O-Glycosylation Induces Amyloid-ß To Form New Fibril Polymorphs Vulnerable for Degradation.

Brain accumulation of amyloid-ß (Aß) peptides (resulting from a disrupted balance between biosynthesis and clearance) occurs during the progression of Alzheimer's disease (AD). Aß peptides have diverse posttranslational modifications (PTMs) that variously modulate Aß aggregation into fibrils, but understanding the mechanistic roles of PTMs in these processes remains a challenge. Here, we chemically synthesized three homogeneously modified isoforms of Aß (1-42) peptides bearing Tyr10 O-glycosylation, an unusual PTM initially identified from the cerebrospinal fluid samples of AD patients. We discovered that O-glycans significantly affect both the aggregation and degradation of Aß42. By combining cryo-EM and various biochemical assays, we demonstrate that a Galß1-3GalNAc modification redirects Aß42 to form a new fibril polymorphic structure that is less stable and more vulnerable to Aß-degrading enzymes (e.g., insulin-degrading enzyme). Thus, beyond showing how particular O-glycosylation modifications affect Aß42 aggregation at the molecular level, our study provides powerful experimental tools to support further investigations about how PTMs affect Aß42 fibril aggregation and AD-related neurotoxicity.

A Cu-bis(imidazole) Substrate Intermediate Is the Catalytically Competent Center for Catechol Oxidase Activity of Copper Amyloid-ß.

Interaction of copper ions with Aß peptides alters the redox activity of the metal ion and can be associated with neurodegeneration. Many studies deal with the characterization of the copper binding mode responsible for the reactivity. Oxidation experiments of dopamine and related catechols by copper(II) complexes with the N-terminal amyloid-ß peptides Aß16 and Aß9, and the Aß16[H6A] and Aß16[H13A] mutant forms, both in their free amine and N-acetylated forms show that efficient reactivity requires the oxygenation of a CuI-bis(imidazole) complex with a bound substrate. Therefore, the active intermediate for catechol oxidation differs from the proposed "in-between state" for the catalytic oxidation of ascorbate. During the catechol oxidation process, hydrogen peroxide and superoxide anion are formed but give only a minor contribution to the reaction.

Mutual structural effects of unmodified and pyroglutamylated amyloid ß peptides during aggregation.

Amyloid ß (Aß) peptide aggregates are linked to Alzheimer's disease (AD). Posttranslationally pyroglutamylated Aß (pEAß) occurs in AD brains in significant quantities and is hypertoxic, but the underlying structural and aggregation properties remain poorly understood. Here, the structure and aggregation of Aß1-40 and pEAß3-40 are analyzed separately and in equimolar combination. Circular dichroism data show that Aß1-40 , pEAß3-40 , and their combination assume α-helical structure in dry state and transition to unordered structure in aqueous buffer. Aß1-40 and the 1:1 combination gradually acquire ß-sheet structure while pEAß3-40 adopts an α-helix/ß-sheet conformation. Thioflavin-T fluorescence studies suggest that the two peptides mutually inhibit fibrillogenesis. Fourier transform infrared (FTIR) spectroscopy identifies the presence of ß-turn and α-helical structures in addition to ß-sheet structure in peptides in aqueous buffer. The kinetics of transitions from the initial α-helical structure to ß-sheet structure were resolved by slow hydration of dry peptides by D2 O vapor, coupled with isotope-edited FTIR. These data confirmed the mutual suppression of ß-sheet formation by the two peptides. Remarkably, pEAß3-40 maintained a significant fraction of α-helical structure in the combined sample, implying a reduced ß-sheet propensity of pEAß3-40 . Altogether, the data imply that the combination of unmodified and pyroglutamylated Aß peptides resists fibrillogenesis and favors the prefibrillar state, which may underlie hypertoxicity of pEAß.

Deuterium solid-state NMR quadrupolar order rotating frame relaxation with applications to amyloid-ß fibrils.

