Inhibition of beta-amyloid peptide aggregation and neurotoxicity by alpha-d-mannosylglycerate, a natural extremolyte

The aggregation of soluble beta-amyloid (Abeta) peptide into oligomers/fibrils is one of the key pathological features in Alzheimer's disease (AD). The use of naturally occurring small molecules for inhibiting protein aggregation has recently attracted many interests due to their effectiveness for treating protein folding diseases such as AD, Parkinson's, Huntington's disease, and other amyloidosis diseases. alpha-d-Mannosylglycerate (MG), a natural extremolyte identified in microorganisms growing under extremely high temperatures up to 100 degrees C, had been shown to protect proteins against various stress conditions such as heat, freezing, thawing, and drying. Here, we report the effectiveness of MG on the suppression of Alzheimer's Abeta aggregation and neurotoxicity to human neuroblastoma cells. According to our study--carried out by using thioflavin-T induced fluorescence, atomic force microscopy, and cell viability assay--MG had significant inhibitory effect against Abeta amyloid formation and could reduce the toxicity of amyloid aggregates to human neuroblastoma cells while MG itself was innocuous to cells. On the other hand, the structural analogs of MG such as alpha-d-mannosylglyceramide, mannose, methylmannoside, glycerol, showed negligible effect on Abeta aggregate formation. The results suggest that MG could be a potential drug candidate for treating Alzheimer's disease.     

Beta-amyloid-derived pentapeptide RIIGLa inhibits Abeta(1-42) aggregation and toxicity

Pr-IIGL(a), a derivative of the tetrapeptide beta-amyloid 31-34 (Abeta(31-34)), exerts controversial effects: it is toxic in a neuroblastoma culture, but it protects glial cells from the cytotoxic action of Abeta(1-42). For an understanding of this phenomenon, a new pentapeptide, RIIGL(a) was synthetized, and both compounds were studied by different physicochemical and biological methods. Transmission electron microscopic (TEM) studies revealed that Pr-IIGL(a) forms fibrillar aggregates, whereas RIIGL(a) does not form fibrils. Congo red binding studies furnished the same results. Aggregated Pr-IIGL(a) acts as a cytotoxic agent in neuroblastoma cultures, but RIIGL(a) does not display inherent toxicity. RIIGL(a) co-incubated with Abeta(1-42) inhibits the formation of mature amyloid fibres (TEM studies) and reduces the cytotoxic effect of fibrillar Abeta(1-42). These results indicate that RIIGL(a) is an effective inhibitor of both the aggregation and the toxic effects of Abeta(1-42) and can serve as a lead compound for the design of novel neuroprotective peptidomimetics.     

An FT-IR study of the beta-amyloid conformation: standardization of aggregation grade

The aggregation of beta-amyloid peptides is very important for their neurotoxic effect; standardization of the aggregation grade is necessary for biological experiments. Measurement of aggregation with physicochemical methods is a difficult task. The present work revealed that FT-IR can be used for studying the aggregation properties of beta-amyloid peptides and the effects of environmental variables (solvent, pH, ions, and temperature) on aggregation. In dimethyl sulfoxide or hexafluoroisopropanol, amyloid peptides are in a monomeric state; on dilution with phosphate buffer just before measurement is made, aggregation begins. A detailed two-dimensional FT-IR correlation spectroscopic study was made of the conformational transitions that occur during the aggregation of beta-amyloid peptides. Two processes (random/helix-to-beta-sheet and aggregation of beta-sheets) and multiple conformational states were observed before the most stable form was attained. beta-Amyloid peptides undergo decomposition in basic buffers containing Ca(2+); this process should be avoided during aging experiments.     

Metal ions differentially influence the aggregation and deposition of Alzheimer's beta-amyloid on a solid template

