Xanthi Antoniou

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.

c-Jun N-terminal kinase regulates soluble Aβ oligomers and cognitive impairment in AD mouse model.

Background: Neuropathological mechanisms in Alzheimer disease (AD) are partially unknown. Results: Chronic JNK inhibition with a cell-permeable peptide (CPP) rescues memory deficits, LTP impairment, and reduces Aβ oligomers in a mouse model that mimics AD. Conclusion: JNK is crucial in AD neurodegenerative mechanisms. Significance: CPPs offer an important tool to interfere with neurodegeneration. JNK is a promising target against AD. Alzheimer disease (AD) is characterized by cognitive impairment that starts with memory loss to end in dementia. Loss of synapses and synaptic dysfunction are closely associated with cognitive impairment in AD patients. Biochemical and pathological evidence suggests that soluble Aβ oligomers correlate with cognitive impairment. Here, we used the TgCRND8 AD mouse model to investigate the role of JNK in long term memory deficits. TgCRND8 mice were chronically treated with the cell-penetrating c-Jun N-terminal kinase inhibitor peptide (D-JNKI1). D-JNKI1, preventing JNK action, completely rescued memory impairments (behavioral studies) as well as the long term potentiation deficits of TgCRND8 mice. Moreover, D-JNKI1 inhibited APP phosphorylation in Thr-668 and reduced the amyloidogenic cleavage of APP and Aβ oligomers in brain parenchyma of treated mice. In conclusion, by regulating key pathogenic mechanisms of AD, JNK might hold promise as innovative therapeutic target.

Crosstalk between JNK and SUMO Signaling Pathways: deSUMOylation Is Protective against H2O2-Induced Cell Injury

Background Oxidative stress is a key feature in the pathogenesis of several neurological disorders. Following oxidative stress stimuli a wide range of pathways are activated and contribute to cellular death. The mechanism that couples c-Jun N-terminal kinase (JNK) signaling, a key pathway in stress conditions, to the small ubiquitin-related modifier (SUMO), an emerging protein in the field, is largely unknown. Methodology/Principal Findings With this study we investigated if SUMOylation participates in the regulation of JNK activation as well as cellular death in a model of H2O2 induced-oxidative stress. Our data show that H2O2 modulates JNK activation and induces cellular death in neuroblastoma SH-SY5Y cells. Inhibition of JNKs action with the D-JNKI1 peptide rescued cells from death. Following H2O2, SUMO-1 over-expression increased phosphorylation of JNK and exacerbated cell death, although only in conditions of mild oxidative stress. Furthermore inhibition of SUMOylation, following transfection with SENP1, interfered with JNK activation and rescued cells from H2O2 induced death. Importantly, in our model, direct interaction between these proteins can occur. Conclusions/Significance Taken together our results show that SUMOylation may significantly contribute to modulation of JNK activation and contribute to cell death in oxidative stress conditions.

JNK3 as a Therapeutic Target for Neurodegenerative Diseases

c-Jun N-terminal kinases (JNKs) and in particular JNK3 the neuronal specific isoform, have been recognized as important enzymes in the pathology of diverse neurological disorders. Indeed, several efforts have been made to design drugs that inhibit JNK signaling. The success that characterized the new generation of cell permeable peptides raise the hope in the field of neurodegeneration for new therapeutic routes. However, in order to design new and more efficient therapeutical approaches careful re-examination of current knowledge is required. Scaffold proteins are key endogenous regulators of JNK signaling: they can modulate spatial and temporal activation of the JNK signaling and can thus provide the basis for the design of more specific inhibitors. This review focuses on delineating the role of scaffold proteins on the regulation of JNK signaling in neurons. Furthermore the possibility to design a new JNK3 cell permeable peptide inhibitor by targeting the β-arrestin-JNK3 interaction is discussed.