We describe a new method for measuring molecular dynamics based on the deuterium solid-state nuclear magnetic resonance (NMR) quadrupolar order rotating frame relaxation rate R1ρ,Q under static conditions. The observed quadrupolar order coherence is created using the broad-band Jeener-Broekaert excitation and is locked with a weak radio frequency (RF) field. We describe the experimental and theoretical approaches and show applications to a selectively deuterated valine side chain of the phosphorylated amyloid-ß (1-40) fibrils phosphorylated at the serine-8 position. The R1ρ,Q rate is sensitive to the rotameric exchange mode. For biological samples, the low spin-lock field in the 5- to 10-kHz range has the advantage of avoiding sample heating and dehydration. Thus, it provides an alternative to approaches based on single-quantum coherence, which require larger spin-lock fields.

Site-specific glycation of Aß1-42 affects fibril formation and is neurotoxic.

Aß1-42 is involved in Alzheimer's disease (AD) pathogenesis and is prone to glycation, an irreversible process where proteins accumulate advanced glycated end products (AGEs). Nϵ-(Carboxyethyl)lysine (CEL) is a common AGE associated with AD patients and occurs at either Lys-16 or Lys-28 of Aß1-42. Methyglyoxal is commonly used for the unspecific glycation of Aß1-42, which results in a complex mixture of AGE-modified peptides and makes interpretation of a causative AGE at a specific amino acid residue difficult. We address this issue by chemically synthesizing defined CEL modifications on Aß1-42 at Lys-16 (Aß-CEL16), Lys-28 (Aß-CEL28), and Lys-16 and -28 (Aß-CEL16&28). We demonstrated that double-CEL glycations at Lys-16 and Lys-28 of Aß1-42 had the most profound impact on the ability to form amyloid fibrils. In silico predictions indicated that Aß-CEL16&28 had a substantial decrease in free energy change, which contributes to fibril destabilization, and a increased aggregation rate. Single-CEL glycations at Lys-28 of Aß1-42 had the least impact on fibril formation, whereas CEL glycations at Lys-16 of Aß1-42 delayed fibril formation. We also tested these peptides for neuronal toxicity and mitochondrial function on a retinoic acid-differentiated SH-SY5Y human neuroblastoma cell line (RA-differentiated SH-SY5Y). Only Aß-CEL16 and Aß-CEL28 were neurotoxic, possibly through a nonmitochondrial pathway, whereas Aß-CEL16&28 showed no neurotoxicity. Interestingly, Aß-CEL16&28 had depolarized the mitochondrial membrane potential, whereas Aß-CEL16 had increased mitochondrial respiration at complex II. These results may indicate mitophagy or an alternate route of metabolism, respectively. Therefore, our results provides insight into potential therapeutic approaches against neurotoxic CEL-glycated Aß1-42.

Aluminium Binding to Modified Amyloid-ß Peptides: Implications for Alzheimer's Disease.

Aluminium (Al) is clearly neurotoxic and considerable evidence exists that Al may play a role in the aetiology or pathogenesis of Alzheimer's disease (AD). Nevertheless, the link between AD pathology and Al is still open to debate. Therefore, we investigated here the interaction of aluminium ions with two Aß peptide fragments and their analogues. First, we synthesised by the Fmoc/tBu solid-phase peptide synthesis (SPPS) strategy using an automated peptide synthesiser two new peptides starting from the Aß(1-16) native peptide fragment. For this purpose, the three histidine residues (H6, H13, and H14) of the Aß(1-16) peptide were replaced by three alanine and three serine residues to form the modified peptides Aß(1-16)A36,13,14 and Aß(1-16)S36,13,14 (primary structures: H-1DAEFRADSGYEVAAQK16-NH2 and H-1DAEFRSDSGYEVSSQK16-NH2). In addition, the Aß(9-16) peptide fragment (H-9GYEVHHQK16-NH2) and its glycine analogues, namely Aß(9-16)G110, (H-9GGEVHHQK16-NH2), Aß(9-16)G213,14 (H-9GYEVGGQK16-NH2), and Aß(9-16)G310,13,14 (H-9GGEVGGQK16-NH2), were manually synthesised in order to study Al binding to more specific amino acid residues. Both the peptides and the corresponding complexes with aluminium were comparatively investigated by mass spectrometry (MS), circular dichroism spectroscopy (CD), atomic force microscopy (AFM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). Al-peptide molecular ions and Al-fragment ions were unambiguously identified in the MS and MS/MS spectra. AFM images showed dramatic changes in the film morphology of peptides upon Al binding. Our findings from the investigation of N-terminal 1-16 and even 9-16 normal and modified sequences of Aß peptides suggest that they have the capability to be involved in aluminium ion binding associated with AD.

