Amyloid Abeta Peptide Research Articles

Weaker N-Terminal Interactions for the Protective over the Causative Aβ Peptide Dimer Mutants.

Knowing that abeta amyloid peptide (Aβ42) dimers are the smallest and most abundant neurotoxic oligomers for Alzheimer's disease (AD), we used molecular simulations with advanced sampling methods (replica-exchange) to characterize and compare interactions between the N-termini (residues 1-16) of wild type (WT-WT) and five mutant dimers under constrained and unconstrained conditions. The number of contacts and distances between the N-termini, and contact maps of their conformational landscape illustrate substantial differences for a single residue change. The N-terminal contacts are significantly diminished for the dimers containing the monomers that protect against (WT-A2T) as compared with those that predispose toward (A2V-A2V) AD and for the control WT-WT dimers. The reduced number of N-terminal contacts not only occurs at or near the second residue mutations but also is distributed through to the 10th residue. These findings provide added support to the accumulating evidence for the "N-terminal hypothesis of AD" and offer an alternate mechanism for the cause of protection from the A2T mutant.

Affinity-mass spectrometry approaches for elucidating structures and interactions of protein-ligand complexes.

Affinity-based approaches in combination with mass spectrometry for molecular structure identification in biological complexes such as protein-protein, and protein-carbohydrate complexes have become popular in recent years. Affinity-mass spectrometry involves immobilization of a biomolecule on a chemically activated support, affinity binding of ligand(s), dissociation of the complex, and mass spectrometric analysis of the bound fraction. In this chapter the affinity-mass spectrometric methodologies will be presented for (1) identification of the epitope structures in the Abeta amyloid peptide, (2) identification of oxidative modifications in proteins such as nitration of tyrosine, (3) determination of carbohydrate recognition domains, and as (4) development of a biosensor chip-based mass spectrometric system for concomitant quantification and identification of protein-ligand complexes.

Disaggregation of Amyloid Plaque in Brain of Alzheimer’s Disease Transgenic Mice with Daily Subcutaneous Administration of a Tetravalent Bispecific Antibody That Targets the Transferrin Receptor and the Abeta Amyloid Peptide

Anti-amyloid antibodies (AAA) are under development as new therapeutics that disaggregate the amyloid plaque in brain in Alzheimer's disease (AD). However, the AAAs are large molecule drugs that do not cross the blood-brain barrier (BBB), in the absence of BBB disruption. In the present study, an AAA was re-engineered for receptor-mediated transport across the BBB via the endogenous BBB transferrin receptor (TfR). A single chain Fv (ScFv) antibody form of an AAA was fused to the carboxyl terminus of each heavy chain of a chimeric monoclonal antibody (mAb) against the mouse TfR, and this produced a tetravalent bispecific antibody designated the cTfRMAb-ScFv fusion protein. Unlike a conventional AAA, which has a plasma half-time of weeks, the cTfRMAb-ScFv fusion protein is cleared from plasma in mice with a mean residence time of about 3 h. Therefore, a novel protocol was developed for the treatment of one year old presenilin (PS)-1/amyloid precursor protein (APP) AD double transgenic PSAPP mice, which were administered daily subcutaneous (sc) injections of 5 mg/kg of the cTfRMAb-ScFv fusion protein for 12 consecutive weeks. At the end of the treatment, brain amyloid plaques were quantified with confocal microscopy using both Thioflavin-S staining and immunostaining with the 6E10 antibody against Abeta amyloid fibrils. Fusion protein treatment caused a 57% and 61% reduction in amyloid plaque in the cortex and hippocampus, respectively. No increase in plasma immunoreactive Abeta amyloid peptide, and no cerebral microhemorrhage, was observed. Chronic daily sc treatment of the mice with the fusion protein caused no immune reactions and only a low titer antidrug antibody response. In conclusion, re-engineering AAAs for receptor-mediated BBB transport allows for reduction in brain amyloid plaque without cerebral microhemorrhage following daily sc treatment for 12 weeks.

