In vitro and in vivo Staining Characteristics of Small,Fluorescent, Aβ42‐Binding D‐Enantiomeric Peptides in Transgenic AD Mouse Models

Plaque visualisation : We identified three different D ‐enantiomeric peptides that bind to Alzheimer's amyloid β (Aβ1‐42). As there is currently no definitive pre‐mortem diagnosis for Alzheimer's disease, we investigated the peptides' suitability as molecular probes for in vivo imaging in transgenic mouse models.

     

Sialic Acid Hydroxamate: A Potential Antioxidant and Inhibitor of Metal‐Induced β‐Amyloid Aggregates

Current methods for Alzheimer's treatment require a three‐component system: metal chelators, antioxidants, and amyloid β (Aβ)‐peptide‐binding scaffolds. We report sialic acid (Sia) hydroxamate as a potential radical scavenger and metal chelator to inhibit Aβ aggregation. A cell viability assay revealed that Sia hydroxamate can protect HeLa and glioblastoma (LN229) cells from oxidative damage induced by the Fenton reaction. Sedimentation and turbidity assays showed profound protection of neuroblastoma SH‐SY5Y cells from metal‐induced Aβ aggregation and neural toxicity.     

A Cyclic KLVFF‐Derived Peptide Aggregation Inhibitor Induces the Formation of Less‐Toxic Off‐Pathway Amyloid‐β Oligomers

Inhibition of amyloid‐β (Aβ) aggregation could be a target of drug development for the treatment of currently incurable Alzheimer's disease. We previously reported that a head‐to‐tail cyclic peptide of KLVFF (cyclic‐KLVFF), a pentapeptide fragment corresponding to the Aβ16–20 region (which plays a critical role in the generating Aβ fibrils), possesses potent inhibitory activity against Aβ aggregation. Here we found that the inhibitory activity of cyclic‐KLVFF was significantly improved by incorporating an additional phenyl group at the β‐position of the Phe4 side chain (inhibitor 3 ). Biophysical and biochemical analyses revealed the rapid formation of 3 ‐embedded oligomer species when Aβ1–42 was mixed with 3 . The oligomer species is an “off‐pathway” species with low affinity for cross‐β‐sheet‐specific dye thioflavin T and oligomer‐specific A11 antibodies. The oligomer species had a sub‐nanometer height and little capability of aggregation to amyloid fibrils. Importantly, the toxicity of the oligomer species was significantly lower than that of native Aβ oligomers. These insights will be useful for further refinement of cyclic‐KLVFF‐based aggregation inhibitors.     

Spatiotemporal Control of Amyloid‐Like Aβ Plaque Formation Using a Multichannel Organic Electronic Device

We herein report on an iontronic device to drive and control Aβ1‐40 and Aβ1‐42 fibril formation. This system allows kinetic control of Aβ aggregation by regulation of H⁺ flows. The formed aggregates show both nanometer‐sized fibril structure and microscopic growth, thus mimicking senile plaques, at the H⁺‐outlet. Mechanistically we observed initial accumulation of Aβ1‐40 likely driven by electrophoretic migration which preceded nucleation of amyloid structures in the accumulated peptide cluster.

     

Carnosine Inhibits Aβ42 Aggregation by Perturbing the H‐Bond Network in and around the Central Hydrophobic Cluster