Observation of ß-Amyloid Peptide Oligomerization by Pressure-Jump NMR Spectroscopy.

Brain tissue of Alzheimer's disease patients invariably contains deposits of insoluble, fibrillar aggregates of peptide fragments of the amyloid precursor protein (APP), typically 40 or 42 residues in length and referred to as Aß40 and Aß42. However, it remains unclear whether these fibrils or oligomers constitute the toxic species. Depending on sample conditions, oligomers can form in a few seconds or less. These oligomers are invisible to solution NMR spectroscopy, but they can be rapidly (<1 s) resolubilized and converted to their NMR-visible monomeric constituents by raising the hydrostatic pressure to a few kbar. Hence, utilizing pressure-jump NMR, the oligomeric state can be studied at residue-specific resolution by monitoring its signals in the monomeric state. Oligomeric states of Aß40 exhibit a high degree of order, reflected by slow longitudinal 15N relaxation (T1 > 5 s) for residues 18-21 and 31-34, whereas the N-terminal 10 residues relax much faster (T1 ≤ 1.5 s), indicative of extensive internal motions. Transverse relaxation rates rapidly increase to ca. 1000 s-1 after the oligomerization is initiated.

Native Ion Mobility-Mass Spectrometry Reveals the Formation of ß-Barrel Shaped Amyloid-ß Hexamers in a Membrane-Mimicking Environment.

The mechanisms behind the Amyloid-ß (Aß) peptide neurotoxicity in Alzheimer's disease are intensely studied and under debate. One suggested mechanism is that the peptides assemble in biological membranes to form ß-barrel shaped oligomeric pores that induce cell leakage. Direct detection of such putative assemblies and their exact oligomeric states is however complicated by a high level of heterogeneity. The theory consequently remains controversial, and the actual formation of pore structures is disputed. We herein overcome the heterogeneity problem by employing a native mass spectrometry approach and demonstrate that Aß(1-42) peptides form coclusters with membrane mimetic detergent micelles. The coclusters are gently ionized using nanoelectrospray and transferred into the mass spectrometer where the detergent molecules are stripped away using collisional activation. We show that Aß(1-42) indeed oligomerizes over time in the micellar environment, forming hexamers with collision cross sections in agreement with a general ß-barrel structure. We also show that such oligomers are maintained and even stabilized by addition of lipids. Aß(1-40) on the other hand form significantly lower amounts of oligomers, which are also of lower oligomeric state compared to Aß(1-42) oligomers. Our results thus support the oligomeric pore hypothesis as one important cell toxicity mechanism in Alzheimer's disease. The presented native mass spectrometry approach is a promising way to study such potentially very neurotoxic species and how they could be stabilized or destabilized by molecules of cellular or therapeutic relevance.

High-speed atomic force microscopy reveals structural dynamics of amyloid ß1-42 aggregates.