Pathogenic prions deviate PrPC signaling in neuronal cells and impair A-beta clearance

The subversion of the normal function exerted by the cellular prion protein (PrPC) in neurons by pathogenic prions is assumed to have a central role in the pathogenesis of transmissible spongiform encephalopathies. Using two murine models of prion infection, the 1C11 neuronal cell line and neurospheres, we document that prion infection is associated with the constitutive activation of signaling targets normally coupled with PrPC, including the Fyn kinase, the mitogen-associated protein kinases ERK1/2 and the CREB transcription factor. PrPC-dependent signaling overactivation in infected cells is associated with the recruitment of p38 and JNK stress-associated kinases. Downstream from CREB, prion-infected cells exhibit reduced activity of the matrix metalloprotease (MMP)-9. As MMP-9 catalyzes the degradation of the amyloid A-beta peptide, the decrease in MMP-9 activity in prion-infected cells causes a significant impairment of the clearance of A-beta, leading to its accumulation. By exploiting two 1C11-infected clones accumulating high or moderate levels of prions, we show that the prion-induced changes are correlated with the level of infectivity. Of note, a dose-dependent increase in A-beta levels was also found in the cerebrospinal fluid of mice inoculated with these infected clones. By demonstrating that pathogenic prions trigger increases in A-beta levels through the deviation of PrPC signaling, our data argue that A-beta may exacerbate prion-induced toxicity.

Receptor-Mediated Abeta Amyloid Antibody Targeting to Alzheimer’s Disease Mouse Brain

The goal of this work is the reduction in the Abeta amyloid peptide burden in brain of Alzheimer's disease (AD) transgenic mice without the concomitant elevation in plasma Abeta amyloid peptide. An anti-Abeta amyloid antibody (AAA) was re-engineered as a fusion protein with a blood-brain barrier (BBB) molecular Trojan horse. The AAA was engineered as a single chain Fv (ScFv) antibody, and the ScFv was fused to the heavy chain of a chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR), and this fusion protein was designated cTfRMAb-ScFv. The cTfRMAb-ScFv protein penetrates mouse brain from blood via transport on the BBB TfR, and the brain uptake is 3.5% of injected dose/gram brain following an intravenous administration. Double transgenic APPswe,PSEN1dE9 mice were studied at 12 months of age. The mice were shown to have extensive Abeta amyloid plaques in cerebral cortex based on immunocytochemistry. The mice were treated every 3-4 days by intravenous injections of either saline or the cTfRMAb-ScFv fusion protein at an injection dose of 1 mg/kg for 12 consecutive weeks. The brain Aβ¹⁻⁴² concentration was reduced 40% in the fusion protein treated mice, without any elevation in plasma Aβ¹⁻⁴² concentration. No cerebral microhemorrhage was observed in the treated mice. These results show that brain-penetrating antibody pharmaceutics can be developed for brain disorders such as AD following the re-engineering of the antibody as a fusion protein that is transported across the BBB via receptor-mediated transport.

IgG‐single chain Fv fusion protein therapeutic for alzheimer's disease: Expression in CHO cells and pharmacokinetics and brain delivery in the rhesus monkey

Monoclonal antibodies (MAb) directed against the Abeta amyloid peptide of Alzheimer's disease (AD) are potential new therapies for AD, since these antibodies disaggregate brain amyloid plaque. However, the MAb is not transported across the blood-brain barrier (BBB). To enable BBB transport, a single chain Fv (ScFv) antibody against the Abeta peptide of AD was re-engineered as a fusion protein with the MAb against the human insulin receptor (HIR). The HIRMAb acts as a molecular Trojan horse to ferry the ScFv therapeutic antibody across the BBB. Chinese hamster ovary (CHO) cells were stably transfected with a tandem vector encoding the heavy and light chains of the HIRMAb-ScFv fusion protein. A high secreting line was isolated following methotrexate amplification and dilutional cloning. The HIRMAb-ScFv fusion protein in conditioned serum-free medium was purified by protein A affinity chromatography. The fusion protein was stable as a liquid formulation, and retained high-affinity binding of both the HIR and the Abeta amyloid peptide. The HIRMAb-ScFv fusion protein was radiolabeled with the (125)I-Bolton-Hunter reagent, followed by measurement of the pharmacokinetics of plasma clearance and brain uptake in the adult Rhesus monkey. The HIRMAb-ScFv fusion protein was rapidly cleared from plasma and was transported across the primate BBB in vivo. In conclusion, the HIRMAb-ScFv fusion protein is a new class of antibody-based therapeutic for AD that has been specifically engineered to cross the human BBB.