The abnormal deposition and aggregation of beta-amyloid (Abeta) on brain tissues are considered to be one of the characteristic neuropathological features of Alzheimer's disease (AD). Environmental conditions such as metal ions, pH, and cell membranes are associated with Abeta deposition and plaque formation. According to the amyloid cascade hypothesis of AD, the deposition of Abeta42 oligomers as diffuse plaques in vivo is an important earliest event, leading to the formation of fibrillar amyloid plaques by the further accumulation of soluble Abeta under certain environmental conditions. In order to characterize the effect of metal ions on amyloid deposition and plaque growth on a solid surface, we prepared a synthetic template by immobilizing Abeta oligomers onto a N-hydroxysuccinimide ester-activated solid surface. According to our study using ex situ atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FT-IR), and thioflavin T (ThT) fluorescence spectroscopy, Cu2+ and Zn2+ ions accelerated both Abeta40 and Abeta42 deposition but resulted only in the formation of "amorphous" aggregates. In contrast, Fe3+ induced the deposition of "fibrillar" amyloid plaques at neutral pH. Under mildly acidic environments, the formation of fibrillar amyloid plaques was not induced by any metal ion tested in this work. Using secondary ion mass spectroscopy (SIMS) analysis, we found that binding Cu ions to Abeta deposits on a solid template occurred by the possible reduction of Cu ions during the interaction of Abeta with Cu2+. Our results may provide insights into the role of metal ions on the formation of fibrillar or amorphous amyloid plaques in AD.     

Characterizations of distinct amyloidogenic conformations of the Abeta (1-40) and (1-42) peptides

Major constituents of the amyloid plaques found in the brain of Alzheimer's patients are the 39-43 residue beta-amyloid (Abeta) peptides. Extensive in vitro as well as in vivo biochemical studies have shown that the 40- and 42-residue Abeta peptides play major roles in the neurodegenerative pathology of Alzheimer's disease. Although the two Abeta peptides share common aggregation properties, the 42-residue peptide is more amyloidogenic and more strongly associated with amyloid pathology. Thus, characterizations of the two Abeta peptides are of critical importance in understanding the molecular mechanism of Abeta amyloid formation. In this report, we present combined CD and NMR studies of the monomeric states of the two peptides under both non-amyloidogenic (<5 degrees C) and amyloid-forming conditions (>5 degrees C) at physiological pH. Our CD studies of the Abeta peptides showed that initially unfolded Abeta peptides at low temperature (<5 degrees C) gradually underwent conformational changes to more beta-sheet-like monomeric intermediate states at stronger amyloidogenic conditions (higher temperatures). Detailed residue-specific information on the structural transition was obtained by using NMR spectroscopy. Residues in the N-terminal (3-12) and 20-22 regions underwent conformational changes to more extended structures at the stronger amyloidogenic conditions. Almost identical structural transitions of those residues were observed in the two Abeta peptides, suggesting a similar amyloidogenic intermediate for the two peptides. The 42-residue Abeta (1-42) peptide was, however, more significantly structured at the C-terminal region (39-42), which may lead to the different aggregation propensity of the two peptides.     

Aluminum, copper, iron and zinc differentially alter amyloid-Aβ(1-42) aggregation and toxicity

Amyloid-β(1-42) (Aβ) is believed to play a crucial role in the ethiopathogenesis of Alzheimer's Disease (AD). In particular, its interactions with biologically relevant metal ions may lead to the formation of highly neurotoxic complexes. Here we describe the species that are formed upon reacting Aβ with several biometals, namely copper, zinc, iron, and with non-physiological aluminum to assess whether different metal ions are able to differently drive Aβ aggregation. The nature of the resulting Aβ-metal complexes and of the respective aggregates was ascertained through a number of biophysical techniques, including electrospray ionization mass spectrometry, dynamic light scattering, fluorescence, transmission electron microscopy and by the use of conformation-sensitive antibodies (OC, αAPF). Metal binding to Aβ is shown to confer highly different chemical properties to the resulting complexes; accordingly, their overall aggregation behaviour was deeply modified. Both aluminum(III) and iron(III) ions were found to induce peculiar aggregation properties, ultimately leading to the formation of annular protofibrils and of fibrillar oligomers. Notably, only Aβ-aluminum was characterized by the presence of a relevant percentage of aggregates with a mean radius slightly smaller than 30 nm. In contrast, both zinc(II) and copper(II) ions completely prevented the formation of soluble fibrillary aggregates. The biological effects of the various Aβ-metal complexes were studied in neuroblastoma cell cultures: Aβ-aluminum turned out to be the only species capable of triggering amyloid precursor and tau181 protein overproduction. Our results point out that Al can effectively interact with Aβ, forming "structured" aggregates with peculiar biophysical properties which are associated with a high neurotoxicity.     

