Tiziana Borsello

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.

Synthetic amyloid-β oligomers impair long-term memory independently of cellular prion protein

Inability to form new memories is an early clinical sign of Alzheimer’s disease (AD). There is ample evidence that the amyloid-β (Aβ) peptide plays a key role in the pathogenesis of this disorder. Soluble, bio-derived oligomers of Aβ are proposed as the key mediators of synaptic and cognitive dysfunction, but more tractable models of Aβ−mediated cognitive impairment are needed. Here we report that, in mice, acute intracerebroventricular injections of synthetic Aβ1–42 oligomers impaired consolidation of the long-term recognition memory, whereas mature Aβ1–42 fibrils and freshly dissolved peptide did not. The deficit induced by oligomers was reversible and was prevented by an anti-Aβ antibody. It has been suggested that the cellular prion protein (PrPC) mediates the impairment of synaptic plasticity induced by Aβ. We confirmed that Aβ1–42 oligomers interact with PrPC, with nanomolar affinity. However, PrP-expressing and PrP knock-out mice were equally susceptible to this impairment. These data suggest that Aβ1–42 oligomers are responsible for cognitive impairment in AD and that PrPC is not required.

JNK Signalling: A Possible Target to Prevent Neurodegeneration

The c-Jun N-terminal kinases (JNK) belong to the subfamily of mitogen-activated protein kinase (MAPK). JNK is an important transducing enzyme that is involved in many facets of cellular regulation including gene expression, cell proliferation and programmed cell death. The activation of JNK pathways is critical for naturally occurring cell death during development as well as for pathological death associated with neurodegenerative diseases. Initial research concentrated on defining the components and organization of JNK signalling cascades, but more recent studies see JNK as a target to prevent cell death. Several in vitro and in vivo studies have reported alterations of JNK pathways potentially associated with neuronal death in Parkinsons and Alzheimers disease. So efforts are now aimed at developing chemical inhibitors of this pathway. These have proved effective in vivo, reducing brain damage and some of the symptoms of arthritis in animal models. An alternative cell penetrating peptide approach is now available, with the identification of the JNK permeable peptide inhibitor, which modifies JNK action rather than activation, preventing neuronal death with unprecedented specificity and efficacy in several experimental conditions, including two animal models of ischemia. In this review we examine in detail the role of JNK in neurodegeneration, particularly in Alzheimers and Parkinsons disease. The possibility of intervention on the JNK pathway as a therapeutic approach is also illustrated.

N‐methyl‐d‐aspartate‐triggered neuronal death in organotypic hippocampal cultures is endocytic, autophagic and mediated by the c‐Jun N‐terminal kinase pathway

Acute excitotoxic neuronal death was studied in rat organotypic hippocampal slices exposed to 100u2003µmN‐methyl‐d‐aspartate. Fulgurant death of pyramidal neurons occurred in the CA1 and CA3 regions and was already detectable within 2u2003h of the N‐methyl‐d‐aspartate administration. Morphologically, the neuronal death was neither apoptotic nor necrotic but had the hallmarks of autophagic neuronal death, as shown by acid phosphatase histochemistry in both CA1 and CA3 and by electron microscopy in CA1. The dying neurons also manifested strong endocytosis of horseradish peroxidase or microperoxidase, occurring probably by a fluid phase mechanism, and followed, surprisingly, by nuclear entry. In addition to these autophagic and endocytic characteristics, there were indications that the c‐Jun N‐terminal kinase pathway was activated. Its target c‐Jun was selectively phosphorylated in CA1, CA3 and the dentate gyrus and c‐Fos, the transcription of which is under the positive control of c‐Jun N‐terminal kinase target Elk1, was selectively up‐regulated in CA1 and CA3. All these effects, the neuronal death itself and the associated autophagy and endocytosis, were totally prevented by a cell‐permeable inhibitor of the interaction between c‐Jun N‐terminal kinase and certain of its targets. These results show that pyramidal neurons undergoing excitotoxic death in this situation are autophagic and endocytic and that both the cell death and the associated autophagy and endocytosis are under the control of the c‐Jun N‐terminal kinase pathway.

An N-terminal Fragment of the Prion Protein Binds to Amyloid-β Oligomers and Inhibits Their Neurotoxicity in Vivo

Background: The cellular prion protein (PrPC) could be a toxicity-transducing receptor for amyloid-β (Aβ) oligomers. Results: N1, a naturally occurring fragment of PrPC, binds Aβ oligomers, inhibits their polymerization into fibrils, and suppresses their neurotoxic effects in vitro and in vivo. Conclusion: N1 binds tightly to Aβ oligomers and blocks their neurotoxicity. Significance: Administration of exogenous N1 or related peptides may represent an effective therapy for Alzheimer disease. A hallmark of Alzheimer disease (AD) is the accumulation of the amyloid-β (Aβ) peptide in the brain. Considerable evidence suggests that soluble Aβ oligomers are responsible for the synaptic dysfunction and cognitive deficit observed in AD. However, the mechanism by which these oligomers exert their neurotoxic effect remains unknown. Recently, it was reported that Aβ oligomers bind to the cellular prion protein with high affinity. Here, we show that N1, the main physiological cleavage fragment of the cellular prion protein, is necessary and sufficient for binding early oligomeric intermediates during Aβ polymerization into amyloid fibrils. The ability of N1 to bind Aβ oligomers is influenced by positively charged residues in two sites (positions 23–31 and 95–105) and is dependent on the length of the sequence between them. Importantly, we also show that N1 strongly suppresses Aβ oligomer toxicity in cultured murine hippocampal neurons, in a Caenorhabditis elegans-based assay, and in vivo in a mouse model of Aβ-induced memory dysfunction. These data suggest that N1, or small peptides derived from it, could be potent inhibitors of Aβ oligomer toxicity and represent an entirely new class of therapeutic agents for AD.

