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.     

Oligomeric aggregates of amyloid β peptide 1–42 activate ERK/MAPK in SH-SY5Y cells via the α7 nicotinic receptor

The production and aggregation of amyloid β peptides (Aβ) has been linked to the development and progression of Alzheimer's disease. It is apparent that the various structural forms of Aβ can affect cell signalling pathways and the activity of neurons differently. In this study, we investigated the effects of oligomeric and fibrillar aggregates of Aβ 1–42 (Aβ42) and non-aggregated peptide upon activation of the ERK/MAPK signalling pathway. In SH-SY5Y cells, acute exposure to oligomeric Aβ42 led to phosphorylation of ERK1/2 at concentrations as low as 1 nM and up to 100 nM. These changes were detected as early as 5 min following exposure to 100 nM oligomeric Aβ42, reaching a maximum level after 10 min. Phosphorylation of ERK1/2 subsequently declined to and remained at basal levels after 30 min to 2 h of exposure. Fibrillar aggregates of Aβ42 did not significantly induce phosphorylation of ERK1/2 and non-aggregated Aβ42 did not activate the pathway. The effects of oligomeric Aβ42 to increase ERK phosphorylation above basal levels were inhibited by MLA, a specific antagonist of the α7 nAChR. U0126, an inhibitor of MEK, the upstream activator of ERK1/2, completely blocked induction of ERK1/2 phosphorylation. Oligomeric aggregates of Aβ42 are the principal structural form of the peptide that activates ERK/MAPK in SH-SY5Y cells and these effects are mediated by the α7 nAChR.     

α‐secretase mediated conversion of the amyloid precursor protein derived membrane stub C99 to C83 limits Aβ generation

The Swedish mutation within the amyloid precursor protein (APP) causes early‐onset Alzheimer’s disease due to increased cleavage of APP by BACE1. While β‐secretase shedding of Swedish APP (APPswe) largely results from an activity localized in the late secretory pathway, cleavage of wild‐type APP occurs mainly in endocytic compartments. However, we show that liberation of Aβ from APPswe is still dependent on functional internalization from the cell surface. Inspite the unchanged overall β‐secretase cleaved soluble APP released from APP_swe secretion, mutations of the APPswe internalization motif strongly reduced C99 levels and substantially decreased Aβ secretion. We point out that α‐secretase activity‐mediated conversion of C99 to C83 is the main cause of this Aβ reduction. Furthermore, we demonstrate that α‐secretase cleavage of C99 even contributes to the reduction of Aβ secretion of internalization deficient wild‐type APP. Therefore, inhibition of α‐secretase cleavage increased Aβ secretion through diminished conversion of C99 to C83 in APP695, APP695swe or C99 expressing cells.     

Hyposensitivity to the amnesic effects of scopolamine or amyloid β25–35 peptide in heterozygous acetylcholinesterase knockout (AChE) mice

We examined the sensitivity of AChE^+/− mice to the amnesic effects of scopolamine and amyloid β peptide. AChE^+/− and AChE^+/+ littermates, tested at 5–9 weeks of age, failed to show any difference in locomotion, exploration and anxiety in the open-field test, or in-place learning in the water-maze. However, when treated with the muscarinic receptor antagonist scopolamine (0.5, 5 mg/kg s.c.) 20 min before each water-maze training session, learning impairments were observed at both doses in AChE^+/+ mice, but only at the highest dose in AChE^+/− mice. The central injection of Aβ_25–35 peptide (9 nmol) induced learning deficits only in AChE^+/+ but not in AChE^+/− mice. Therefore, the hyper-activity of cholinergic systems in AChE^+/− mice did not result in increased memory abilities, but prevented the deleterious effects of muscarinic blockade or amyloid toxicity.     

