Prion protein stabilizes amyloid-ß (Aß) oligomers and enhances Aß neurotoxicity in a Drosophila model of Alzheimer's disease.

The cellular prion protein (PrPC) can act as a cell-surface receptor for ß-amyloid (Aß) peptide; however, a role for PrPC in the pathogenesis of Alzheimer's disease (AD) is contested. Here, we expressed a range of Aß isoforms and PrPC in the Drosophila brain. We found that co-expression of Aß and PrPC significantly reduces the lifespan, disrupts circadian rhythms, and increases Aß deposition in the fly brain. In contrast, under the same conditions, expression of Aß or PrPC individually did not lead to these phenotypic changes. In vitro studies revealed that substoichiometric amounts of PrPC trap Aß as oligomeric assemblies and fragment-preformed Aß fibers. The ability of membrane-anchored PrPC to trap Aß as cytotoxic oligomers at the membrane surface and fragment inert Aß fibers suggests a mechanism by which PrPC exacerbates Aß deposition and pathogenic phenotypes in the fly, supporting a role for PrPC in AD. This study provides a second animal model linking PrPC expression with Aß toxicity and supports a role for PrPC in AD pathogenesis. Blocking the interaction of Aß and PrPC represents a potential therapeutic strategy.

N-Terminally Truncated Amyloid-ß(11-40/42) Cofibrillizes with its Full-Length Counterpart: Implications for Alzheimer's Disease.

Amyloid-ß peptide (Aß) isoforms of different lengths and aggregation propensities coexist in vivo. These different isoforms are able to nucleate or frustrate the assembly of each other. N-terminally truncated Aß(11-40) and Aß(11-42) make up one fifth of plaque load yet nothing is known about their interaction with full-length Aß(1-40/42) . We show that in contrast to C-terminally truncated isoforms, which do not co-fibrillize, deletions of ten residues from the N terminus of Aß have little impact on its ability to co-fibrillize with the full-length counterpart. As a consequence, N-terminally truncated Aß will accelerate fiber formation and co-assemble into short rod-shaped fibers with its full-length Aß counterpart. This has implications for the assembly kinetics, morphology, and toxicity of all Aß isoforms.

The Rapid Exchange of Zinc(2+) Enables Trace Levels to Profoundly Influence Amyloid-ß Misfolding and Dominates Assembly Outcomes in Cu(2+)/Zn(2+) Mixtures.

The misfolding and self-assembly of amyloid-ß (Aß) into oligomers and fibres is fundamental to Alzheimer's disease pathology. Alzheimer's disease is a multifaceted disease. One factor that is thought to have a significant role in disease aetiology is Zn(2+) homeostasis, which is disrupted in the brains of Alzheimer's disease sufferers and has been shown to modulate Alzheimer's symptoms in animal models. Here, we investigate how the kinetics of Aß fibre growth are affected at a range of Zn(2+) concentrations and we use transmission electron microscopy to characterise the aggregate assemblies formed. We demonstrate that for Aß(1-40), and Aß(1-42), as little as 0.01mol equivalent of Zn(2+) (100nM) is sufficient to greatly perturb the formation of amyloid fibres irreversibly. Instead, Aß(1-40) assembles into short, rod-like structures that pack tightly together into ordered stacks, whereas Aß(1-42) forms short, crooked assemblies that knit together to form a mesh of disordered tangles. Our data suggest that a small number of Zn(2+) ions are able to influence a great many Aß molecules through the rapid exchange of Zn(2+) between Aß peptides. Surprisingly, although Cu(2+) binds to Aß 10,000 times tighter than Zn(2+), the effect of Zn(2+) on Aß assembly dominates in Cu(2+)/Zn(2+) mixtures, suggesting that trace levels of Zn(2+) must have a profound effect on extracellular Aß accumulation. Trace Zn(2+) levels profoundly influence Aß assembly even at concentrations weaker than its affinity for Aß. These observations indicate that inhibitors of fibre assembly do not necessarily have to be at high concentration and affinity to have a profound impact.

A Comparison of Three Fluorophores for the Detection of Amyloid Fibers and Prefibrillar Oligomeric Assemblies. ThT (Thioflavin T); ANS (1-Anilinonaphthalene-8-sulfonic Acid); and bisANS (4,4'-Dianilino-1,1'-binaphthyl-5,5'-disulfonic Acid).

