Christophe Bonny

A peptide inhibitor of c-Jun N-terminal kinase protects against excitotoxicity and cerebral ischemia

Neuronal death in cerebral ischemia is largely due to excitotoxic mechanisms, which are known to activate the c-Jun N-terminal kinase (JNK) pathway. We have evaluated the neuroprotective power of a cell-penetrating, protease-resistant peptide that blocks the access of JNK to many of its targets. We obtained strong protection in two models of middle cerebral artery occlusion (MCAO): transient occlusion in adult mice and permanent occlusion in 14-d-old rat pups. In the first model, intraventricular administration as late as 6 h after occlusion reduced the lesion volume by more than 90% for at least 14 d and prevented behavioral consequences. In the second model, systemic delivery reduced the lesion by 78% and 49% at 6 and 12 h after ischemia, respectively. Protection correlated with prevention of an increase in c-Jun activation and c-Fos transcription. In view of its potency and long therapeutic window, this protease-resistant peptide is a promising neuroprotective agent for stroke.

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-α-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.

Fatty Acids Decrease IDX-1 Expression in Rat Pancreatic Islets and Reduce GLUT2, Glucokinase, Insulin, and Somatostatin Levels

IDX-1 (islet/duodenum homeobox-1) is a transcription factor expressed in the duodenum and pancreatic beta and delta cells. It is required for embryonic development of the pancreas and transactivates theGlut2, glucokinase, insulin, and somatostatin genes. Here we show that exposure of isolated rat pancreatic islets to palmitic acid induced a ∼70% decrease in IDX-1 mRNA and protein expression as well as 40 and 65% decreases in the binding activity of IDX-1 for its cognate cis-regulatory elements of theGlut2 and insulin promoters, respectively. The inhibitory effect of palmitic acid required its mitochondrial oxidation since it was prevented by the carnitine palmitoyltransferase I inhibitor bromopalmitic acid. The palmitic acid effect on IDX-1 was correlated with decreases in GLUT2 and glucokinase expression of 40 and 25%, respectively, at both the mRNA and protein levels. Insulin and somatostatin mRNA expression was also decreased by 40 and 60%, whereas glucagon mRNA expression was not modified. After 48 h of exposure to fatty acids, total islet insulin, somatostatin, and glucagon contents were decreased by 85, 55, and 65%, respectively. At the same time, total hormone release was strongly stimulated (13-fold) for glucagon, whereas its was only marginally increased for insulin and somatostatin (1.5- and 1.7-fold, respectively). These results indicate that elevated fatty acid levels 1) negatively regulateIdx-1 expression; 2) decrease the expression of genes transactivated by IDX-1 such as those for GLUT2, glucokinase, insulin, and somatostatin; and 3) lead to an important increase in glucagon synthesis and secretion. Fatty acids thus have pleiotropic effects on pancreatic islet gene expression, and the negative control ofIdx-1 expression may be an initial event in the development of these multiple defects.

The gene MAPK8IP1, encoding islet-brain-1, is a candidate for type 2 diabetes.

Type 2 diabetes is a polygenic and genetically heterogenous disease. The age of onset of the disease is usually late and environmental factors may be required to induce the complete diabetic phenotype. Susceptibility genes for diabetes have not yet been identified. Islet-brain-1 (IB1, encoded by MAPK8IP1), a novel DNA-binding transactivator of the glucose transporter GLUT2 (encoded by SLC2A2), is the homologue of the c-Jun amino-terminal kinase-interacting protein-1 (JIP-1; refs ). We evaluated the role of IB1 in β-cells by expression of a MAPK8IP1 antisense RNA in a stable insulinoma β-cell line. A 38% decrease in IB1 protein content resulted in a 49% and a 41% reduction in SLC2A2 and INS (encoding insulin) mRNA expression, respectively. In addition, we detected MAPK8IP1 transcripts and IB1 protein in human pancreatic islets. These data establish MAPK8IP1 as a candidate gene for human diabetes. Sibpair analyses performed on 149 multiplex French families with type 2 diabetes excluded MAPK8IP1 as a major diabetogenic locus. We did, however, identify in one family a missense mutation located in the coding region of MAPK8IP1 (S59N) that segregated with diabetes. In vitro , this mutation was associated with an inability of IB1 to prevent apoptosis induced by MAPK/ERK kinase kinase 1 (MEKK1) and a reduced ability to counteract the inhibitory action of the activated c-JUN amino-terminal kinase (JNK) pathway on INS transcriptional activity. Identification of this novel non-maturity onset diabetes of the young (MODY) form of diabetes demonstrates that IB1 is a key regulator of β-cell function.