Soluble Aβ oligomer-induced synaptopathy: c-Jun N-terminal kinase's role

Dear Editor, Among the neurodegenerative diseases, Alzheimer disease (AD) is the most common and severe age-related dementia for which there is currently no available treatment. Many studies support the assumption that AD is a spine pathology (Selkoe, 2002; Sivanesan et al., 2013) and that soluble amyloid-b (Ab) oligomers are causative of AD synaptopathy. Diverse lines of evidence indicate that Aboligomers induce formation of pore-like structures on the membrane (Arispe et al., 1993; Lashuel et al., 2002) and interfere with glutamatergic transmission. The Ab oligomers result in a decreased number of AMPA receptors (AMPA-r) and NMDA receptors (NMDA-r), as well as PSD-95 at the postsynaptic membrane, and thus reduce the strength and plasticity of excitatory synapses (Chapman et al., 1999; Walsh et al., 2002). However, the underlying intracellular mechanisms regulating synaptic changes are only partially known. By understanding the pathophysiological mechanisms leading to synaptic dysfunction and the progression of this dysfunction, better interference in the pathogenesis of AD can be achieved. We present an in vitro model to study the temporal sequence of dendritic spine modifications induced by soluble Ab oligomers, and to analyse the intracellular signalling pathways leading to AD synaptopathy. This model allows synaptic alterations to be followed in living neurons before and after treatment and reduces bias due to cell variability. This model also permits testing of pharmaceuticals that are designed to reverse the biochemical and structural alterations of synapses induced by Ab oligomers. Brainbow hippocampal neurons, which express fluorescent proteins, were used to visualize dendritic spines and study synaptic plasticity (Figure 1A and B). To obtain isolated cells, fluorescent neurons were seeded on a layer of non-fluorescent neurons (ratio: 1/16) (Figure 1A and B). In this way we avoided the need for transfection, infection protocols, and low density cultures that are not well tolerated by neurons. Neurons were treated with a subtoxic dose (Figure 1E) of soluble Ab1–42 oligomers in order to induce synaptic changes without any signs of neuronal death (Figure 1E). The preparation of synthetic Ab1–42 that were used to induce in vitro synaptic dysfunction had been previously characterized. Oligomeric assemblies were only observed in peptide preparations after a 24-h incubation at 48C (referred to as oligomers). Immediately after dissolution, the majority of Ab1–42 remained as unassembled monomeric structures (Figure 1C and D and Supplementary Figure S1). Subtoxic concentrations of soluble Ab oligomers induced alterations in the postsynaptic density (PSD) composition of dendritic spines, while monomers had no effect on synaptic plasticity (Supplementary Figure S1F). Exposing the neurons for 3 h to 1 mM Ab oligomers induced changes in the PSD region, leading to a 68% and 61% drop of GluN2A and GluN2B subunits of NMDA-r, respectively; a decrease of 70% and 65% of GluA1 and GluA2 subunits of AMPA-r, respectively; a 53% loss of PSD-95; and a 76% loss of drebrin (Figure 1F and Supplementary Figure S2A). To assess dendritic spine modifications in vitro, we analysed changes in spine density and morphology in neurons exposed to soluble Ab oligomers. Application of soluble Ab oligomers (1 mM) for 3 h caused a 25% decrease in total spine number compared with that before Ab application (Figure 1J and K). The decrease involved all types of spines in a proportional manner. The number of mushroom, stubby, and thin spines decreased by 32%, 25%, and 22%, respectively (Figure 1J and L). Moreover, Ab oligomer treatment induced a 67% reduction in new spine formation compared with control conditions, and led to spine shrinkage (Supplementary Figure S2E). The number of mushroom spines that became stubby or thin was significantly increased by Ab oligomer treatment, while the number of spines that became mushroom was decreased (Supplementary Figure S2E). The morphological changes were consistent with the observed biochemical alterations since thin and stubby spines have a less extended PSD region and lower contents of glutamate receptors as well as postsynaptic markers, in comparison with mushroom spines (Tackenberg et al., 2009). To analyse the pathways involved in Ab oligomer-induced synaptopathy, we evaluated synaptic changes induced after 30 min and 3 h of Ab oligomer exposure (1 mM) and correlated them to the activation of two stress signalling pathways, c-Jun N-terminal kinase (JNK) and caspase-3. After 30 min there was no sign of molecular changes: NMDA-r and AMPA-r subunits, PSD-95, and drebrin levels were unaffected by the Ab oligomer treatment (Figure 1F). However, JNK was already activated at this stage, as indicated by a 2.36-fold increase of the P-JNK/JNK ratio compared with control conditions (Figure 1G and Supplementary Figure S2B). There was no indication of caspase-3 cleavage after 30 min of Ab oligomer exposure (Figure 1H and Supplementary Figure S2C). Exposing neurons to soluble Ab oligomers for 3 h induced a biochemical perturbation of PSD (Figure 1F) with a 4.33-fold increase of caspase-3 cleavage (Figure 1H) (Li et al., 2010; D’Amelio et al., 2011), while JNK activity remained elevated by 2.26 folds (Figure 1G). Our results showed that JNK activation was triggered by Ab oligomers before PSD alterations were induced and JNK activation persisted up to 3 h, at which doi:10.1093/jmcb/mjt015 Journal of Molecular Cell Biology (2013), 5, 277–279 | 277 Published online April 18, 2013