Amyloid fiber formation is a key event in many misfolding disorders. The ability to monitor the kinetics of fiber formation and other prefibrillar assemblies is therefore crucial for understanding these diseases. Here we compare three fluorescent probes for their ability to monitor fiber formation, ANS (1-anilinonaphthalene-8-sulfonic acid) and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid) along with the more widely used thioflavin T (ThT). For this, we have used two highly amyloidogenic peptides: amyloid-ß (Aß) from Alzheimer's disease and islet amyloid polypeptide (IAPP) associated with type II diabetes. Using a well-plate reader, we show all three fluorophores can report the kinetics of fiber formation. Indeed, bis-ANS is markedly more sensitive to fiber detection than ThT and has a submicromolar affinity for Aß fibers. Furthermore, we show that fluorescence detection is very sensitive to the presence of excess fluorophore. In particular, beyond a 1:1 stoichiometry these probes demonstrate marked fluorescence quenching, for both Aß and IAPP. Indeed, the fiber-associated fluorescence signal is almost completely quenched in the presence of excess ThT. There is also intense interest in the detection of prefibrillar amyloid assemblies, as oligomers and protofibrils are believed to be highly cytotoxic. We generate stable, fiber-free, prefibrillar assemblies of Aß and survey their fluorescence with ANS and bis-ANS. Fluorescence from ANS has often been used as a marker for oligomers; however, we show ANS can fluoresce more strongly in the presence of fibers and should therefore be used as a probe for oligomers with caution.

Cu²âº accentuates distinct misfolding of Aß1₋40 and Aß1₋42 peptides, and potentiates membrane disruption.

Central to Alzheimer's disease is the misfolding of amyloid-beta (Aß) peptide, which generates an assorted population of amorphous aggregates, oligomers and fibres. Metal ion homoeostasis is disrupted in the brains of sufferers of Alzheimer's disease and causes heightened Alzheimer's disease phenotype in animal models. In the present study, we demonstrate that substochiometric Cu²âº affects the misfolding pathway of Aß1₋40, and the more toxic Aß1₋42, in markedly different ways. Cu²âº accelerates Aß1₋40 fibre formation. In contrast, for Aß1₋42, substoichiometric levels of Cu²âº almost exclusively promote the formation of oligomeric and protofibrillar assemblies. Indeed, mature Aß1₋42 fibres are disassembled into oligomers when Cu²âº is added. These Cu²âº stabilized oligomers of Aß1₋42 interact with the lipid bilayer, disrupting the membrane and increasing permeability. Our investigation of Aß1₋40/Aß1₋42 mixtures with Cu²âº revealed that Aß1₋40 neither contributed to nor perturbed formation of Aß1₋42 oligomers, although Cu²âº-Aß1₋42 does frustrate Cu²âº-Aß1₋40 fibre growth. Small amounts of Cu²âº accentuate differences in the propensity of Aß1₋40 and Aß1₋42 to form synaptotoxic oligomers, providing an explanation for the connection between disrupted Cu²âº homoeostasis and elevated Aß1₋42 neurotoxicity in Alzheimer's disease.

Bioactivity and structural properties of chimeric analogs of the starfish SALMFamide neuropeptides S1 and S2.

The starfish SALMFamide neuropeptides S1 (GFNSALMFamide) and S2 (SGPYSFNSGLTFamide) are the prototypical members of a family of neuropeptides that act as muscle relaxants in echinoderms. Comparison of the bioactivity of S1 and S2 as muscle relaxants has revealed that S2 is ten times more potent than S1. Here we investigated a structural basis for this difference in potency by comparing the bioactivity and solution conformations (using NMR and CD spectroscopy) of S1 and S2 with three chimeric analogs of these peptides. A peptide comprising S1 with the addition of S2's N-terminal tetrapeptide (Long S1 or LS1; SGPYGFNSALMFamide) was not significantly different to S1 in its bioactivity and did not exhibit concentration-dependent structuring seen with S2. An analog of S1 with its penultimate residue substituted from S2 (S1(T); GFNSALTFamide) exhibited S1-like bioactivity and structure. However, an analog of S2 with its penultimate residue substituted from S1 (S2(M); SGPYSFNSGLMFamide) exhibited loss of S2-type bioactivity and structural properties. Collectively, our data indicate that the C-terminal regions of S1 and S2 are the key determinants of their differing bioactivity. However, the N-terminal region of S2 may influence its bioactivity by conferring structural stability in solution. Thus, analysis of chimeric SALMFamides has revealed how neuropeptide bioactivity is determined by a complex interplay of sequence and conformation.

Structural analysis of the starfish SALMFamide neuropeptides S1 and S2: the N-terminal region of S2 facilitates self-association.