Aggregation of the amyloid‐β peptide (Aβ) into fibrillar structures is a hallmark of Alzheimer's disease. Thus, preventing self‐assembly of the Aβ peptide is an attractive therapeutic strategy. Here, we used experimental techniques and atomistic simulations to investigate the influence of carnosine, a dipeptide naturally occurring in the brain, on Aβ aggregation. Scanning force microscopy, circular dichroism and thioflavin T fluorescence experiments showed that carnosine does not modify the conformational features of Aβ42 but nonetheless inhibits amyloid growth. Molecular dynamics (MD) simulations indicated that carnosine interacts transiently with monomeric Aβ42 by salt bridges with charged side chains, and van der Waals contacts with residues in and around the central hydrophobic cluster (¹⁷LVFFA²¹). NMR experiments on the nonaggregative fragment Aβ12–28 did not evidence specific intermolecular interactions between the peptide and carnosine, in agreement with MD simulations. However, a close inspection of the spectra revealed that carnosine interferes with the local propensity of the peptide to form backbone hydrogen bonds close to the central hydrophobic cluster (residues E22, S26 and N27). Finally, MD simulations of aggregation‐prone Aβ heptapeptide segments show that carnosine reduces the propensity to form intermolecular backbone hydrogen bonds in the region 18–24. Taken together, the experimental and simulation results (cumulative MD sampling of 0.2 ms) suggest that, despite the inability of carnosine to form stable contacts with Aβ, it might block the pathway toward toxic aggregates by perturbing the hydrogen bond network near residues with key roles in fibrillogenesis.     

Copper and Zinc Binding to Amyloid‐β: Coordination,Dynamics, Aggregation,Reactivity and Metal‐Ion Transfer

The metal ions copper, zinc and iron have been shown to be involved in Alzheimer's disease (AD). Cu, Zn and Fe ions are proposed to be implicated in two key steps of AD pathology: 1) aggregation of the peptide amyloid‐β (Aβ), and 2) production of reactive oxygen species (ROS) induced by Aβ. There is compelling evidence that Cu and Zn bind directly to Aβ in AD. This formation of Cu/Zn–Aβ complexes is thought to be aberrant as they have been detected only in AD, but not under healthy conditions. In this context, the understanding of how these metal ions interact with Aβ, their influence on structure and oligomerization become an important issue for AD. Moreover, the mechanism of ROS production by Cu–Aβ in relation to its aggregations state, as well as the metal‐transfer reaction from and to Aβ are crucial in order to understand why Aβ oligomers are highly toxic and why Aβ seems to bind Cu and Zn only in AD.     

Retro‐Enantio N‐Methylated Peptides as β‐Amyloid Aggregation Inhibitors

An emerging and attractive target for the treatment of Alzheimer's disease is to inhibit the aggregation of β‐amyloid protein (Aβ). We applied the retro‐enantio concept to design an N‐methylated peptidic inhibitor of the Aβ42 aggregation process. This inhibitor, inrD, as well as the corresponding all‐L (inL) and all‐D (inD) analogues were assayed for inhibition of Aβ42 aggregation. They were also screened in neuroblastoma cell cultures to assess their capacity to inhibit Aβ42 cytotoxicity and evaluated for proteolytic stability. The results reveal that inrD and inD inhibit Aβ42 aggregation more effectively than inL, that inrD decreases Aβ42 cytotoxicity to a greater extent than inL and inD, and that as expected, both inD and inrD are stable to proteases. Based on these results, we propose that the retro‐enantio approach should be considered in future designs of peptide inhibitors of protein aggregation.     

The Natural Products Beauveriolide I and III: a New Class of β‐Amyloid‐Lowering Compounds

Attacking Alzheimer's by ACAT : The aggregation of β‐amyloid peptides, especially Aβ₄₂, into senile plaques is a hallmark of Alzheimer's disease (AD). We show that the fungal natural products beauveriolides I and III can potently decrease Aβ secretion from cells expressing human amyloid precursor protein; this offers a potential new scaffold for the development of compounds with proven bioavailability for the treatment of AD.

