Sleep inhibition induced by amyloid-ß oligomers is mediated by the cellular prion protein.

Sleep is severely impaired in patients with Alzheimer's disease. Amyloid-ß deposition in the brain of Alzheimer's disease patients is a key event in its pathogenesis and is associated with disrupted sleep, even before the appearance of cognitive decline. Because soluble amyloid-ß oligomers are the key mediators of synaptic and cognitive dysfunction in Alzheimer's disease and impair long-term memory in rodents, the first aim of this study was to test the hypothesis that amyloid-ß oligomers would directly impair sleep in mice. The cellular prion protein is a cell surface glycoprotein of uncertain function. Because cellular prion protein binds oligomeric amyloid-ß with high affinity and mediates some of its neurotoxic effects, the second aim of the study was to test whether amyloid-ß oligomer-induced sleep alterations were mediated by cellular prion protein. To address these aims, wild-type and cellular prion protein-deficient mice were given acute intracerebroventricular injections (on different days, at lights on) of vehicle and synthetic amyloid-ß oligomers. Compared to vehicle, amyloid-ß oligomers significantly reduced the amount of time spent in non-rapid eye movement sleep by wild-type mice during both the light and dark phases of the light-dark cycle. The amount of time spent in rapid eye movement sleep was reduced during the dark phase. Sleep was also fragmented by amyloid-ß oligomers, as the number of transitions between states increased in post-injection hours 9-24. No such effects were observed in cellular prion protein-deficient mice. These results show that amyloid-ß oligomers do inhibit and fragment sleep, and that these effects are mediated by cellular prion protein.

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

BACKGROUND: Amyloid-ß oligomers (AßO) are species mainly involved in the synaptic and cognitive dysfunction in Alzheimer's 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 AßO-mediated mouse model of AD, we explored whether the memory alteration AßOs 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 AßO-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-1ß receptor antagonist) to prevent impairment in the novel object recognition task, and compared AßO's effects in TLR4 knockout mice. RESULTS: A single AßO injection rapidly activated glial cells and increased pro-inflammatory cytokine expression. Both anti-inflammatory drugs prevented the AßO-mediated impairment in memory establishment. A selective TLR4 receptor antagonist abolished AßO's 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 AßO's 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.

The cell-permeable Aß1-6A2VTAT(D) peptide reverts synaptopathy induced by Aß1-42wt.

Alzheimer disease (AD) is the most prevalent form of dementia. Loss of hippocampal synapses is the first neurodegenerative event in AD. Synaptic loss has been associated with the accumulation in the brain parenchyma of soluble oligomeric forms of amyloid ß peptide (Aß1-42wt). Clinical observations have shown that a mutation in the APP protein (A673V) causes an early onset AD-type dementia in homozygous carriers while heterozygous carriers are unaffected. This mutation leads to the formation of mutated Aß peptides (Aß1-42A2V) in homozygous patients, while in heterozygous subjects both Aß1-42wt and Aß1-42A2V are present. To better understand the impact of the A673V mutation in AD, we analyzed the synaptotoxic effect of oligomers formed by aggregation of different Aß peptides (Aß1-42wt or Aß1-42A2V) and the combination of the two Aß1-42MIX (Aß1-42wt and Aß1-42A2V) in an in vitro model of synaptic injury. We showed that Aß1-42A2V oligomers are more toxic than Aß1-42wt oligomers in hippocampal neurons, confirming the results previously obtained in cell lines. Furthermore, we reported that oligomers obtained by the combination of both wild type and mutated peptides (Aß1-42MIX) did not exert synaptic toxicity. We concluded that the combination of Aß1-42wt and Aß1-42A2V peptides hinders the toxicity of Aß1-42A2V and counteracts the manifestation of synaptopathy in vitro. Finally we took advantage of this finding to generate a cell-permeable peptide for clinical application, by fusing the first six residues of the Aß1-42A2V to the TAT cargo sequence (Aß1-6A2VTAT(D)). Noteworthy, the treatment with Aß1-6A2VTAT(D) confers neuroprotection against both in vitro and in vivo synaptopathy models. Therefore Aß1-6A2VTAT(D) may represent an innovative therapeutic tool to prevent synaptic degeneration in AD.

The pentraxins PTX3 and SAP in innate immunity, regulation of inflammation and tissue remodelling.

Pentraxins are a superfamily of fluid phase pattern recognition molecules conserved in evolution and characterized by a cyclic multimeric structure. C-reactive protein (CRP) and serum amyloid P component (SAP) constitute the short pentraxin arm of the superfamily. CRP and SAP are produced in the liver in response to IL-6 and are acute phase reactants in humans and mice respectively. In addition SAP has been shown to affect tissue remodelling and fibrosis by stabilizing all types of amyloid fibrils and by regulating monocyte to fibrocyte differentiation. Pentraxin 3 (PTX3) is the prototype of the long pentraxin arm. Gene targeted mice and genetic and epigenetic studies in humans suggest that PTX3 plays essential non-redundant roles in innate immunity and inflammation as well as in tissue remodelling. Recent studies have revealed the role of PTX3 as extrinsic oncosuppressor, able to tune cancer-related inflammation. In addition, at acidic pH PTX3 can interact with provisional matrix components promoting inflammatory matrix remodelling. Thus acidification during tissue repair sets PTX3 in a tissue remodelling and repair mode, suggesting that matrix and microbial recognition are common, ancestral features of the humoral arm of innate immunity.

