i.e., activation of Bax, release of cytochrome c, activation of procaspases, and cleavage of fodrin). Fluorescein isothiocyanate (FITC)-conjugated d-JNKI-1 peptide applied onto an intact cochlear RWM diffuses through this membrane and penetrates cochlear tissues with the exception of the stria vascularis. A time sequence of fluorescence measurements demonstrates that FITC-labeled d-JNKI-1 remains in cochlear tissues for as long as 3 weeks. In addition to blocking JNK-mediated activation of a mitochondrial cell death pathway, RWM-delivered d-JNKI-1 prevents hair cell death and development of a permanent shift in hearing threshold that is caused by sound trauma in a dose-dependent manner (EC50 = 2.05 microM). The therapeutic window for protection of the cochlea from sound trauma with RWM delivery of d-JNKI-1 extended out to 12 h after sound exposure. These results show that the mitogen-activated protein kinase/JNK signaling pathway plays a crucial role in sound trauma-initiated hair cell death. Blocking this signaling pathway with RWM delivery of d-JNKI-1 may have significant therapeutic value as a therapeutic intervention to protect the human cochlea from the effects of sound trauma.

Animals↗

PubMed2006

Blocking c-Jun-N-terminal kinase signaling can prevent hearing loss induced by both electrode insertion trauma and neomycin ototoxicity.

Neomycin ototoxicity and electrode insertion trauma both involve activation of the mitogen activated protein kinase (MAPK)/c-Jun-N-terminal kinase (JNK) cell death signal cascade. This article discusses mechanisms of cell death on a cell biology level (e.g. necrosis and apoptosis) and proposes the blocking of JNK signaling as a therapeutic approach for preventing the development of a permanent hearing loss that can be initiated by either neomycin ototoxicity or electrode insertion trauma. Blocking of JNK molecules incorporates the use of a peptide inhibitor (i.e. D-JNKI-1), which is specific for all three isoforms of JNK and has been demonstrated to prevent loss of hearing following either electrode insertion trauma or loss of both hearing and hair cells following exposure to an ototoxic level of neomycin. We present previously unpublished results that control for the effect of perfusate washout of aminoglycoside antibiotic by perfusion of the scala tympani with an inactive form of D-JNKI-1 peptide, i.e. JNKI-1(mut) peptide, which was not presented in the original J. Neurosci. article that tested locally delivered D-JNKI-1 peptide against both noise- and neomycin-induced hearing loss (i.e. Wang, J., Van De Water, T.R., Bonny, C., de Ribaupierre, F., Puel, J.L., Zine, A. 2003a. A peptide inhibitor of c-Jun N-terminal kinase protects against both aminoglycoside and acoustic trauma-induced auditory hair cell death and hearing loss. J. Neurosci. 23, 8596-8607). D-JNKI-1 is a cell permeable peptide that blocks JNK signaling at the level of the three JNK molecular isoforms, which when blocked prevents the increases in hearing thresholds and the loss of auditory hair cells. This unique therapeutic approach may have clinical application for preventing: (1) hearing loss caused by neomycin ototoxicity; and (2) the progressive component of electrode insertion trauma-induced hearing loss.

Animals↗

PubMed2006

Homogeneous and nonradioactive high-throughput screening platform for the characterization of kinase inhibitors in cell lysates.

Protein kinases are directly implicated in many human diseases; therefore, kinase inhibitors show great promises as new therapeutic drugs. In an effort to facilitate the screening and the characterization of kinase inhibitors, a novel application of the AlphaScreen technology was developed to monitor JNK activity from (1) purified kinase preparations and (2) endogenous kinase from cell lysates preactivated with different cytokines. The authors confirmed that both adenosine triphosphate (ATP) competitive as well as peptide-based JNK inhibitors were able to block the activity of both recombinant and HepG2 endogenous JNK activity. Using the same luminescence technique adapted for binding studies, the authors characterized peptide inhibitor mechanisms by measuring the binding affinity of the inhibitors for JNK. Because of the versatility of the technology, this cell-based JNK kinase assay could be adapted to other kinases and would represent a powerful tool to evaluate endogenous kinase activity and test a large number of potential inhibitors in a more physiologically relevant environment.

Binding Sites↗

PubMed2006

Brief reoxygenation episodes during chronic hypoxia enhance posthypoxic recovery of LV function: role of mitogen-activated protein kinase signaling pathways.