Using bacterial inclusion bodies to screen for amyloid aggregation inhibitors

ABSTRACT: BACKGROUND: The amyloid-beta peptide (Abeta42) is the main component of the inter-neuronal amyloid plaques characteristic of Alzheimer's disease (AD). The mechanism by which Abeta42 and other amyloid peptides assemble into insoluble neurotoxic deposits is still not completely understood and multiple factors have been reported to trigger their formation. In particular, the presence of endogenous metal ions has been linked to the pathogenesis of AD and other neurodegenerative disorders. RESULTS: Here we describe a rapid and high-throughput screening method to identify molecules able to modulate amyloid aggregation. The approach exploits the inclusion bodies (IBs) formed by Abeta42 when expressed in bacteria. We have shown previously that these aggregates retain amyloid structural and functional properties. In the present work we demonstrate that their in vitro refolding is selectively sensitive to the presence of aggregation-promoting metal ions, allowing the detection of inhibitors of metal-promoted amyloid aggregation with potential therapeutic interest. CONCLUSIONS: Because IBs can be produced at high levels and easily purified, the method overcomes one of the main limitations in screens to detect amyloid modulators: the use of expensive and usually highly insoluble synthetic peptides.     

Destabilization of non-pathological variants of ataxin-3 by metal ions results in aggregation/fibrillogenesis

Ataxin-3 (AT3), a protein that causes spinocerebellar ataxia type 3, has a C-terminus containing a polyglutamine stretch, the length of which can be expanded in its pathological variants. Here, we report on the role of Cu(2+), Mn(2+), Zn(2+) and Al(3+) in the induction of defective protein structures and subsequent aggregation/fibrillogenesis of three different non-pathological forms of AT3, i.e. murine (Q6), human non-expanded (Q26) and human moderately expanded (Q36). AT3 variants showed an intrinsic propensity to misfolding/aggregation; on the other hand, Zn(2+) and Al(3+) strongly stimulated the amplitude and kinetics of these conformational conversions. While both metal ions induced a time-dependent aggregation into amyloid-like fibrillar forms, only small oligomers and/or short protofibrillar species were detected for AT3s alone. The rate and extent of the metal-induced aggregation/fibrillogenesis processes increased with the size of the polyglutamine stretch. Mn(2+) and Cu(2+) had no effect on (Q6) or actually prevented (Q26 and Q36) the AT3 structural transitions. The observation that Zn(2+) and Al(3+) promote AT3 fibrillogenesis is consistent with similar results found for other amyloidogenic molecules, such as beta-amyloid and prion proteins. Plausibly, these metal ions are a major common factor/cofactor in the etiopathogenesis of neurodegenerative diseases. Studies of liposomes as membrane models showed dramatic changes in the structural properties of the lipid bilayer in the presence of AT3, which were enhanced after supplementing the protein with Zn(2+) and Al(3+). This suggests that cell membranes could be a potential primary target in the ataxin-3 pathogenesis and metals could be a biological factor capable of modulating their interaction with AT3.     

Aluminum increases levels of beta-amyloid and ubiquitin in neuroblastoma but not in glioma cells

Several epidemiological studies suggest the involvement of aluminum (Al) in the pathogenesis of Alzheimer's disease (AD). There is an increase in the levels of Abeta and ubiquitin in the pathological lesions of AD. Therefore, we have investigated whether aluminum (Al) treatment alters the levels of Abeta and ubiquitin in murine neuroblastoma (NBP2) and rat glioma (C-6) cell cultures. At a low concentration (10 microM), aluminum sulfate stimulated the level of immunoreactive Abeta and ubiquitin in NBP2 cells without changing the levels of the amyloid precursor protein (APP). However, at higher concentrations (100 and 500 microM), aluminum failed to elicit any significant effect on beta-amyloid, whereas ubiquitin levels continued to increase. No changes in the Abeta and ubiquitin content were found in the C-6 glioma cells following treatment with Al at any of the concentrations tested. Exposure of cells to aluminum salts did not alter the rate of proliferation in either of the two cell lines. These data suggest that one of the mechanisms by which Al may play a role in AD is by promoting the formation of Abeta and ubiquitin in neurons.     