A New Series of 1,3‐Dihidro‐Imidazo[1,5‐c]thiazole‐5,7‐Dione Derivatives: Synthesis and Interaction with Aβ(25‐35) Amyloid Peptide

Deposition of senile plaques composed of fibrillar aggregates of Abeta-amyloid peptide is a characteristic hallmark of Alzheimer's disease. A widely employed approach in the study of anti-Alzheimer agents involves the identification of substances able to prevent amyloid aggregation, or to disaggregate the amyloid fibrils through a direct structural interaction with the soluble or aggregated forms of the peptide. Here, we report the synthesis of a set of 1,3-dihydro-3,6-disubstituted-imidazo[1,5-c]thiazole-5,7-dione derivatives supporting different alkyl, aryl and alkylamine side chains. The ability of these compounds to interact with the Abeta(25-35) peptide was evaluated using circular dichroism, nuclear magnetic resonance and thioflavin fluorescence spectroscopy. A molecular model for Abeta(25-35)-ligand interactions was calculated by molecular docking procedures. Our data show that the ability of the synthesized compounds to modify the structural behaviour of Abeta(25-35) varies as a function of the overall structural features of the ligands rather contributions from specific individual substituents.

Internalization of Abeta amyloid peptides in neuronal cells depending of their sequence and their structural assembly

Internalization of Abeta amyloid peptides in neuronal cells depending of their sequence and their structural assembly

Design and synthesis of new trehalose‐conjugated pentapeptides as inhibitors of Aβ(1–42) fibrillogenesis and toxicity

Aggregation of the amyloid Abeta peptide and its accumulation into insoluble deposits (plaques) are believed to be the main cause of neuronal dysfunction associated with Alzheimer's disease (AD); small molecules that can interfere with the Abeta amyloid fibril formation are therefore of interest for a potential therapeutic strategy. Three new trehalose-conjugated peptides of the well known beta-sheet breaker peptide iAbeta5p, were synthesized. The disaccharide was covalently attached to different sites of the LPFFD peptide chain, i.e. at the N-terminus, C-terminus or at the Asp side chain. CD spectroscopy in different solvents was used to assess changes in the peptide conformation of these compounds. The effects of these glycopeptides on the self-assembly and morphology of Abeta aggregates were investigated by ThT fluorescence assay and dynamic Scanning Force Microscopy, respectively. All the synthesized compounds were tested as inhibitors of Abeta toxicity toward pure cultures of rat cortical neurons.

On Enhancers and Inhibitors of Elastin-Derived Amyloidogenesis

The main aim of this study is to better understand the self-aggregation mechanism of amyloid-like elastin-derived fibers in order to design and produce new powerful drugs that will inhibit the onset of 'amyloidosis'. Atomic force microscopy (AFM), Congo Red birefringence assay and Thioflavin T fluorescence measurements were used to demonstrate the amyloid-like behavior of some fragments of elastin protein (exon 30 [EX30] and exon 28 [EX28]). Turbidimetry on apparent absorbance technique was used to investigate the effect either of enhancers or of inhibitors on the amyloidogenic elastin-like peptides. Circular-dichroism spectroscopy was used to study the secondary structures of the peptides. We used Congo Red birefringence assay, Thioflavin T fluorescence measurements and AFM measurements that are used commonly to demonstrate the formation of amyloids. The elastin fibrillogenesis is amyloid-like. Then, the elastin amyloidogenesis is inhibited by particular pentapeptides. We have reported herein that the fibrillogenesis of elastin-derived EX28 and EX30 polypeptides is facilitated significantly by the effect of sodium taurocholate bile salt and is inhibited by a classical inhibitor of Abeta-amyloid peptide, such as KLVFF, as well as by novel inhibitors, designed by us on the basis of some elastin sequences.

Substrate specificity of human kallikreins 1 and 6 determined by phage display.