Children with congenital cyanotic heart defects have worse outcomes after surgical repair of their heart defects compared with noncyanotic ones. Institution of extracorporeal circulation in these children exposes the cyanotic heart to reoxygenation injury. Mitogen-activated protein kinase (MAPK) signaling cascades are major regulators of cardiomyocyte function in acute hypoxia and reoxygenation. However, their roles in chronic hypoxia are incompletely understood. We determined myocardial activation of the three major MAPKs, c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase-1/2 (ERK1/2), and p38-MAPK in adult rats exposed to hypoxia (FIO2=0.10) for varying periods of time. Myocardial function was analyzed in isolated perfused hearts. Acute hypoxia stimulated JNK and p38-MAPK activation. Chronic hypoxia (2 weeks) was associated with increased p38-MAPK (but not JNK) activation, increased apoptosis, and impaired posthypoxic recovery of LV function. Brief normoxic episodes (1 h/day) during chronic hypoxia abolished p38-MAPK activation, stimulated MEK-ERK1/2 activation modestly, and restored posthypoxic LV function. In vivo p38-MAPK inhibition by SB203580 or SB202190 in chronically hypoxic rats restored posthypoxic LV function. These results indicate that sustained hypoxemia maintains p38-MAPK in a chronically activated state that predisposes to myocardial impairment upon reoxygenation. Brief normoxic episodes during chronic hypoxia prevent p38-MAPK activation and restore posthypoxic recovery of myocardial function.

Animals↗

PubMed2006

A peptide c-Jun N-terminal kinase (JNK) inhibitor blocks mechanical allodynia after spinal nerve ligation: respective roles of JNK activation in primary sensory neurons and spinal astrocytes for neuropathic pain development and maintenance.

Optimal management of neuropathic pain is a major clinical challenge. We investigated the involvement of c-Jun N-terminal kinase (JNK) in neuropathic pain produced by spinal nerve ligation (SNL) (L5). SNL induced a slow (>3 d) and persistent (>21 d) activation of JNK, in particular JNK1, in GFAP-expressing astrocytes in the spinal cord. In contrast, p38 mitogen-activated protein kinase activation was found in spinal microglia after SNL, which had fallen to near basal level by 21 d. Intrathecal infusion of a JNK peptide inhibitor, D-JNKI-1, did not affect normal pain responses but potently prevented and reversed SNL-induced mechanical allodynia, a major symptom of neuropathic pain. Intrathecal D-JNKI-1 also suppressed SNL-induced phosphorylation of the JNK substrate, c-Jun, in spinal astrocytes. However, SNL-induced upregulation of GFAP was not attenuated by spinal D-JNKI-1 infusion. Furthermore, SNL induced a rapid (<12 h) but transient activation of JNK in the L5 (injured) but not L4 (intact) DRG. JNK activation in the DRG was mainly found in small-sized C-fiber neurons. Infusion of D-JNKI-1 into the L5 DRG prevented but did not reverse SNL-induced mechanical allodynia. Finally, intrathecal administration of an astroglial toxin, l-alpha-aminoadipate, reversed mechanical allodynia. Our data suggest that JNK activation in the DRG and spinal cord play distinct roles in regulating the development and maintenance of neuropathic pain, respectively, and that spinal astrocytes contribute importantly to the persistence of mechanical allodynia. Targeting the JNK pathway in spinal astroglia may present a new and efficient way to treat neuropathic pain symptoms.

Animals↗

PubMed2006

A unique set of SH3-SH3 interactions controls IB1 homodimerization.

Islet-brain 1 (IB1 or JIP-1) is a scaffold protein that interacts with components of the c-Jun N-terminal kinase (JNK) signal-transduction pathway. IB1 is expressed at high levels in neurons and in pancreatic beta-cells, where it controls expression of several insulin-secretory components and secretion. IB1 has been shown to homodimerize, but neither the molecular mechanisms nor the function of dimerization have yet been characterized. Here, we show that IB1 homodimerizes through a novel and unique set of Src homology 3 (SH3)-SH3 interactions. X-ray crystallography studies show that the dimer interface covers a region usually engaged in PxxP-mediated ligand recognition, even though the IB1 SH3 domain lacks this motif. The highly stable IB1 homodimer can be significantly destabilized in vitro by three individual point mutations directed against key residues involved in dimerization. Each mutation reduces IB1-dependent basal JNK activity in 293T cells. Impaired dimerization also results in a reduction in glucose transporter type 2 expression and in glucose-dependent insulin secretion in pancreatic beta-cells. Taken together, these results indicate that IB1 homodimerization through its SH3 domain has pleiotropic effects including regulation of the insulin secretion process.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Blocking stress signaling pathways with cell permeable peptides.