     

Biophysical Studies of the Amyloid β‐Peptide: Interactions with Metal Ions and Small Molecules

Alzheimer's disease is the most common of the protein misfolding (“amyloid”) diseases. The deposits in the brains of afflicted patients contain as a major fraction an aggregated insoluble form of the so‐called amyloid β‐peptides (Aβ peptides): fragments of the amyloid precursor protein of 39–43 residues in length. This review focuses on biophysical studies of the Aβ peptides: that is, of the aggregation pathways and intermediates observed during aggregation, of the molecular structures observed along these pathways, and of the interactions of Aβ with Cu and Zn ions and with small molecules that modify the aggregation pathways. Particular emphasis is placed on studies based on high‐resolution and solid‐state NMR methods. Theoretical studies relating to the interactions are also included. An emerging picture is that of Aβ peptides in aqueous solution undergoing hydrophobic collapse together with identical partners. There then follows a relatively slow process leading to more ordered secondary and tertiary (quaternary) structures in the growing aggregates. These aggregates eventually assemble into elongated fibrils visible by electron microscopy. Small molecules or metal ions that interfere with the aggregation processes give rise to a variety of aggregation products that may be studied in vitro and considered in relation to observations in cell cultures or in vivo. Although the heterogeneous nature of the processes makes detailed structural studies difficult, knowledge and understanding of the underlying physical chemistry might provide a basis for future therapeutic strategies against the disease. A final part of the review deals with the interactions that may occur between the Aβ peptides and the prion protein, where the latter is involved in other protein misfolding diseases.     

Development of Alkaline Phosphatase-Fused Mouse Prion Protein and Its Application in Toxic Aβ Oligomer Detection

Amyloid β (Aβ) oligomers play a key role in the progression of Alzheimer’s disease (AD). Multiple forms of Aβ assemblies have been identified by in vitro and in vivo analyses; however, it is uncertain which oligomer is highly neurotoxic. Thus, understanding the pathogenesis of AD by detecting toxic Aβ oligomers is crucial. In this study, we report a fusion protein of cellular prion protein (PrPc) and alkaline phosphatase (ALP) from Escherichia coli as a sensing element for toxic Aβ oligomers. Since the N-terminus domain of PrPc (residue 23–111) derived from mice is known to bind to toxic Aβ oligomers in vitro, we genetically fused PrPc_23–111 to ALP. The developed fusion protein, PrP–ALP, retained both the binding ability of PrPc and enzymatic activity of ALP. We showed that PrP–ALP strongly bound to high molecular weight (HMW) oligomers but showed little or no affinity toward monomers. The observation that PrP–ALP neutralized the toxic effect of Aβ oligomers indicated an interaction between PrP–ALP and toxic HMW oligomers. Based on ALP activity, we succeeded in detecting Aβ oligomers. PrP–ALP may serve as a powerful tool for detecting toxic Aβ oligomers that may be related to AD progression.     

Modulation of Amyloid‐β Aggregation by Histidine‐Coordinating Cobalt(III) Schiff Base Complexes

Oligomers of the Aβ42 peptide are significant neurotoxins linked to Alzheimer's disease (AD). Histidine (His) residues present at the N terminus of Aβ42 are believed to influence toxicity by either serving as metal–ion binding sites (which promote oligomerization and oxidative damage) or facilitating synaptic binding. Transition metal complexes that bind to these residues and modulate Aβ toxicity have emerged as therapeutic candidates. Cobalt(III) Schiff base complexes (Co–sb) were evaluated for their ability to interact with Aβ peptides. HPLC‐MS, NMR, fluorescence, and DFT studies demonstrated that Co–sb complexes could interact with the His residues in a truncated Aβ16 peptide representing the Aβ42 N terminus. Coordination of Co–sb complexes altered the structure of Aβ42 peptides and promoted the formation of large soluble oligomers. Interestingly, this structural perturbation of Aβ correlated to reduced synaptic binding to hippocampal neurons. These results demonstrate the promise of Co–sb complexes in anti‐AD therapeutic approaches.     