The human tissue kallikrein (KLK) family contains 15 secreted serine proteases that are expressed in a wide range of tissues and have been implicated in different physiological functions and disease states. Of these, KLK1 has been shown to be involved in the regulation of multiple physiological processes such as blood pressure, smooth muscle contraction, and vascular cell growth. KLK6 is overexpressed in breast and ovarian cancer tissues and has been shown to cleave peptide derived from human myelin protein and Abeta amyloid peptide in vitro. Here we analyzed the substrate specificity of KLK1 and KLK6, by substrate phage display using a random octapeptide library. Consistent with earlier biochemical data, KLK1 was shown to exhibit both trypsin- and chymotrypsin-like selectivities with Tyr/Arg preferred at site P1, Ser/Arg strongly preferred at P1', and Phe/Leu at P2. KLK6 displayed trypsin-like activity, with the P1 position occupied only by Arg and a strong preference for Ser in P1'. Docking simulations of consensus peptide provide information on the identity of the enzyme residues that are responsible for substrate binding. Bioinformatic analysis suggested several putative KLK6 protein substrates, such as ionotropic glutamate receptor (GluR) and synphilin.

Inhibitory effects of short-term administration of dl-α-lipoic acid on oxidative vulnerability induced by Aβ amyloid fibrils (25–35) in mice

Abeta amyloid peptide is believed to induce oxidative stress leading to inflammation, which is postulated to play a significant role in the toxicity of Alzheimer's disease (AD). This study was designed to investigate the inhibitory effects of DL-alpha lipoic acid (LA), a potential free radical scavenger, on oxidative vulnerability induced by intraperitoneal injection of Abeta25-35 amyloid fibrils in mice. Mice were divided into three groups: control, Abeta amyloid toxicity induced (AT), and LA treated (ATL). Blood Plasma was separated, liver, spleen and brain were dissected and analysis of oxidants, antioxidants, ATPases, glial fibrillary acidic protein (GFAP) and nuclear factor kappa-B (NFkappaB) were carried out. Results show biochemical parameters such as reactive oxygen species (ROS) and lipid peroxidation (LPO) were significantly lowered (P < 0.05) and levels of antioxidants and ATPase (P < 0.05) were significantly increased (P < 0.05) in hepatocytes, splenocytes and astrocytes of the ATL group. Moreover, our histological results revealed a decreased GFAP immunoreactivity in the neocortical region and NFkappaB immunoreactivity in neocortex, liver and spleen. This study reiterates LA as a potent free radical scavenger to combat oxidative vulnerability in the treatment for Abeta amyloid toxicity.

Lipids revert inert Aβ amyloid fibrils to neurotoxic protofibrils that affect learning in mice

Although soluble oligomeric and protofibrillar assemblies of Aβ-amyloid peptide cause synaptotoxicity and potentially contribute to Alzheimer's disease (AD), the role of mature Aβ-fibrils in the amyloid plaques remains controversial. A widely held view in the field suggests that the fibrillization reaction proceeds ‘forward' in a near-irreversible manner from the monomeric Aβ peptide through toxic protofibrillar intermediates, which subsequently mature into biologically inert amyloid fibrils that are found in plaques. Here, we show that natural lipids destabilize and rapidly resolubilize mature Aβ amyloid fibers. Interestingly, the equilibrium is not reversed toward monomeric Aβ but rather toward soluble amyloid protofibrils. We characterized these ‘backward' Aβ protofibrils generated from mature Aβ fibers and compared them with previously identified ‘forward' Aβ protofibrils obtained from the aggregation of fresh Aβ monomers. We find that backward protofibrils are biochemically and biophysically very similar to forward protofibrils: they consist of a wide range of molecular masses, are toxic to primary neurons and cause memory impairment and tau phosphorylation in mouse. In addition, they diffuse rapidly through the brain into areas relevant to AD. Our findings imply that amyloid plaques are potentially major sources of soluble toxic Aβ-aggregates that could readily be activated by exposure to biological lipids.

Cholesterol depletion reduces aggregation of amyloid-beta peptide in hippocampal neurons

A key event in the pathogenesis of Alzheimer's disease is the conversion of soluble amyloid Abeta-peptide into toxic aggregates. Here, we studied the effect of cholesterol depletion on the formation of insoluble Abeta. We found that reduction of neuronal cholesterol by approximately 25% reduced the neuronal formation of insoluble Abeta without affecting the secretion of soluble Abeta. Moreover, we demonstrate that Abeta-oligomers from Alzheimer's disease brains associate with a detergent-resistant membrane fraction in a cholesterol-dependent manner. These results suggest a key role for cholesterol in aggregation of Abeta.