Cells are continuously adapting to changes in their environment by activating extracellular stimuli-dependent signal transduction cascades. These cascades, or signaling pathways, culminate both in changes in genes expression and in the functional regulation of pre-existing proteins. The Mitogen-Activated Protein Kinases (MAPKs) constitute a structurally related class of signaling proteins whose distinctive feature is their ability to directly phosphorylate, and thereby modulate, the activity of the transcription factors that are targets of the initial stimuli. The specificity of activation of MAPK signaling modules is determined, at least for an important part, by the specificity of the protein-protein contacts that are required for the propagation of the signal. We will discuss how we may interfere with MAPK signaling by using short cell-permeable peptides able to block, through a competitive mechanisms, relevant protein-protein contacts, and their effects on signaling and cell function.

Animals↗

PubMed2006

D-JNKI-1 treatment prevents the progression of hearing loss in a model of cochlear implantation trauma.

HYPOTHESES: 1) Hearing loss caused by electrode insertion trauma has both acute and delayed components; and 2) the delayed component of trauma-initiated hearing loss can be prevented by a direct delivery of a peptide inhibitor of the c-Jun N-terminal kinase cell death signal cascade, that is, D-JNKI-1, immediately after the electrode insertion within the cochlea. BACKGROUND: Acute trauma to the macroscopic elements of the cochlea from electrode insertion is well known. The impact of trauma-induced oxidative stress within injured cochlear tissues and the efficacy of drugs (e.g., D-JNKI-1) to prevent apoptosis of damaged hair cells is not well defined. METHODS: Hearing function was tested by pure-tone evoked auditory brainstem responses (ABRs) and distortion products of otoacoustic emissions (DPOAEs). D-JNKI-1 in artificial perilymph (AP) or AP alone was delivered into the scala tympani immediately after electrode trauma and for 7 days. Controls were nontreated contralateral and D-JNKI-1-treated ears without electrode insertion trauma. RESULTS: There was no increase in the hearing thresholds of either the contralateral control ears or in the D-JNKI-1 without trauma animals. There was a progressive increase in ABR thresholds and decrease in DPOAE amplitudes after electrode insertion trauma in untreated and in AP-treated cochleae. Treatment with D-JNKI-1 prevented the progressive increase in ABR thresholds and decrease in DPOAE amplitudes that occur after electrode insertion trauma. CONCLUSION: Hearing loss caused by cochlear implant electrode insertion trauma in guinea pigs has both acute and delayed components. The delayed component can be prevented by treating the cochlea with D-JNKI-1.

Analysis of Variance↗

PubMed2005

[Role of intracellular signalling pathways in the development of type 1 diabetes].

Type I diabetes develops following an attack of the immune system that destroys the cells secreting insulin. Death of these cells however requires many intracellular mechanisms that are dependent of the insulin-secreting cells themselves. The pivotal role that the insulin-secreting cells play in their own destruction starts to be uncovered, and these new mechanisms are the target for a whole battery of future drugs.

Animals↗

PubMed2005

D-TAT transporter as an ocular peptide delivery system.

BACKGROUND: Future treatment for genetic diseases may involve the replacement of malfunctioning genes through virus-mediated gene therapy. However, this approach is plagued with many problems, both ethical and scientific. Therefore, alternative treatments based on new molecules may represent a safer option. Molecular treatment of many eye diseases will need to bring active molecules into the photoreceptors. Recently, the trans-activator protein (TAT) human immunodeficiency virus type 1 (HIV-1) transcriptional factor has proven to be effective in transporting molecules across cellular membranes. The half-life of these molecules does not exceed 48 hours. The potential use of the retro-inverso form of the TAT (D-TAT) peptide, the protein transducing domain of the HIV-1 transcriptional factor, as a molecular transporter was investigated. METHODS: FITC-labelled D-TAT (D-TAT FITC) was applied to the 661W murine photoreceptor cell line in culture. The labelled peptide was also injected into the vitreous body or the subretinal space of adult mice. Cells and cryosections of eyes were analysed under fluorescence microscopy at various time points after peptide treatment. Coimmunostaining with various antibodies was performed in order to characterize the transduces cells. RESULTS: D-TAT was effective in transducing photoreceptor cells in culture. Transduction of D-TAT FITC was also effective when injected into the vitreous or subretinal space and was observed for a longer period of time than L-TAT FITC. CONCLUSIONS: The retro-inverso form of the TAT sequence is effective in transducing cells from various compartments of the eye. After 14 days, the D-TAT FITC was clearly visible in the retina whereas L-TAT FITC had almost disappeared. The D-TAT peptide represents an interesting molecular transporter that, when coupled to a specific effector, may have potential therapeutic future, especially when a long-lasting action is needed.

