Massimo Messa

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.

The Molecular Assembly of Amyloid Aβ Controls Its Neurotoxicity and Binding to Cellular Proteins

Accumulation of β-sheet-rich peptide (Aβ) is strongly associated with Alzheimers disease, characterized by reduction in synapse density, structural alterations of dendritic spines, modification of synaptic protein expression, loss of long-term potentiation and neuronal cell death. Aβ species are potent neurotoxins, however the molecular mechanism responsible for Aβ toxicity is still unknown. Numerous mechanisms of toxicity were proposed, although there is no agreement about their relative importance in disease pathogenesis. Here, the toxicity of Aβ 1–40 and Aβ 1–42 monomers, oligomers or fibrils, was evaluated using the N2a cell line. A structure-function relationship between peptide aggregation state and toxic properties was established. Moreover, we demonstrated that Aβ toxic species cross the plasma membrane, accumulate in cells and bind to a variety of internal proteins, especially on the cytoskeleton and in the endoplasmatic reticulum (ER). Based on these data we suggest that numerous proteins act as Aβ receptors in N2a cells, triggering a multi factorial toxicity.

The Peculiar Role of the A2V Mutation in Amyloid-β (Aβ) 1–42 Molecular Assembly

Background: A2V mutation is associated with early onset AD-type dementia in homozygous individuals. Results: A2V mutation leads to a peculiar kinetics of Aβ oligomerization. Conclusion: The Aβ N-terminal region plays an important role in the molecular assembly. Significance: in the homozygous condition the A2V mutation led to aggregation, whereas in the heterozygous state the evolution and kinetics of the aggregation process was hindered. We recently reported a novel Aβ precursor protein mutation (A673V), corresponding to position 2 of Aβ1–42 peptides (Aβ1–42A2V), that caused an early onset AD-type dementia in a homozygous individual. The heterozygous relatives were not affected as an indication of autosomal recessive inheritance of this mutation. We investigated the folding kinetics of native unfolded Aβ1–42A2V in comparison with the wild type sequence (Aβ1–42WT) and the equimolar solution of both peptides (Aβ1–42MIX) to characterize the oligomers that are produced in the early phases. We carried out the structural characterization of the three preparations using electron and atomic force microscopy, fluorescence emission, and x-ray diffraction and described the soluble oligomer formation kinetics by laser light scattering. The mutation promoted a peculiar pathway of oligomerization, forming a connected system similar to a polymer network with hydrophobic residues on the external surface. Aβ1–42MIX generated assemblies very similar to those produced by Aβ1–42WT, albeit with slower kinetics due to the difficulties of Aβ1–42WT and Aβ1–42A2V peptides in building up of stable intermolecular interaction.

Curcumin derivatives as new ligands of Aβ peptides

Curcumin derivatives with high chemical stability, improved solubility and carrying a functionalized appendage for the linkage to other entities, have been synthesized in a straightforward manner. All compounds retained Curcumin ability to bind Aβ peptide oligomers without inducing their aggregation. Moreover all Curcumin derivatives were able to stain very efficiently Aβ deposits.

Toll-like receptor 4-dependent glial cell activation mediates the impairment in memory establishment induced by β-amyloid oligomers in an acute mouse model of Alzheimer's disease

BACKGROUND Amyloid-β oligomers (AβO) are species mainly involved in the synaptic and cognitive dysfunction in Alzheimers disease. Although their action has been described mainly at neuronal level, it is now clear that glial cells govern synaptic activity in their resting state, contributing to new learning and memory establishment. In contrast, when activated, they may lead to synaptic and cognitive dysfunction. Using a reliable acute AβO-mediated mouse model of AD, we explored whether the memory alteration AβOs induce relies on the activation of glial cells, and if Toll-like receptor 4 (TLR4), pivotal in the initiation of an immune response, is involved. METHODS C57 naïve mice were given a single intracerebroventricular injection of synthetic AβO-containing solution (1μM), which induces substantial impairment in the establishment of recognition memory. Then, first we assessed glial cell activation at different times post-injection by western blot, immunohistochemistry and ELISA in the hippocampus. After that we explored the efficacy of pre-treatment with anti-inflammatory drugs (indomethacin and an IL-1β receptor antagonist) to prevent impairment in the novel object recognition task, and compared AβOs effects in TLR4 knockout mice. RESULTS A single AβO injection rapidly activated glial cells and increased pro-inflammatory cytokine expression. Both anti-inflammatory drugs prevented the AβO-mediated impairment in memory establishment. A selective TLR4 receptor antagonist abolished AβOs action on memory, and in TLR4 knockout mice it had no effect on either memory or glial activation. CONCLUSIONS These data provide new information on AβOs mechanism of action, indicating that besides direct action at the synapses, they also act through the immune system, with TLR4 playing a major role. This suggests that in a potential therapeutic setting inflammation must be considered as well.