The peculiar role of the A2V mutation in amyloid-ß (Aß) 1-42 molecular assembly.

We recently reported a novel Aß precursor protein mutation (A673V), corresponding to position 2 of Aß1-42 peptides (Aß1-42A2V), 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 Aß1-42A2V in comparison with the wild type sequence (Aß1-42WT) and the equimolar solution of both peptides (Aß1-42MIX) 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. Aß1-42MIX generated assemblies very similar to those produced by Aß1-42WT, albeit with slower kinetics due to the difficulties of Aß1-42WT and Aß1-42A2V peptides in building up of stable intermolecular interaction.

An N-terminal fragment of the prion protein binds to amyloid-ß oligomers and inhibits their neurotoxicity in vivo.

A hallmark of Alzheimer disease (AD) is the accumulation of the amyloid-ß (Aß) peptide in the brain. Considerable evidence suggests that soluble Aß 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 Aß 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 Aß polymerization into amyloid fibrils. The ability of N1 to bind Aß 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 Aß oligomer toxicity in cultured murine hippocampal neurons, in a Caenorhabditis elegans-based assay, and in vivo in a mouse model of Aß-induced memory dysfunction. These data suggest that N1, or small peptides derived from it, could be potent inhibitors of Aß oligomer toxicity and represent an entirely new class of therapeutic agents for AD.

Natural compounds against neurodegenerative diseases: molecular characterization of the interaction of catechins from green tea with Aß1-42, 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 Aß1-42, 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.

ß-amyloid 1-42 induces physiological transcriptional regulation of BACE1.

The pathogenesis of Alzheimer's disease (AD) is only partially understood. ß-amyloid (Aß) is physiologically generated by sequential cleavage of its precursor protein by the ß- and the γ-secretase and it is normally disposed of. In Alzheimer's disease, Aß is excessively produced or less dismissed, but the hypothesis on its physiological and pathological role are heterogeneous and often discordant. It has been described a positive feedback loop from the γ- to the ß-secretase cleavages of Aß precursor protein, which is activated by mutations of Presenilin 1 (PS1), the catalytic core of the γ-secretase. These findings show that Aß precursor protein as well the activity of the γ-secretase are required to obtain the up-regulation of ß-secretase which is induced by Presenilin 1 mutations. Then, Aß 1-42 is the Aß precursor protein derivative that up-regulates the expression of ß-secretase, and c-jun N-terminal kinase (JNK)/c-Jun and ERK1/2 are involved. Here, we describe the activation of ß-secretase and c-jun N-terminal kinase related proteins by monomeric Aß 1-42, defining the conditions that most efficiently strike the described signaling without producing toxicity. Taken together these data imply that monomeric Aß 1-42, at non-toxic concentrations and time frames, are able to induce a signaling pathway that leads to transcriptional activation of ß-secretase.

Amyloid-ß peptides in interaction with raft-mime model membranes: a neutron reflectivity insight.

The role of first-stage ß-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 ß-amyloid (Aß) 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 Aß(1-42), its most acknowledged membrane-active state, is embedded as such into the external leaflet of the membrane. Conversely, the Aß(1-42) unstructured early-oligomers deeply penetrate the membrane, likely mimicking the interaction at neuronal cell surfaces, when the Aß(1-42) is cleaved from APP protein and the membrane constitutes a template for its further structural evolution. Moreover, the smaller Aß(1-6) fragment, the N-terminal portion of Aß, was also used. Aß 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.

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 Aß peptides and it markedly enhances the aggregation and fibrillogenic properties of both Aß1-40 and Aß1-42. However, co-incubation of mutated and wild-type (wt) Aß 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 Aß variant may offer grounds for the development of therapeutic strategies for AD based on modified Aß peptides. Indeed, the interaction between mutated Aß1-6 and wt full-length Aß 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.

Curcumin derivatives as new ligands of Aß 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 Aß peptide oligomers without inducing their aggregation. Moreover all Curcumin derivatives were able to stain very efficiently Aß deposits.

The molecular assembly of amyloid aß controls its neurotoxicity and binding to cellular proteins.

Accumulation of ß-sheet-rich peptide (Aß) is strongly associated with Alzheimer's 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. Aß species are potent neurotoxins, however the molecular mechanism responsible for Aß 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 Aß 1-40 and Aß 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 Aß 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 Aß receptors in N2a cells, triggering a multi factorial toxicity.

Overcoming synthetic Abeta peptide aging: a new approach to an age-old problem.

Investigations of amyloidogenic diseases use synthetic peptides for cell-free and in vitro studies. However, amyloidogenic peptides often show intrinsic variability that markedly affects the reproducibility of experiments. Proof of physicochemical and biological variability with different batches of amyloidogenic peptides have been reported in literature. Here, we show that differences can be observed even within the same batch of Abeta1-42 peptide after storing lyophilised samples at -20 degrees C. This change (referred to as 'peptide aging') was reproduced with Abeta1-40 peptide samples by using a series of lyophilisation cycles, showing that lyophilisation, rather than preserving the physicochemical and biological features of Abeta peptides, introduces wide variability. To counteract synthetic peptide aging, we set up a procedure involving the sequential use of trifluoroacetic acid, formic acid and sodium hydroxide solutions that disaggregate preformed seeds and enriched Abeta peptide solutions into monomers and low-molecular-weight oligomers. This procedure enabled us to obtain reproducible physicochemical and biological features of Abeta peptides, irrespective of their age.