Animals↗

PubMed2005

Targeting the JNK pathway as a therapeutic protective strategy for nervous system diseases.

The c-Jun N-terminal kinases (JNKs) are members of the family of mitogen activated protein kinases (MAPKs). While the functions of the JNKs under physiological conditions are diverse and not completely understood, there is increasing evidence that JNKs are potent effectors of apoptosis in both the brain and the mammalian inner ear following a variety of injuries. The activation of the inducible transcription factor c-Jun by N-terminal phosphorylation is a central event in JNK-mediated neural and inner ear hair cell death. A cell permeable peptide designed specifically to inhibit JNK signaling has proven successful in in vivo models of both neuronal degeneration following cerebral ischemia and auditory hair cell degeneration following exposure to either acoustic trauma or a toxic level of an aminoglycoside antibiotic. Here we discuss the evidence supporting the application of JNK inhibitors to prevent cellular degeneration in several central nervous system (CNS) and peripheral nervous system (PNS) diseases with an emphasis on traumatic ischemic damage to the CNS and acquired deafness in the PNS receptors.

Animals↗

PubMed2004

Antitumorigenic effect of proteasome inhibitors on insulinoma cells.

Malignant insulinoma is a critical cancer form with a poor prognosis. Because cure by surgery is infrequent, effective chemotherapy is in demand. Induction of cell death in tumor cells by proteasome inhibitors is emerging as a potential strategy in cancer therapy. Here we investigated whether inhibition of the proteasome has an antitumorigenic potential in insulinoma cells. Exposure of mouse betaTC3 insulinoma cells to the proteasome inhibitor N-Acetyl-Leu-Leu-Nle-CHO (ALLN) reduced cell viability, activated caspase-3, induced apoptosis, and suppressed insulin release. Treatment with ALLN also resulted in phosphorylation of c-jun N-terminal kinase (JNK) and an increase in in vitro phosphorylation of c-jun. In insulinoma cells with impaired JNK signaling, ALLN-induced apoptosis was significantly suppressed. Another proteasome inhibitor, lactacystin, also stimulated JNK activation, caused activation of caspase-3, suppressed cell viability, and induced apoptosis in betaTC3 and rat INS-1E cells. Both ALLN and lactacystin caused a marked decrease in the cellular amount of the JNK scaffold protein JNK-interacting protein 1/islet-brain-1. In primary pancreatic rat islet cells, proteasome inhibition reduced insulin secretion but had no impact on cell viability and even partially protected against the toxic effect of proinflammatory cytokines. Our findings demonstrate that proteasome inhibitors possess antitumorigenic and antiinsulinogenic effects on insulinoma cells.

Acetylcysteine↗

PubMed2004

A RasGAP-derived cell permeable peptide potently enhances genotoxin-induced cytotoxicity in tumor cells.

Treatment of many cancers relies on the combined action of several genotoxins, but the detrimental effect of these drugs on normal cells can cause severe side effects. One major challenge in anticancer therapy is therefore to increase the selectivity of current treatments toward cancer cells in order to spare normal cells. We have recently demonstrated that a RasGAP caspase cleavage fragment is able to sensitize HeLa cells towards cisplatin-induced apoptosis. Here, we extend this observation by showing that this fragment also enhances cell death induced by adriamycin and mitoxantrone, two other widely used genotoxins. Furthermore, we have delineated a short sequence within this fragment that still bears the genotoxin-sensitization property. The peptide encoded by this sequence, when fused to the TAT cell permeation sequence, potently sensitized a number of tumors cells, but not normal cells, towards apoptosis induced by cisplatin, adriamycin and mitoxantrone. This sensitization effect was not mediated through modulation of NFkappaB activity or activation of the JNK and p38 MAPK pathways. Our results demonstrate the feasibility in enhancing the efficacy of currently used drugs to selectively kill cancer cells using peptides derived from pro-apoptotic caspase substrate fragments.

Apoptosis↗

PubMed2004

D-JNKI1, a cell-penetrating c-Jun-N-terminal kinase inhibitor, protects against cell death in severe cerebral ischemia.