Aggregation of amyloidogenic proteins into insoluble amyloid fibrils is implicated in various neurodegenerative diseases. This process involves protein assembly into oligomeric intermediates and fibrils with highly polymorphic molecular structures. These structural differences may be responsible for different disease presentations. For this reason, elucidation of the structural features and assembly kinetics of amyloidogenic proteins has been an area of intense study. We report here the results of high-speed atomic force microscopy (HS-AFM) studies of fibril formation and elongation by the 42-residue form of the amyloid ß-protein (Aß1-42), a key pathogenetic agent of Alzheimer's disease. Our data demonstrate two different growth modes of Aß1-42, one producing straight fibrils and the other producing spiral fibrils. Each mode depends on initial fibril nucleus structure, but switching from one growth mode to another was occasionally observed, suggesting that fibril end structure fluctuated between the two growth modes. This switching phenomenon was affected by buffer salt composition. Our findings indicate that polymorphism in fibril structure can occur after fibril nucleation and is affected by relatively modest changes in environmental conditions.

Platinum(II) O,S Complexes Inhibit the Aggregation of Amyloid Model Systems.

Platinum(II) complexes with different cinnamic acid derivatives as ligands were investigated for their ability to inhibit the aggregation process of amyloid systems derived from Aß, Yeast Prion Protein Sup35p and the C-terminal domain of nucleophosmin 1. Thioflavin T binding assays and circular dichroism data indicate that these compounds strongly inhibit the aggregation of investigated peptides exhibiting IC50 values in the micromolar range. MS analysis confirms the formation of adducts between peptides and Pt(II) complexes that are also able to reduce amyloid cytotoxicity in human SH-SY5Y neuroblastoma cells. Overall data suggests that bidentate ligands based on ß-hydroxy dithiocinnamic esters can be used to develop platinum or platinoid compounds with anti-amyloid aggregation properties.

Key aromatic/hydrophobic amino acids controlling a cross-amyloid peptide interaction versus amyloid self-assembly.

The interaction of the intrinsically disordered polypeptide islet amyloid polypeptide (IAPP), which is associated with type 2 diabetes (T2D), with the Alzheimer's disease amyloid-ß (Aß) peptide modulates their self-assembly into amyloid fibrils and may link the pathogeneses of these two cell-degenerative diseases. However, the molecular determinants of this interaction remain elusive. Using a systematic alanine scan approach, fluorescence spectroscopy, and other biophysical methods, including heterocomplex pulldown assays, far-UV CD spectroscopy, the thioflavin T binding assay, transmission EM, and molecular dynamics simulations, here we identified single aromatic/hydrophobic residues within the amyloid core IAPP region as hot spots or key residues of its cross-interaction with Aß40(42) peptide. Importantly, we also find that none of these residues in isolation plays a key role in IAPP self-assembly, whereas simultaneous substitution of four aromatic/hydrophobic residues with Ala dramatically impairs both IAPP self-assembly and hetero-assembly with Aß40(42). Furthermore, our experiments yielded several novel IAPP analogs, whose sequences are highly similar to that of IAPP but have distinct amyloid self- or cross-interaction potentials. The identified similarities and major differences controlling IAPP cross-peptide interaction with Aß40(42) versus its amyloid self-assembly offer a molecular basis for understanding the underlying mechanisms. We propose that these insights will aid in designing intervention strategies and novel IAPP analogs for the management of type 2 diabetes, Alzheimer's disease, or other diseases related to IAPP dysfunction or cross-amyloid interactions.

Energy Landscapes for the Aggregation of Aß17-42.

The aggregation of the Aß peptide (Aß1-42) to form fibrils is a key feature of Alzheimer's disease. The mechanism is thought to be a nucleation stage followed by an elongation process. The elongation stage involves the consecutive addition of monomers to one end of the growing fibril. The aggregation process proceeds in a stop-and-go fashion and may involve off-pathway aggregates, complicating experimental and computational studies. Here we present exploration of a well-defined region in the free and potential energy landscapes for the Aß17-42 pentamer. We find that the ideal aggregation process agrees with the previously reported dock-lock mechanism. We also analyze a large number of additional stable structures located on the multifunnel energy landscape, which constitute kinetic traps. The key contributors to the formation of such traps are misaligned strong interactions, for example the stacking of F19 and F20, as well as entropic contributions. Our results suggest that folding templates for aggregation are a necessity and that aggregation studies could employ such species to obtain a more detailed description of the process.