IB1, a JIP-1-related nuclear protein present in insulin-secreting cells.

JIP-1 is a cytoplasmic inhibitor of the c-Jun amino-terminal kinase activated pathway recently cloned from a mouse brain cDNA library. We report herein the expression cloning of a rat cDNA encoding a JIP-1-related nuclear protein from a pancreatic β-cell cDNA library that we named IB1 for Islet-Brain 1. IB1 was isolated by its ability to bind to GTII, a cis-regulatory element of the GLUT2 promoter. The IB1 cDNA encodes a 714-amino acid protein, which differs from JIP-1 by the insertion of 47 amino acids in the carboxyl-terminal part of the protein. The remaining 667 amino acids are 97% identical to JIP-1. The 47-amino acid insertion contains a truncated phosphotyrosine interaction domain and a putative helix-loop-helix motif. Recombinant IB1 (amino acids 1–714 and 280–714) was shown to bind in vitro to GTII. Functionally IB1 transactivated the GLUT2 gene. IB1 was localized within the cytoplasm and the nucleus of insulin-secreting cells or COS-7 cells transfected with an expression vector encoding IB1. Using a heterologous GAL4 system, we localized an activation domain of IB1 within the first 280 amino acids of the protein. These data demonstrate that IB1 is a DNA-binding protein related to JIP-1, which is highly expressed in pancreatic β-cells where it functions as a transactivator of the GLUT2 gene.

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 triphenyltetrazolium 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 mm3 (n= 14) to 85±27 mm3 (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.

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 μM). 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.

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.

Role of the JNK pathway in NMDA-mediated excitotoxicity of cortical neurons

Excitotoxic insults induce c-Jun N-terminal kinase (JNK) activation, which leads to neuronal death and contributes to many neurological conditions such as cerebral ischemia and neurodegenerative disorders. The action of JNK can be inhibited by the D-retro-inverso form of JNK inhibitor peptide (D-JNKI1), which totally prevents death induced by N-methyl-D-aspartate (NMDA) in vitro and strongly protects against different in vivo paradigms of excitotoxicity. To obtain optimal neuroprotection, it is imperative to elucidate the prosurvival action of D-JNKI1 and the death pathways that it inhibits. In cortical neuronal cultures, we first investigate the pathways by which NMDA induces JNK activation and show a rapid and selective phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7), whereas the only other known JNK activator, mitogen-activated protein kinase kinase 4 (MKK4), was unaffected. We then analyze the action of D-JNKI1 on four JNK targets containing a JNK-binding domain: MAPK-activating death domain-containing protein/differentially expressed in normal and neoplastic cells (MADD/DENN), MKK7, MKK4 and JNK-interacting protein-1 (IB1/JIP-1).

Glucose and leptin induce apoptosis in human {beta}-cells and impair glucose-stimulated insulin secretion through activation of c-Jun N-terminal kinases

c‐Jun N‐terminal kinases (SAPK/JNKs) are activated by inflammatory cytokines, and JNK sig naling is involved in insulin resistance and β‐ cell secre tory function and survival. Chronic high glucose con centrations and leptin induce interleukin‐1 Vβ (IL‐1β) secretion from pancreatic islets, an event that is possi bly causal in promoting β‐ cell dysfunction and death. The present study provides evidence that chronically elevated concentrations of leptin and glucose induce P‐cell apoptosis through activation of the JNK pathway in human islets and in insulinoma (INS 832/13) cells. JNK inhibition by the dominant inhibitor JNK‐binding domain of IB1/JIP‐1 (JNKi) reduced JNK activity and apoptosis induced by leptin and glucose. Exposure of human islets to leptin and high glucose concentrations leads to a decrease of glucose‐induced insulin secretion, which was partly restored by JNKi. We detected an interplay between the JNK cascade and the caspase 1/IL‐1 β‐ converting enzyme in human islets. The caspase 1 gene, which contains a potential activating protein‐1 binding site, was up‐regulated in pancreatic sections and in isolated islets from type 2 diabetic patients. Similarly, cultured human islets exposed to high glucose‐ and leptin‐induced caspase 1 and JNK inhibition prevented this up‐regulation. Therefore, JNK inhibition may protect β‐ cells from the deleterious effects of high glucose and leptin in diabetes.— Maedler, K., Schulthess, F. T., Bielman, C., Berney, T., Bonny, C., Prentki, M., Donath, M. Y., Roduit, R. Glucose and leptin induce apoptosis in human β‐ cells and impair glucose‐stimulated insulin secretion through activation of c‐Jun N‐terminal kinases. FASEB J. 22, 1905–1913 (2008)