BACKGROUND AND PURPOSE: In 2 models of severe ischemic injury, we have evaluated the neuroprotective action of D-JNKI1, a cell-penetrating and protease-resistant peptide selectively inhibiting the c-Jun-N-terminal kinase (JNK). METHODS: Hippocampal slices from newborn rats were subjected to oxygen (5%) and glucose (1 mmol/L) deprivation for 30 minutes. Cell death was evaluated with propidium iodide, and the evoked potential responses were recorded in the CA1 region after stimulation in CA3. Male ICR-CD1 mice were subjected to permanent endoluminal "suture" middle cerebral artery occlusion (MCAo). The lesion size was determined after 24 hours by triphenyl-tetrazolium chloride staining, and neurological scores and rotarod treadmill performance were used to evaluate the neurological outcome. RESULTS: In vitro, D-JNKI administration 6 hours after oxygen glucose deprivation reduced cell death at 24 hours from 21%+/-8% (n=10) to 5%+/-3% (n=7, P<0.01). This protective effect was still seen at 48 hours, paralleled by an improved amplitude of the evoked potential response. In vivo in the mouse, D-JNKI1 administration 3 hours after ischemia significantly reduced the infarct volume from 162+/-27 mm(3) (n=14) to 85+/-27 mm(3) (n=9, P<0.001). The functional outcome was also improved. CONCLUSIONS: JNK inhibition prevents cell death induced by oxygen and glucose deprivation in hippocampal slice cultures in vitro and by permanent suture MCAo in vivo. D-JNKI1 is a powerful neuroprotectant in models of both mild and severe cerebral ischemia, with an extended therapeutic window. Further investigations are needed to identify the relevant JNK target(s) mediating ischemic neuronal death.

Animals↗

PubMed2004

Profiling treatment-specific post-translational modifications in a complex proteome with subtractive substrate phage display.

Proteolytic activation of zymogens or controlled degradation of inhibitory factors is part of a major regulatory system on the post-translational level to regulate treatment induced cellular stress responses. The identification of differential activity based substrates is thus of high interest to prioritize and validate candidate targets for drug discovery. Here we present a novel subtractive substrate phage display screening method for the selection of treatment induced post-translational peptide modifications in complex proteomes. We investigated this approach with tumor cells in response to a protease activating anticancer treatment modality using subtractive and iterative screening of cellular extracts derived from control and treated cells. Specific phage were identified that served as substrates for proteolytic activities in response to treatment related activity changes and could be distinguished from substrates for unspecific proteolytic background activities. Novel, selected peptide substrates were investigated in vitro and in vivo and showed high substrate specificity and functional biological significance.

Amino Acid Sequence↗

PubMed2004

Use of cell-permeable peptides to prevent neuronal degeneration.

The loss of neurons is a hallmark of neurodegenerative disorders and evidence suggests that this occurs through an apoptotic mechanism. Following an insult, neuronal cells activate signal transduction pathways that lead to cell death and the establishment of the pathological state. The mechanisms underlying the cell-death response involve protein kinases, which phosphorylate many substrates and culminate in changes in gene expression. Traditionally, attempts at blocking such signaling targeted the phosphorylation of the substrates. However, preventing the interaction between two proteins using specific peptides might block the function of key mediators in signaling cascades. A cell-permeable peptide designed specifically to inhibit c-Jun N-terminal kinase action proved successful in in vivo models of neuronal degeneration following ischemia. Here, the recent findings that highlight the potential of this approach for therapeutic application are reviewed.

Animals↗

PubMed2004

Intracellular stress signaling pathways activated during human islet preparation and following acute cytokine exposure.

Pancreatic islet transplantation may successfully restore normoglycemia in type 1 diabetic patients. However, successful grafting requires transplantation of a sufficient number of islets, usually requiring two or more donors. During the isolation process and following clinical transplantation, islets are subjected to severe adverse conditions that impair survival and ultimately contribute to graft failure. Here, we have mapped the major intracellular stress-signaling pathways that may mediate human islet loss during isolation and following cytokine attack. We found that the isolation procedure potently recruits two pathways consisting of |mitogen-activated protein kinase kinase (MKK)7 --> Jun NH(2)-terminal kinase (JNK)/p38 --> c-fos| and the |nuclear factor-kappaB (NF-kappaB) --> iNOS| module. Cytokines activate the |NF-kappaB --> iNOS| and |MKK4/MKK3/6 --> JNK/p38| pathways without recruitment of c-fos. Culturing the islets for 48 h after isolation allows for the activated pathways to return to background levels, with expression of MKK7 becoming undetectable. These data indicate that isolation and cytokines recruit different death pathways. Therefore, strategies might be rationally developed to avoid possible synergistic activation of these pathways in mediating islet loss during isolation and following grafting.

Apoptosis↗