Efficient synthesis and characterisation of the amyloid beta peptide, Aß1-42, using a double linker system.

The amyloidogenic Aß42 peptide was efficiently prepared using a double linker system, markedly improving solubility and chromatographic peak resolution, thus enabling full characterisation using standard techniques. The tag was readily cleaved with sodium hydroxide and removed by aqueous extraction, affording Aß42 in high purity and yield for biophysical characterisation studies.

Synthesized Aß42 Caused Intracellular Oxidative Damage, Leading to Cell Death, via Lysosome Rupture.

Neuronal cellular accumulation of amyloid beta peptide (Aß) has been implicated in the pathogenesis of Alzheimer's disease (AD). Intracellular accumulation of Aß42, a toxic form of Aß, was observed as an early event in AD patients. However, its contribution and the cellular mechanism of cell death remained unclear. We herein revealed the mechanism by which Aß42 incorporated into cells leads to cell death by using chemically synthesized Aß42 variants. The Aß42 variant Aß42 (E22P) which has an increased tendency to oligomerize, accumulated in lysosomes at an earlier stage than wild-type Aß42, leading to higher ROS production and lysosomal membrane oxidation, and resulting in cell death. On the other hand, Aß42 (E22V), which is incapable of oligomerization, did not accumulate in cells or affect the cell viability. Moreover, intracellular localization of EGFP-Galectin-3, a ß-galactoside binding lectin, showed that accumulation of oligomerized Aß42 in lysosomes caused lysosomal membrane permeabilization (LMP). Overexpression of lysosome-localized LAMP1-fused peroxiredoxin 1 and treatment with U18866A, an inhibitor of cholesterol export from lysosomes that causes an increase in lysosomal membrane stability, attenuated Aß42-mediated LMP and cell death. Our findings show that lysosomal ROS generation by toxic conformer of Aß led to cell death via LMP, and suggest that these events are potential targets for AD prevention.Key words: Amyloid-beta (Aß), Cell death, Lysosome, Lysosomal membrane permeabilization, Reactive oxygen species (ROS).

Clusterin Binds to Aß1-42 Oligomers with High Affinity and Interferes with Peptide Aggregation by Inhibiting Primary and Secondary Nucleation.

The aggregation of amyloid ß protein (Aß) is a fundamental pathogenic mechanism leading to the neuronal damage present in Alzheimer disease, and soluble Aß oligomers are thought to be a major toxic culprit. Thus, better knowledge and specific targeting of the pathways that lead to these noxious species may result in valuable therapeutic strategies. We characterized some effects of the molecular chaperone clusterin, providing new and more detailed evidence of its potential neuroprotective effects. Using a classical thioflavin T assay, we observed a dose-dependent inhibition of the aggregation process. The global analysis of time courses under different conditions demonstrated that clusterin has no effect on the elongation rate but mainly interferes with the nucleation processes (both primary and secondary), reducing the number of nuclei available for further fibril growth. Then, using a recently developed immunoassay based on surface plasmon resonance, we obtained direct evidence of a high-affinity (KD= 1 nm) interaction of clusterin with biologically relevant Aß1-42oligomers, selectively captured on the sensor chip. Moreover, with the same technology, we observed that substoichiometric concentrations of clusterin prevent oligomer interaction with the antibody 4G8, suggesting that the chaperone shields hydrophobic residues exposed on the oligomeric assemblies. Finally, we found that preincubation with clusterin antagonizes the toxic effects of Aß1-42oligomers, as evaluated in a recently developedin vivomodel inCaenorhabditis elegans.These data substantiate the interaction of clusterin with biologically active regions exposed on nuclei/oligomers of Aß1-42, providing a molecular basis for the neuroprotective effects of the chaperone.