Demonstration of aluminum in amyloid fibers in the cores of senile plaques in the brains of patients with Alzheimer’s disease

Aluminum (Al) exposure has been reported to be a risk factor for Alzheimer’s disease (senile dementia of Alzheimer type), although the role of Al in the etiology of Alzheimer’s disease remains controversial. We examined the presence of Al in the Alzheimer’s brain using energy-dispersive X-ray spectroscopy combined with transmission electron microscopy (TEM-EDX). TEM-EDX analysis allows simultaneous imaging of subcellular structures with high spatial resolution and analysis of small quantities of elements contained in the same subcellular structures. We identified senile plaques by observation using TEM and detected Al in amyloid fibers in the cores of senile plaques located in the hippocampus and the temporal lobe by EDX. Phosphorus and calcium were also present in the amyloid fibers. No Al could be detected in the extracellular space in senile plaques or in the cytoplasm of nerve cells. In this study, we demonstrated colocalization of Al and beta-amyloid (Abeta) peptides in amyloid fibers in the cores of senile plaques. The results support the following possibilities in the brains of patients with Alzheimer’s disease: Al could be involved in the aggregation of Abeta peptides to form toxic fibrils; Al might induce Abeta peptides into the beta-sheet structure; and Al might facilitate iron-mediated oxidative reactions, which cause severe damage to brain tissues.     

Neurotoxicity of acetylcholinesterase amyloid beta-peptide aggregates is dependent on the type of Abeta peptide and the AChE concentration present in the complexes.

Alzheimer's disease (AD) is a neurodegenerative disorder whose hallmark is the presence of senile plaques and neurofibrillary tangles. Senile plaques are mainly composed of amyloid beta-peptide (Abeta) fibrils and several proteins including acetylcholinesterase (AChE). AChE has been previously shown to stimulate the aggregation of Abeta1-40 into amyloid fibrils. In the present work, the neurotoxicity of different amyloid aggregates formed in the absence or presence of AChE was evaluated in rat pheochromocytoma PC12 cells. Stable AChE-Abeta complexes were found to be more toxic than those formed without the enzyme, for Abeta1-40 and Abeta1-42, but not for amyloid fibrils formed with AbetaVal18-Ala, a synthetic variant of the Abeta1-40 peptide. Of all the AChE-Abeta complexes tested the one containing the Abeta1-40 peptide was the most toxic. When increasing concentrations of AChE were used to aggregate the Abeta1-40 peptide, the neurotoxicity of the complexes increased as a function of the amount of enzyme bound to each complex. Our results show that AChE-Abeta1-40 aggregates are more toxic than those of AChE-Abeta1-42 and that the neurotoxicity depends on the amount of AChE bound to the complexes, suggesting that AChE may play a key role in the neurodegeneration observed in Alzheimer brain.     

Enalaprilat Inhibits Zinc-Dependent Oligomerization of Metal-Binding Domain of Amyloid-beta Isoforms and Protects Human Neuroblastoma Cells from Toxic Action of these Isoforms

Intact amyloid-β peptides (Aβ) may undergo prion-like aggregation when they interact with chemically or structurally modified variants of Aβ present in extracellular pathohistological inclusions (amyloid plaques). This aggregation is regarded as one of the key molecular mechanisms of Alzheimer’s disease (AD) pathogenesis. Zinc ions are involved in the pathological dimerization and oligomerization of natural Aβ isoforms, and zinc-induced oligomers can also initiate the pathological aggregation of Aβ. Based on the earlier found molecular mechanism of zinc-dependent oligomerization of Aβ, it has been suggested that the targeted inhibition of the 11EVHH14 site in one Aβ molecule from zinc-mediated interactions with the same site of another Aβ molecule can effectively inhibit the oligomerization and aggregation of Aβ. Taking into account the similarity in the structural organization of zinc-binding sites within Aβ and angiotensin-converting enzyme (ACE), we hypothesized that inhibitors of the ACE active sites could specifically interact with the 11EVHH14 site of Aβ. Using a surface plasmon resonance biosensor and nuclear magnetic resonance spectroscopy, we have found that the ACE inhibitor enalaprilat effectively inhibits zinc-dependent dimerization of the metal-binding domains of intact Aβ and Aβ with isomerized Asp7 (isoAβ). We have also found that enalaprilat protects SH-SY5Y human neuroblastoma cells from the toxic effects of Aβ(1–42) and isoAβ(1–42), which are among the most common components of amyloid plaques. The results confirm the role of zincdependent oligomerization of Aβ in AD pathogenesis and make it possible one to consider enalaprilat as a prototype of antiaggregation agents for treating AD.     