Interpretation of the dissolution of insoluble peptide sequences based on the acid-base properties of the solvent.

The dissolution process of model insoluble peptide sequences was investigated in view of the electron acceptor (AN) and electron donor (DN) solvent properties. The Alzheimer's disease-inducing (1-42) Abeta-amyloid peptide and its (1-21) fragment, the (66-97) transmembrane bradykinin B2 receptor sequence, and the strongly aggregated VVLGAAIV were selected as models of insoluble peptides. Solvents presenting similar AN and DN values failed, despite their polarities, to dissociate peptide chains (free in solution or bound to a polymer). The maximum solubility of these aggregated sequences was attained in solvents presenting the highest possible (AN-DN) values (in positive or negative mode). The AN-DN values ranged from approximately -20 to +80 and, notably, the lowest dissociation power was ascribed to solvents presenting values of approximately +40. The strong hydrogen bond donor water is located in this region, indicating that, for dissociation of specific insoluble segments, the solvent should appropriately combine its acid/base strength with the potential for van der Waals interactions. We also observed a sequence-dependent pH effect on peptide solubility confirmed through circular dichroism spectroscopy. This approach also revealed a complex but, in many cases, consistent influence of peptide conformation on its solubility degree, even when structure-inducing solvents were added. In conclusion, the random method of selecting solvents to dissolve insoluble and intractable peptide sequences, still in use by some, could be partially supplanted by the strategy described herein, which may be also applicable to other solute dissociation processes.

Substrate Specificity of Human Kallikrein 6: SALT AND GLYCOSAMINOGLYCAN ACTIVATION EFFECTS

Human kallikrein 6 (hK6) is abundantly expressed in the central nervous system and is implicated in demyelinating disease. This study provided biochemical data about the substrate specificity and activation of hK6 by glycosaminoglycans and by kosmotropic salts, which followed the Hofmeister series. The screening of fluorescence resonance energy transfer (FRET) peptide families derived from Abz-KLRSSKQ-EDDnp resulted in the finding that Abz-AFRFSQ-EDDnp (where Abz is ortho-aminobenzoic acid and EDDnp is N-[2,4-dinitrophenyl]ethylenediamine)) is the best synthetic substrate described so far for hK6 (kcat/Km 38,667 s(-1) mm(-1)). It is noteworthy that the AFRFS sequence was found as a motif in the amino-terminal domain of seven human ionotropic glutamate receptor subunits. We also examined the hK6 hydrolytic activity on FRET peptides derived from human myelin basic protein, precursor of the Abeta amyloid peptide, reactive center loop of alpha1-antichymotrypsin, plasminogen, and maturation and inactivation cleavage sites of hK6, which were described earlier as natural substrates for hK6. The best substrates were derived from myelin basic protein. The hK6 maturation cleavage site was poorly hydrolyzed, and no evidence was found to support a two-step self-activation process reported previously. Finally, we assayed FRET peptides derived from sequences that span the cleavage sites for activation of protease-activated receptors (PAR) 1-4, and only the substrate with the PAR 2 sequence was hydrolyzed. These results further supported the hypothesis that hK6 expressed in the central nervous system is involved in normal myelin turnover/demyelination processes, but it is unlikely to self-activate. This report also suggested the possible modulation of ionotropic glutamate receptors and activation of PAR 2 by hK6.

The amyloid-β precursor protein: integrating structure with biological function

Proteolytic processing of the amyloid-beta precursor protein (APP) generates the Abeta amyloid peptide of Alzheimer's disease. The biological function of APP itself remains, however, unclear. In the current review, we study in detail the different subdomains of APP and try to assign functional significance to particular structures identified in the protein.