Water Distribution, Dynamics, and Interactions with Alzheimer's ß-Amyloid Fibrils Investigated by Solid-State NMR.

Water is essential for protein folding and assembly of amyloid fibrils. Internal water cavities have been proposed for several amyloid fibrils, but no direct structural and dynamical data have been reported on the water dynamics and site-specific interactions of water with the fibrils. Here we use solid-state NMR spectroscopy to investigate the water interactions of several Aß40 fibrils. 1H spectral lineshapes, T2 relaxation times, and two-dimensional (2D) 1H-13C correlation spectra show that there are five distinct water pools: three are peptide-bound water, while two are highly dynamic water that can be assigned to interfibrillar water and bulk-like matrix water. All these water pools are associated with the fibrils on the nanometer scale. Water-transferred 2D correlation spectra allow us to map out residue-specific hydration and give evidence for the presence of a water pore in the center of the three-fold symmetric wild-type Aß40 fibril. In comparison, the loop residues and the intramolecular strand-strand interface have low hydration, excluding the presence of significant water cavities in these regions. The Osaka Aß40 mutant shows lower hydration and more immobilized water than wild-type Aß40, indicating the influence of peptide structure on the dynamics and distribution of hydration water. Finally, the highly mobile interfibrillar and matrix water exchange with each other on the time scale of seconds, suggesting that fibril bundling separates these two water pools, and water molecules must diffuse along the fibril axis before exchanging between these two environments. These results provide insights and experimental constraints on the spatial distribution and dynamics of water pools in these amyloid fibrils.

Thermodynamics of Polypeptide Supramolecular Assembly in the Short-Chain Limit.

The self-assembly of peptides into ordered supramolecular structures, such as fibrils and crystals, is of relevance in such diverse areas as molecular medicine and materials science. However, little information is available about the fundamental thermodynamic driving forces of these types of self-assembly processes. Here, we investigate in detail the thermodynamics of assembly of diphenylalanine (FF). This dipeptide forms the central motif of the Aß peptides, which are associated with Alzheimer's disease through their presence in amyloid plaques in the nervous systems of affected individuals. We identify the molecular origins of the self-assembly of FF in aqueous solution, and we evaluate these findings in the context of the aggregation free energies of longer peptides that are able to form amyloid fibrils. We find that the thermodynamics of FF assembly displays the typical characteristics of hydrophobic desolvation processes, and detailed analysis of the temperature dependence of the kinetics of assembly within the framework of crystallization theories reveals that the transition state from solution to crystalline aggregates is enthalpically unfavorable and entropically favorable, qualitatively similar to what has been found for longer sequences. This quantitative comparison of aggregating peptides of very different lengths is the basis of an in-depth understanding of the relationship between sequence and assembly behavior.

α-Helix Mimetics as Modulators of Aß Self-Assembly.

A key molecular species in Alzheimer's disease (AD) is the Aß42 alloform of Aß peptide, which is dominant in the amyloid plaques deposited in the brains of AD patients. Recent studies have decisively demonstrated that the prefibrillar soluble oligomers are the neurotoxic culprits and are associated with the pathology of AD. Nascent Aß42 is predominantly disordered but samples α-helical conformations covering residues 15-24 and 29-35 in the presence of micelles and structure-inducing solvents. In this report, a focused library of oligopyridylamide based α-helical mimetics was designed to target the central α-helix subdomain of Aß (Aß13-26). A tripyridylamide, ADH-41, was identified as one of the most potent antagonists of Aß fibrillation. Amyloid-assembly kinetics, transmission electron microscopy (TEM), and atomic force microscopy (AFM) show that ADH-41 wholly suppresses the aggregation of Aß at a substoichiometric dose. Dot blot and ELISA assays demonstrate the inhibition of the putative neurotoxic Aß oligomers. ADH-41 targets Aß in a sequence and structure-specific manner, as it did not have any effect on the aggregation of islet amyloid polypeptide (IAPP), a peptide which shares sequence similarity with Aß. Spectroscopic studies using NMR and CD confirm induction of α-helicity in Aß mediated by ADH-41. Calorimetric and fluorescence titrations yielded binding affinity in the low micromolar range. ADH-41 was also effective at inhibiting the seed-catalyzed aggregation of Aß probably by modulating the Aß conformation into a fiber incompetent structure. Overall, we speculate that ADH-41 directs Aß into off-pathway structures, and thereby alters various solution based functions of Aß. Cell-based assays to assess the effect of ADH-41 on Aß are underway and will be presented in due course.