Copper complexing decreases the ability of amyloid beta peptide to cross the BBB and enter brain parenchyma

The amyloid hypothesis states that amyloid beta protein (Abeta) plays a major causal role in the onset of Alzheimer's disease. Toxicity of Abeta can be modified by metal ions. Two mechanisms by which such Abeta and metal ions could interact are by enhanced oxidative stress or by altered fibrillation. Specifically, Abeta fibrillation is increased by aluminum (Al) and copper (Cu) and Al also increases Abeta uptake into brain. Here, we determined whether chelation with Cu would alter uptake of the human or rat 1-42 form of Abeta (Abeta42) by brain or alter Abeta-induced oxidative stress in an immortalized line of rat brain endothelial cells (RBE4). We found that Cu enhanced cytotoxicity of rat, but not of human Abeta, had no effect on glutathione (GSH) or cysteine (CYS) levels. Cu significantly decreased homocysteine (HCYS) levels when complexed with Abeta. Cu chelation did not alter Abeta uptake into brain or other tissues (except for kidney) or alter clearance from blood or brain in vivo, but did increase efflux in an in vitro model of the blood-brain barrier (BBB). Chelation to Cu also impaired the capillary to brain transport of Abeta, an effect opposite to that previously found for chelation of Abeta to Al. These results show that metal ions have varied effects on Abeta uptake by brain and that Cu could be protective against the neurotoxic effects of circulating Abeta.     

Therapeutic Strategies for Alzheimer’s Disease

Therapeutic approaches for Alzheimer's disease (AD) are guided by four disease characteristics: amyloid plaques, neurofibrillar tangles (NFT), neurodegeneration, and dementia. Amyloid plaques are composed largely of 4 kDa beta-amyloid (Abeta) peptides, with the more amyloidogenic, 42 amino acid form (Abeta42) as the primary species. Because multiple, rare mutations that cause early-onset, familial AD lead to increased production or aggregation of Abeta42, amyloid therapeutics aim to reduce the amount of toxic Abeta42 aggregates. Amyloid-based therapies include gamma-secretase inhibitors and modulators, BACE inhibitors, aggregation blockers, catabolism inducers, and anti-Abeta biologics. Tangles are composed of paired helical filaments of hyperphosphorylated tau protein. Tau-based therapeutics include kinase inhibitors, microtubule stabilizers, and catabolism inducers. Therapeutic strategies for neurodegeneration target multiple mechanisms, including excitotoxicity, mitochondrial dysfunction, oxidative damage, and inflammation or stimulation of neuronal viability. Although not disease modifying, cognition enhancers are important to treat the symptom of dementia. Strategies for cognition enhancement include cholinesterase inhibitors, and other approaches to enhance the signaling of cholinergic and glutamatergic neurons. In summary, plaques, tangles, neurodegeneration and dementia guide the development of multiple therapeutic approaches for AD and are the subject of this review.     

Physiological cholesterol concentration is a neuroprotective factor against β-amyloid and β-amyloid-metal complexes toxicity

Alzheimer's disease is one of the most common forms of dementia in the elderly. One of its hallmarks is the abnormal aggregation and deposition of β-amyloid (Aβ). Endogenous and exogenous metal ions seem to influence β-amyloid folding process, aggregation and deposition. Besides these variables other elements appear to affect β-amyloid behavior, such as cholesterol. The physiological concentration of cholesterol in the cerebrospinal fluid (CSF) was used in order to determine the extent in which Aβ and Aβ-metal complexes in vitro aggregation and their toxicity on human neuroblastoma cell cultures is affected. Cholesterol did not appear to influence Aβ and Aβ-metal complexes aggregation, but it was effective in protecting neuroblastoma cells against Aβ complexes' toxicity. The Aβ-Al complex seemed to be the most effective in disrupting and damaging membrane external layer, and simultaneously it appears to increase its toxicity on cell cultures; both of these effects are preventable by cholesterol. The presence in physiological concentrations of cholesterol seemed to compensate membrane damage that occurred to neuroblastoma cells. These findings appear to contradict some data reported in literature. We believe that our results might shed some light on the role played by cholesterol at physiological concentrations in both cellular balance and membrane protection.     