JNK regulates APP cleavage and degradation in a model of Alzheimer's disease

Secretion of Amyloid-beta peptide (Abeta) circulating oligomers and their aggregate forms derived by processing of beta-amyloid precursor protein (APP) are a key event in Alzheimers disease (AD). We show that phosphorylation of APP on threonine 668 may play a role in APP metabolism in H4-APP(sw) cell line, a degenerative AD model. We proved that JNK plays a fundamental role in this phosphorylation since its specific inhibition, with the JNK inhibitor peptide (D-JNKI1), induced APP degradation and prevented APP phosphorylation at T668. This results in a significant drop of betaAPPs, Abeta fragments and Abeta circulating oligomers. Moreover the D-JNKI1 treatment produced a switch in the APP metabolism, since the peptide reduced the rate of the amyloidogenic processing in favour of the non-amyloidogenic one. All together our results suggest an important link between APP metabolism and the JNK pathway and contribute to shed light on the molecular signalling pathway of this disease indicating JNK as an innovative target for AD therapy.

Time-course of c-Jun N-terminal kinase activation after cerebral ischemia and effect of D-JNKI1 on c-Jun and caspase-3 activation

The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. The d-retro-inverso form of c-Jun N-terminal kinase-inhibitor (D-JNKI1), a cell-permeable inhibitor of JNK, powerfully reduces neuronal death induced by permanent and transient ischemia, even when administered 6 h after the ischemic insult, offering a clinically relevant window. We investigated the JNK molecular cascade activation in rat cerebral ischemia and the effects of D-JNKI1 on this cascade. c-Jun activation starts after 3 h after ischemia and peaks at 6 h in the ischemic core and in the penumbra at 1 h and at 6 h respectively. The 6 h c-Jun activation peak correlates well with that of P-JNK. We also examined the activation of the two direct JNK activators, MAP kinase kinase 4 (MKK4) and MAP kinase kinase 7 (MKK7). MKK4 showed the same time course as JNK in both core and penumbra, reaching peak activation at 6 h. MKK7 did not show any significant increase of phosphorylation in either core or penumbra. D-JNKI1 markedly prevented the increase of P-c-Jun in both core and penumbra and powerfully inhibited caspase-3 activation in the core. These results confirm that targeting the JNK cascade using the TAT cell-penetrating peptide offers a promising therapeutic approach for ischemia, raising hopes for human neuroprotection, and elucidates the molecular pathways leading to and following JNK activation.

Blocking ADAM10 synaptic trafficking generates a model of sporadic Alzheimer’s disease

We describe here an innovative, non-transgenic animal model of Alzheimers disease. This model mimics early stages of sporadic disease, which represents the vast majority of cases. The model was obtained by interfering with the complex between a disintegrin and metalloproteinase domain containing protein 10 (ADAM10), the main α-secretase candidate, and its partner, synapse-associated protein 97, a protein of the postsynaptic density-membrane associated guanylate kinase family. Association of ADAM10 with synapse-associated protein 97 governs enzyme trafficking and activity at synapses. Interfering with the ADAM10/synapse-associated protein 97 complex for 2 weeks by means of a cell-permeable peptide strategy is sufficient to shift the metabolism of the amyloid precursor protein towards amyloidogenesis and allows the reproduction of initial phases of sporadic Alzheimers disease. After 2 weeks of treatment, we detected progressive Alzheimers disease-like neuropathology, with an increase of β-amyloid aggregate production and of tau hyperphosphorylation, and a selective alteration of N-methyl-d-aspartic acid receptor subunit composition in the postsynaptic compartment of mouse brain. Behavioural and electrophysiological deficits were also induced by peptide treatment.

JNK Plays a Key Role in Tau Hyperphosphorylation in Alzheimer's Disease Models

Alzheimers disease (AD) is a major clinical concern, and the search for new molecules to combat disease progression remains important. One of the major hallmarks in AD pathogenesis is the hyperphosphorylation of tau and subsequent formation of neurofibrillary tangles. Several kinases are involved in this process. Amongst them, c-Jun N-terminal kinases (JNKs) are activated in AD brains and are also associated with the development of amyloid plaques. This study was designed to investigate the contribution of JNK in tau hyperphosphorylation and whether it may represent a potential therapeutic target for the fight against AD. The specific inhibition of JNK by the cell permeable peptide D-JNKI-1 led to a reduction of p-tau at S202/T205 and S422, two established target sites of JNK, in rat neuronal cultures and in human fibroblasts cultures. Similarly, D-JNKI-1 reduced p-tau at S202/T205 in an in vivo model of AD (TgCRND8 mice). Our findings support the fundamental role of JNK in the regulation of tau hyperphosphorylation and subsequently in AD pathogenesis.