Clioquinol Mediates Copper Uptake and Counteracts Copper Efflux Activities of the Amyloid Precursor Protein of Alzheimer's Disease

The key protein in Alzheimer's disease, the amyloid precursor protein (APP), is a ubiquitously expressed copper-binding glycoprotein that gives rise to the Abeta amyloid peptide. Whereas overexpression of APP results in significantly reduced brain copper levels in three different lines of transgenic mice, knock-out animals revealed increased copper levels. A provoked rise in peripheral levels of copper reduced concentrations of soluble amyloid peptides and resulted in fewer pathogenic Abeta plaques. Contradictory evidence has been provided by the efficacy of copper chelation treatment with the drug clioquinol. Using a yeast model system, we show that adding clioquinol to the yeast culture medium drastically increased the intracellular copper concentration but there was no significant effect observed on zinc levels. This finding suggests that clioquinol can act therapeutically by changing the distribution of copper or facilitating copper uptake rather than by decreasing copper levels. The overexpression of the human APP or APLP2 extracellular domains but not the extracellular domain of APLP1 decreased intracellular copper levels. The expression of a mutant APP deficient for copper binding increased intracellular copper levels several-fold. These data uncover a novel biological function for APP and APLP2 in copper efflux and provide a new conceptual framework for the formerly diverging theories of copper supplementation and chelation in the treatment of Alzheimer's disease.

The Integrated Role of Desferrioxamine and Phenserine Targeted to an Iron‐Responsive Element in the APP‐mRNA 5′‐Untranslated Region

The Alzheimer's amyloid precursor protein (APP) is the metalloprotein that is cleaved to generate the pathogenic Abeta peptide. We showed that iron closely regulated the expression of APP by 5'-untranslated region (5'-UTR) sequences in APP mRNA. Iron modulated APP holoprotein expression by a pathway similar to iron control of the translation of the ferritin-L and -H mRNAs by iron-responsive elements in their 5'-UTRs. APP gene transcription is also responsive to copper deficit where the Menkes protein depleted fibroblasts of copper to suppress transcription of APP through metal regulatory and copper regulatory sequences upstream of the APP 5' cap site. APP is a copper-zinc metalloprotein and chelation of Fe(3+) by desferrioxamine and Cu(2+) by clioquinol appeared to provide effective therapy for the treatment of AD in limited patient studies. We have introduced an RNA-based screen for small APP 5'-UTR binding molecules to identify leads that limit APP translation (but not APLP-1 and APLP-2) and amyloid Abeta peptide production. A library of 1200 drugs was screened to identify lead drugs that limited APP 5'-UTR-directed translation of a reporter gene. The efficacy of these leads was confirmed for specificity in a cell-based secondary assay to measure the steady-state levels of APP holoprotein relative to APLP-1/APLP-2 by Western blotting. Several chelators were identified among the APP 5'-UTR directed leads consistent with the presence of an IRE stem-loop in front of the start codon of the APP transcript. The APP 5'-UTR-directed drugs--desferrioxamine (Fe(3+) chelator), tetrathiomolybdate (Cu(2+) chelator), and dimercaptopropanol (Pb(2+) and Hg(2+) chelator)--each suppressed APP holoprotein expression (and lowered Abeta peptide secretion). The novel anticholinesterase phenserine also provided "proof of concept" for our strategy to target the APP 5'-UTR sequence to identify "anti-amyloid" drugs. We further defined the interaction between iron chelation and phenserine action to control APP 5'-UTR-directed translation in neuroblastoma (SY5Y) transfectants. Phenserine was most efficient to block translation under conditions of intracellular iron chelation with desferrioxamine suggesting that this anticholinesterase operated through an iron (metal)-dependent pathway at the APP 5'-UTR site.

Proteasomal Inhibition by α-Synuclein Filaments and Oligomers

A unifying feature of many neurodegenerative disorders is the accumulation of polyubiquitinated protein inclusions in dystrophic neurons, e.g. containing alpha-synuclein, which is suggestive of an insufficient proteasomal activity. We demonstrate that alpha-synuclein and 20 S proteasome components co-localize in Lewy bodies and show that subunits from 20 S proteasome particles, in contrast to subunits of the 19 S regulatory complex, bind efficiently to aggregated filamentous but not monomeric alpha-synuclein. Proteasome binding to insoluble alpha-synuclein filaments and soluble alpha-synuclein oligomers results in marked inhibition of its chymotrypsin-like hydrolytic activity through a non-competitive mechanism that is mimicked by model amyloid-Abeta peptide aggregates. Endogenous ligands of aggregated alpha-synuclein like heat shock protein 70 and glyceraldehyde-6-phosphate dehydrogenase bind filaments and inhibit their anti-proteasomal activity. The inhibitory effect of amyloid aggregates may thus be amenable to modulation by endogenous chaperones and possibly accessible for therapeutic intervention.