Cu（Ⅱ） Potentiation of Alzheimer Aβ1-40 Cytotoxicity and Transition on its Secondary Structure

Mounting evidence has shown that dyshomeostasis of the redox-active biometals such as Cuand Fe can lead to oxidative stress,which plays a key role in the neuropathology of Alzheimer's disease(AD).Here we demonstrate that with the formation of Cu(Ⅱ)·Aβ1-40 complexes,copper markedly potentiatesthe neurotoxicity exhibited by β-amyloid peptide (Aβ).A greater amount of hydrogen peroxide was releasedwhen Cu(Ⅱ)·Aβ1-40 complexes was added to the xanthine oxidase/xanthine system detected by potassiumiodide spectrophotometry.Copper bound to Aβ1-40 was observed by electron paramagnetic resonance(EPR) spectroscopy.Circular dichroism (CD) studies indicated that copper chelation could cause a structuraltransition of Aβ.The addition of copper to Aβ introduced an increase on β-sheet as well as α-helix,whichmay be responsible for the aggregation of Aβ.We hypothesized that Aβ aggregation induced by copper maybe responsible for local injury in AD.The interaction between Cu~(2 ) and Aβ also provides a possible mechanismfor the enrichment of metal ions in amyloid plaques in the AD brain.     

Effect of divalent metal ions on annexin-mediated aggregation of asolectin liposomes

Annexins belong to a family of Ca2+- and phospholipid-binding proteins that can mediate the aggregation of granules and vesicles in the presence of Ca2+. We have studied the effects of different divalent metal ions on annexin-mediated aggregation of liposomes using annexins isolated from rabbit liver and large unilamellar vesicles prepared from soybean asolectin II-S. In the course of these studies, we have found that annexin-mediated aggregation of liposomes can be driven by various earth and transition metal ions other than Ca2+. The ability of metal ions to induce annexin-mediated aggregation decreases in the order: Cd2+ > Ba2+, Sr2+ > Ca2+ > Mn2+ > Ni2+ > Co2+. Annexin-mediated aggregation of vesicles is more selective to metal ions than the binding of annexins to membranes. We speculate that not every type of divalent metal ion can induce conformational change sufficient to promote the interaction of annexins either with two opposing membranes or with opposing protein molecules. Relative concentration ratios of metal ions in the intimate environment may be crucial for the functioning of annexins within specialized tissues and after treatment with toxic metal ions.     

Direct interaction of soluble human recombinant tau protein with Abeta 1-42 results in tau aggregation and hyperphosphorylation by tau protein kinase II

We report here that aggregated beta-amyloid (Abeta) 1-42 promotes tau aggregation in vitro in a dose-dependent manner. When Abeta-mediated aggregated tau was used as a substrate for tau protein kinase II (TPK II), an 8-fold increase in the rate of TPK II-mediated tau phosphorylation was observed. The extent of TPK II-dependent tau phosphorylation increased as a function of time and Abeta 1-42 concentration, and hyperphosphorylated tau was found to be decorated with an Alzheimer's disease-related phosphoepitope (P-Thr-231). In HEK 293 cells co-expressing CT-100 amyloid precursor protein and tau, the release of Abeta 1-42 from these cells was impaired. Taken together, these in vitro results suggest that Abeta 1-42 promotes both tau aggregation and hyperphosphorylation.     

Implication of novel biochemical property of beta-amyloid

Alzheimer disease (AD) is a heterogeneous disorder with a variety of molecular pathologies converging predominantly on abnormal amyloid deposition particularly in the brain. beta-Amyloid aggregation into senile plaques is one of the pathological hallmarks of AD. beta-Amyloid is generated by a proteolytic cleavage of a large membrane protein, amyloid precursor protein (APP). We have observed a new property of beta-amyloid. The amyloid 1-42 beta fragment, when aggregated, possesses proteolytic and esterase-like activity, in vitro. Three independent methods were used to test the new property of beta-amyloid. While esterase activity involves imidazole catalysis, proteolytic activity is consistent with participation of a serine peptidase triad: catalytic Ser, His and Glu (or Asp). Although the amino acid triad is a necessary requirement for the protease reactivity, it is not sufficient since the secondary structure of the protein significantly contributes to the proteolytic activity. The ability of beta-amyloid to cleave peptide or ester bonds could be thus responsible for either inactivation of other proteins and/or APP proteolysis itself. This property may be responsible for early pathogenesis of AD since there is emerging evidence that non-plaque amyloid is elevated in Alzheimer patients.