Good gene, bad gene: New APP variant may be both

APP mutations cause Alzheimer disease (AD) with virtually complete penetrance. We found a novel APP mutation (A673V) in the homozygous state in a patient with early-onset AD-type dementia and in his younger sister showing initial signs of cognitive decline. It is noteworthy that the heterozygous relatives were not affected, suggesting that this mutation is inherited as an autosomal recessive trait. Studies on molecular events for the recessive mutation in causing disease revealed a double synergistic effect: the A673V APP variant shifts APP processing towards the amyloidogenic pathway with increased production of Aβ peptides and it markedly enhances the aggregation and fibrillogenic properties of both Aβ1-40 and Aβ1-42. However, co-incubation of mutated and wild-type (wt) Aβ species resulted in inhibition of amyloidogenesis, consistent with the observation that heterozygous carriers do not develop the disease. The opposite effects of the A673V mutation in the homozygous and heterozygous state on amyloidogenesis account for the autosomal recessive pattern of inheritance, revealing a new scenario in AD genetics and pathogenesis. The anti-amyloidogenic properties of this novel human Aβ variant may offer grounds for the development of therapeutic strategies for AD based on modified Aβ peptides. Indeed, the interaction between mutated Aβ1-6 and wt full-length Aβ prevents amyloid fibril formation. The anti-amyloidogenic effect is further amplified by the use of a mutated six-mer peptide, constructed entirely from D-amino acids to increase the its stability in vivo. Here we reviewed the studies on pathogenic mechanisms associated with the A673V mutation and the first experimental steps toward the development of a novel disease-modifying therapy for AD.

β-Amyloid 1-42 induces physiological transcriptional regulation of BACE1

J. Neurochem. (2012) 122, 1023–1031.

Natural Compounds against Neurodegenerative Diseases: Molecular Characterization of the Interaction of Catechins from Green Tea with Aβ1–42, PrP106–126, and Ataxin-3 Oligomers

By combining NMR spectroscopy, transmission electron microscopy, and circular dichroism we have identified the structural determinants involved in the interaction of green tea catechins with Aβ1-42, PrP106-126, and ataxin-3 oligomers. The data allow the elucidation of their mechanism of action, showing that the flavan-3-ol unit of catechins is essential for interaction. At the same time, the gallate moiety, when present, seems to increase the affinity for the target proteins. These results provide important information for the rational design of new compounds with anti-amyloidogenic activity and/or molecular tools for the specific targeting of amyloid aggregates in vivo.

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

Amyloidβ Peptides in interaction with raft-mime model membranes: a neutron reflectivity insight

The role of first-stage β–amyloid aggregation in the development of the Alzheimer disease, is widely accepted but still unclear. Intimate interaction with the cell membrane is invoked. We designed Neutron Reflectometry experiments to reveal the existence and extent of the interaction between β–amyloid (Aβ) peptides and a lone customized biomimetic membrane, and their dependence on the aggregation state of the peptide. The membrane, asymmetrically containing phospholipids, GM1 and cholesterol in biosimilar proportion, is a model for a raft, a putative site for amyloid-cell membrane interaction. We found that the structured-oligomer of Aβ(1-42), its most acknowledged membrane-active state, is embedded as such into the external leaflet of the membrane. Conversely, the Aβ(1-42) unstructured early-oligomers deeply penetrate the membrane, likely mimicking the interaction at neuronal cell surfaces, when the Aβ(1-42) is cleaved from APP protein and the membrane constitutes a template for its further structural evolution. Moreover, the smaller Aβ(1-6) fragment, the N-terminal portion of Aβ, was also used. Aβ N-terminal is usually considered as involved in oligomer stabilization but not in the peptide-membrane interaction. Instead, it was seen to remove lipids from the bilayer, thus suggesting its role, once in the whole peptide, in membrane leakage, favouring peptide recruitment.