Unambiguous Assignment of Short‐ and Long‐Range Structural Restraints by Solid‐State NMR Spectroscopy with Segmental Isotope Labeling

We present an efficient method for the reduction of spectral complexity in the solid‐state NMR spectra of insoluble protein assemblies, without loss of signal intensity. The approach is based on segmental isotope labeling by using the split intein DnaE from Nostoc punctiforme. We show that the segmentally ¹³C,¹⁵N‐labeled prion domain of HET‐s exhibits significantly reduced spectral overlap while retaining the wild‐type structure and spectral quality. A large number of unambiguous distance restraints were thus collected from a single two‐dimensional ¹³C,¹³C cross‐correlation spectrum. The observed resonances could be unambiguously identified as intramolecular without the need for preparing a dilute, less sensitive sample.     

Trans-Chalcone Plus Baicalein Synergistically Reduce Intracellular Amyloid Beta (Aβ42) and Protect from Aβ42 Induced Oxidative Damage in Yeast Models of Alzheimer’s Disease

Finding an effective therapeutic to prevent or cure AD has been difficult due to the complexity of the brain and limited experimental models. This study utilized unmodified and genetically modified Saccharomyces cerevisiae as model organisms to find potential natural bioactive compounds capable of reducing intracellular amyloid beta 42 (Aβ₄₂) and associated oxidative damage. Eleven natural bioactive compounds including mangiferin, quercetin, rutin, resveratrol, epigallocatechin gallate (EGCG), urolithin A, oleuropein, rosmarinic acid, salvianolic acid B, baicalein and trans-chalcone were screened for their ability to reduce intracellular green fluorescent protein tagged Aβ₄₂ (GFP-Aβ₄₂) levels. The two most effective compounds from the screens were combined in varying concentrations of each to study the combined capacity to reduce GFP-Aβ₄₂. The most effective combinations were examined for their effect on growth rate, turnover of native Aβ₄₂ and reactive oxygen species (ROS). The bioactive compounds except mangiferin and urolithin A significantly reduced intracellular GFP-Aβ₄₂ levels. Baicalein and trans-chalcone were the most effective compounds among those that were screened. The combination of baicalein and trans-chalcone synergistically reduced GFP-Aβ₄₂ levels. A combination of 15 μM trans-chalcone and 8 μM baicalein was found to be the most synergistic combination. The combination of the two compounds significantly reduced ROS and Aβ₄₂ levels in yeast cells expressing native Aβ₄₂ without affecting growth of the cells. These findings suggest that the combination of baicalein and trans-chalcone could be a promising multifactorial therapeutic strategy to cure or prevent AD. However, further studies are recommended to look for similar cytoprotective activity in humans and to find an optimal dosage.     

Distance measurement between Tyr10 and Met35 in amyloid beta by site-directed spin-labeling ESR spectroscopy: implications for the stronger neurotoxicity of Abeta42 than Abeta40

The neurotoxicity of the 42-mer and 40-mer amyloid beta peptides (Abeta42 and Abeta40) is closely related to the radicalization at both Tyr10 and Met35. Abeta42 is more neurotoxic than Abeta40. Our previous structural analyses of Abeta42 suggested that Tyr10 and Met35 are brought closer together by the turn at positions 22 and 23, and the S-oxidized radical cation at position 35, which is the ultimate toxic radical species, can be produced effectively through oxidation by the phenoxy radical at position 10. To verify this idea, their separation was measured by site-directed spin labeling (MTSSL) by using ESR spectroscopy. Among the three kinds of Abeta42 derivatives, which are doubly or singly spin-labeled at position 10 and 35, only 10,35-MTSSL-Abeta42 showed a clear dipole coupling in continuous-wave ESR; this suggests that the intramolecular spin labels at position 10 and 35 in Abeta42 are located within approximately 15 A. In contrast, 10,35-MTSSL-Abeta40 did not give such signals. The distance between Tyr10 and Met35 in 10,35-MTSSL-Abeta40, which was successfully measured by pulsed ESR spectroscopy was 30 A long. The difference in the distance between Abeta42 and Abeta40 could explain in part the stronger neurotoxicity of Abeta42 compared to Abeta40.