The neuropeptides S1 (GFNSALMFamide) and S2 (SGPYSFNSGLTFamide), which share sequence similarity, were discovered in the starfish Asterias rubens and are prototypical members of the SALMFamide family of neuropeptides in echinoderms. SALMFamide neuropeptides act as muscle relaxants and both S1 and S2 cause relaxation of cardiac stomach and tube foot preparations in vitro but S2 is an order of magnitude more potent than S1. Here we investigated a structural basis for this difference in potency using spectroscopic techniques. Circular dichroism spectroscopy showed that S1 does not have a defined structure in aqueous solution and this was supported by 2D nuclear magnetic resonance experiments. In contrast, we found that S2 has a well-defined conformation in aqueous solution. However, the conformation of S2 was concentration dependent, with increasing concentration inducing a transition from an unstructured to a structured conformation. Interestingly, this property of S2 was not observed in an N-terminally truncated analogue of S2 (short S2 or SS2; SFNSGLTFamide). Collectively, the data obtained indicate that the N-terminal region of S2 facilitates peptide self-association at high concentrations, which may have relevance to the biosynthesis and/or bioactivity of S2 in vivo.

The cellular prion protein traps Alzheimer's Aß in an oligomeric form and disassembles amyloid fibers.

There is now strong evidence to show that the presence of the cellular prion protein (PrP(C)) mediates amyloid-ß (Aß) neurotoxicity in Alzheimer's disease (AD). Here, we probe the molecular details of the interaction between PrP(C) and Aß and discover that substoichiometric amounts of PrP(C), as little as 1/20, relative to Aß will strongly inhibit amyloid fibril formation. This effect is specific to the unstructured N-terminal domain of PrP(C). Electron microscopy indicates PrP(C) is able to trap Aß in an oligomeric form. Unlike fibers, this oligomeric Aß contains antiparallel ß sheet and binds to a oligomer specific conformational antibody. Our NMR studies show that a specific region of PrP(C), notably residues 95-113, binds to Aß oligomers, but only once Aß misfolds. The ability of PrP(C) to trap and concentrate Aß in an oligomeric form and disassemble mature fibers suggests a mechanism by which PrP(C) might confer Aß toxicity in AD, as oligomers are thought to be the toxic form of Aß. Identification of a specific recognition site on PrP(C) that traps Aß in an oligomeric form is potentially a therapeutic target for the treatment of Alzheimer's disease.

Methionine oxidation perturbs the structural core of the prion protein and suggests a generic misfolding pathway.

Oxidative stress and misfolding of the prion protein (PrP(C)) are fundamental to prion diseases. We have therefore probed the effect of oxidation on the structure and stability of PrP(C). Urea unfolding studies indicate that H(2)O(2) oxidation reduces the thermodynamic stability of PrP(C) by as much as 9 kJ/mol. (1)H-(15)N NMR studies indicate methionine oxidation perturbs key hydrophobic residues on one face of helix-C as follows: Met-205, Val-209, and Met-212 together with residues Val-160 and Tyr-156. These hydrophobic residues pack together and form the structured core of the protein, stabilizing its ternary structure. Copper-catalyzed oxidation of PrP(C) causes a more significant alteration of the structure, generating a monomeric molten globule species that retains its native helical content. Further copper-catalyzed oxidation promotes extended ß-strand structures that lack a cooperative fold. This transition from the helical molten globule to ß-conformation has striking similarities to a misfolding intermediate generated at low pH. PrP may therefore share a generic misfolding pathway to amyloid fibers, irrespective of the conditions promoting misfolding. Our observations support the hypothesis that oxidation of PrP destabilizes the native fold of PrP(C), facilitating the transition to PrP(Sc). This study gives a structural and thermodynamic explanation for the high levels of oxidized methionine in scrapie isolates.

Copper(II)-induced secondary structure changes and reduced folding stability of the prion protein.

The cellular isoform of the prion protein PrP(C) is a Cu(2)(+)-binding cell surface glycoprotein that, when misfolded, is responsible for a range of transmissible spongiform encephalopathies. As changes in PrP(C) conformation are intimately linked with disease pathogenesis, the effect of Cu(2+) ions on the structure and stability of the protein has been investigated. Urea unfolding studies indicate that Cu(2+) ions destabilise the native fold of PrP(C). The midpoint of the unfolding transition is reduced by 0.73 ± 0.07 M urea in the presence of 1 mol equiv of Cu(2+). This equates to an appreciable difference in free energy of unfolding (2.02 ± 0.05 kJ mol(-1) at the midpoint of unfolding). We relate Cu(2)(+)-induced changes in secondary structure for full-length PrP(23-231) to smaller Cu(2)(+) binding fragments. In particular, Cu(2+)-induced structural changes can directly be attributed to Cu(2+) binding to the octarepeat region of PrP(C). Furthermore, a ß-sheet-like transition that is observed when Cu ions are bound to the amyloidogenic fragment of PrP (residues 90-126) is due only to local Cu(2+) coordination to the individual binding sites centred at His95 and His110. Cu(2+) binding does not directly generate a ß-sheet conformation within PrP(C); however, Cu(2+) ions do destabilise the native fold of PrP(C) and may make the transition to a misfolded state more favourable.