JNK Contributes to Hif-1α Regulation in Hypoxic Neurons

Hypoxia is an established factor of neurodegeneration. Nowadays, attention is directed at understanding how alterations in the expression of stress-related signaling proteins contribute to age dependent neuronal vulnerability to injury. The purpose of this study was to investigate how Hif-1α, a major neuroprotective factor, and JNK signaling, a key pathway in neurodegeneration, relate to hypoxic injury in young (6DIV) and adult (12DIV) neurons. We could show that in young neurons as compared to mature ones, the protective factor Hif-1α is more induced while the stress protein phospho-JNK displays lower basal levels. Indeed, changes in the expression levels of these proteins correlated with increased vulnerability of adult neurons to hypoxic injury. Furthermore, we describe for the first time that treatment with the D-JNKI1, a JNK-inhibiting peptide, rescues adult hypoxic neurons from death and contributes to Hif-1α upregulation, probably via a direct interaction with the Hif-1α protein.

The JNK signalling transduction pathway in the brain.

Among the numerous intracellular signalling pathways that control brain development and pathogenesis c-Jun N-terminal kinases have a leading role in the Central Nervous System. JNKs regulate a wide range of processes in brain development, plasticity, repair/regeneration, neuronal death and neuroinflammation. Indeed, accumulating evidence underline the potential of JNK targeted molecules towards the treatment of neurodegenerative disorders. The focus of the presenting review is to provide an overview of the reported data linking JNKs to brain function and dysfunction.

Cell permeable peptides: A promising tool to deliver neuroprotective agents in the brain

The inability of most drugs to cross the blood-brain barrier and/or plasma membrane limits their use for biomedical applications in the brain. Cell Permeable Peptides (CPPs) overcome this problem and are effective in vivo, crossing the plasma membrane and the blood-brain barrier. CPPs deliver a wide variety of compounds intracellularly in an active form. In fact, many bioactive cargoes have neuroprotective properties, and due to their ability to block protein-protein interactions, offer exciting perspectives in the clinical setting. In this review we give an overview of the Cell Permeable Peptides strategy to deliver neuroprotectants against neurodegeneration in the CNS.

Role of Glycogen Synthase Kinase-3β in APP Hyperphosphorylation Induced by NMDA Stimulation in Cortical Neurons

The phosphorylation of Amyloid Precursor Protein (APP) at Thr668 plays a key role in APP metabolism that is highly relevant to AD. The c-Jun-N-terminal kinase (JNK), glycogen synthase kinase-3β (GSK-3β) and cyclin-dependent kinase 5 (Cdk5) can all be responsible for this phosphorylation. These kinases are activated by excitotoxic stimuli fundamental hallmarks of AD. The exposure of cortical neurons to a high dose of NMDA (100 μM) for 30’-45’ led to an increase of P-APP Thr668. During NMDA stimulation APP hyperphosphorylation has to be assigned to GSK-3β activity, since addition of L803-mts, a substrate competitive inhibitor of GSK-3β reduced APP phosphorylation induced by NMDA. On the contrary, inhibition of JNK and Cdk5 with D-JNKI1 and Roscovitine respectively did not prevent NMDA-induced P-APP increase. These data show a tight connection, in excitotoxic conditions, between APP metabolism and the GSK-3β signaling pathway.