Comparison of neurotoxicity of different aggregated forms of Aß40, Aß42 and Aß43 in cell cultures.

The abnormal deposition of amyloid-ß (Aß) peptides in the brain is the main neuropathological hallmark of Alzheimer's disease (AD). Amyloid deposits are formed by a heterogeneous mixture of Aß peptides, among which the most studied are Aß40 and Aß42. Aß40 is abundantly produced in the human brain, but the level of Aß42 is remarkably increased in the brain of AD patients. Aside from Aß40 and Aß42, recent data have raised the possibility that Aß43 peptides may be instrumental in AD pathogenesis. Besides its length, whether the Aß aggregated form accounts for the neurotoxicity is also particularly controversial. Aß fibrils are generally considered as key pathogenic substances in AD pathogenesis. Nevertheless, recent data implicated soluble Aß oligomers as the main cause of synaptic dysfunction and memory loss in AD. To further address this uncertainty, we analyzed the neurotoxicity of different Aß species and Aß forms at the cellular level. The results showed that Aß42 could form oligomers significantly faster than Aß40 and Aß43 and Aß42 oligomers showed the greatest level of neurotoxicity. Regardless of the length of Aß peptides, Aß oligomers induced significantly higher cytotoxicity compared with the other two Aß forms. Surprisingly, the neurotoxicity of fibrils in PC12 cells was only marginally but not significantly stronger than monomers, contrary to previous reports. Altogether, our findings demonstrate the high pathogenicity of Aß42 among the three Aß species and support the idea that Aß42 oligomers contribute to the pathological events leading to neurodegeneration in AD. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

MMP-7 cleaves amyloid ß fragment peptides and copper ion inhibits the degradation.

The extracellular deposition of amyloid ß (Aß) is known to be the fundamental cause of Alzheimer's disease (AD). Aß1-42, generated by ß-secretases from the amyloid precursor protein (APP), is the main component of neuritic plaque, and the aggregation of this protein is shown to be dependent to an extent on metal ions such as copper and zinc. However, the mechanism by which Cu2+ affects the physicochemical properties of Aß1-42 or the central nervous system is still under debate. A recent series of studies have demonstrated that both the soluble-type matrix metalloproteinases (MMP-2 and MMP-9) and the membrane-type matrix metalloproteinase (MT1-MMP) are capable of degrading Aß peptides. MMP-7, one of the soluble-type matrix metalloproteinases, is expressed in hippocampal tissue; however, less information is available concerning the pathophysiological roles of this enzyme in the process and/or progress of Alzheimer's disease. In this study, we examined the degradation activity of MMP-7 against various Aß1-42's fragment peptides and the effect of Cu2+. Although Aß22-40 was degraded by MMP-7 regardless of Cu2+, Cu2+ inhibited the degradation of Aß1-19, Aß11-20, and Aß11-29 by MMP-7. These results indicate that MMP-7 is capable of degrading Aß1-42, and that Aß1-42 acquired resistance against MMP-7 cleavage through Cu2+-binding and structure changes. Our results demonstrate that MMP-7 may play an important role in the defensive mechanism against the aggregation of Aß1-42, which gives rise to the pathology of AD.