Alessandra Sclip

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.

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.

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.

c-Jun N-terminal kinase has a key role in Alzheimer disease synaptic dysfunction in vivo

Altered synaptic function is considered one of the first features of Alzheimer disease (AD). Currently, no treatment is available to prevent the dysfunction of excitatory synapses in AD. Identification of the key modulators of synaptopathy is of particular significance in the treatment of AD. We here characterized the pathways leading to synaptopathy in TgCRND8 mice and showed that c-Jun N-terminal kinase (JNK) is activated at the spine prior to the onset of cognitive impairment. The specific inhibition of JNK, with its specific inhibiting peptide D-JNKI1, prevented synaptic dysfunction in TgCRND8 mice. D-JNKI1 avoided both the loss of postsynaptic proteins and glutamate receptors from the postsynaptic density and the reduction in size of excitatory synapses, reverting their dysfunction. This set of data reveals that JNK is a key signaling pathway in AD synaptic injury and that its specific inhibition offers an innovative therapeutic strategy to prevent spine degeneration in AD.

Specific inhibition of the JNK pathway promotes locomotor recovery and neuroprotection after mouse spinal cord injury.

Limiting the development of secondary damage represents one of the major goals of neuroprotective therapies after spinal cord injury. Here, we demonstrate that specific JNK inhibition via a single intraperitoneal injection of the cell permeable peptide D-JNKI1 6h after lesion improves locomotor recovery assessed by both the footprint and the BMS tests up to 4 months post-injury in mice. JNK inhibition prevents c-jun phosphorylation and caspase-3 cleavage, has neuroprotective effects and results in an increased sparing of white matter at the lesion site. Lastly, D-JNKI1 treated animals show a lower increase of erythrocyte extravasation and blood brain barrier permeability, thus indicating protection of the vascular system. In total, these results clearly point out JNK inhibition as a promising neuroprotective strategy for preventing the evolution of secondary damage after spinal cord injury.

Region- and age-dependent reductions of hippocampal long-term potentiation and NMDA to AMPA ratio in a genetic model of Alzheimer's disease.

To characterize the mechanisms underlying region- and age-dependent hippocampal synaptic dysfunction in Alzheimers disease, we used transgenic CRND8 mice, expressing the Swedish-Indiana APP mutation. In 2-month-old mice, no β-amyloid plaques deposition, but the presence of soluble oligomers, were found in CA1 area but not in dentate gyrus (DG). At this age, long-term potentiation (LTP) was reduced selectively in CA1. In 6-month-old mice, the presence of soluble oligomers was accompanied by accumulation of β-amyloid plaques and decreased LTP in CA1 and DG regions. In both regions, the loss of LTP was linked to reduced N-methyl-D-aspartate (NMDA) to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) current ratio. The acetylcholine-esterase inhibitor, neostigmine rescued LTP in CA1 area at early stage of the disease but not after plaques deposition. Conversely, the NMDA receptor antagonist memantine restored LTP selectively in DG at later stages of the disease. Both these effects were associated with a normalization of the NMDA to AMPA ratio. The association between the recovery of LTP and the normalization of the NMDA to AMPA ratio provides information on new possible therapeutic strategies in Alzheimers disease.

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