Nanoporous protein matrix made of amyloid fibrils of β2‐microglobulin

Amyloid fibrils are considered as novel nanomaterials because of their nanoscale width, a regular constituting structure of cross β‐sheet conformation, and considerable mechanical strength. By using an amyloidogenic protein of β₂‐microglobulin (β₂M) related to dialysis‐related amyloidosis, nanoporous protein matrix has been prepared. The β₂M granules made of around 15 monomers showed an average size of 23.1 nm. They formed worm‐like fibrils at pH 7.4 in 20 mM sodium phosphate containing 0.15 M NaCl following vigorous nondirectional shaking incubation, in which they became laterally associated and interwound to generate the porous amyloid fibrillar matrix with an average pore size of 30–50 nm. This nanoporous protein matrix was demonstrated to be selectively disintegrated by reducing agents, such as tris‐(2‐carboxyethyl) phosphine. High surface area with nanopores on the surface has been suggested to make the matrix of β₂M amyloid fibrils particularly suitable for applications in the area of nanobiotechnology including drug delivery and tissue engineering. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The Alzheimer's Peptide Aβ Adopts a Collapsed Coil Structure in Water

Theself-assembly of the soluble peptide Aβ into Alzheimer's disease amyloid is believed to involve a conformational change. Hence the solution conformation of Aβ is of significant interest. In contrast to studies in other solvents, in water Aβ is collapsed into a compact series of loops, strands, and turns and has no α-helical or β-sheet structure. Conformational stabilization is primarily attributed to van der Waals and electrostatic forces. A large conspicuous uninterrupted hydrophobic patch covers 25% of the surface. The compact coil structure appears meta-stable, and because fibrillization leads to formation of intermolecular β-sheet secondary structure, a global conformational rearrangement is highly likely. A molecular hypothesis for amyloidosis includes at least two primary driving forces, changes in solvation thermodynamics during formation of amyloid deposits and relief of internal conformational stress within the soluble precursor during formation of lower-energy amyloid fibrils.     

Kinetic study of β‐amyloid residue accessibility using reductive alkylation and mass spectrometry

Beta‐amyloid peptide (Aβ) is the major protein constituent found in senile plaques in Alzheimer's disease (AD). It is believed that Aβ plays a role in neurodegeneration associated with AD and that its toxicity is related to its structure or aggregation state. In this study, an approach based on chemical modification of primary amines and mass spectrometric (MS) detection was used to identify residues on Aβ peptide that were exposed or buried upon changes in peptide structure associated with aggregation. Results indicate that the N terminus was the most accessible primary amine in the fibril, followed by lysine 28, then lysine 16. A kinetic analysis of the data was then performed to quantify differences in accessibility between these modification sites. We estimated apparent equilibrium unfolding constants for each modified site of the peptide, and determined that the unfolding constant for the N terminus was approximately 100 times greater than that for K28, which was about six times greater than that for K16. Understanding Aβ peptide structure at the residue level is a first step in designing novel therapies for prevention of Aβ structural transitions and/or cell interactions associated with neurotoxicity in Alzheimer's disease. Biotechnol. Bioeng. 2009; 104: 181–192 © 2009 Wiley Periodicals, Inc.     

Evidence of oxidative damage in Alzheimer's disease brain: central role for amyloid β-peptide

Amyloid β-peptide (Aβ) is heavily deposited in the brains of Alzheimer's disease (AD) patients. Free-radical oxidative stress, particularly of neuronal lipids, proteins and DNA, is extensive in those AD brain areas in which Aβ is abundant. Recent research suggests that these observations might be linked, and it is postulated that Aβ-induced oxidative stress leads to neurodegeneration in AD brain. Consonant with this postulate, Aβ leads to neuronal lipid peroxidation, protein oxidation and DNA oxidation by means that are inhibited by free-radical antioxidants. Here, we summarize current research on phospholipid peroxidation, as well as protein and DNA oxidation, in AD brain, and discuss the potential role of Aβ in this oxidative stress.     

A synthetic substrate assay for the gamma-secretase of the β-A4 amyloid of alzheimer's disease

γ-secretase, the endoprotease which releases the C-terminus of βA4 amyloid peptide, cleaves within the hydrophobic transmembrane domain of the amyloid precursor protein. In order to obtain a substrate for γ-secretase, a dodecapeptide which spans the cleavage site was synthesized, labelled with 125-iodine and conjugated to an agarose gel. A radiometric solid-phase assay was developed using this immobilized substrate. Peptide products were separated by reverse-phase HPLC and TLC to allow characterization of the cleavage site(s).     

Biphasic modulation of protein kinase C and enhanced cell toxicity by amyloid beta peptide and anoxia in neuronal cultures

A major feature of Alzheimer's disease is the deposition of the amyloid beta peptide (Abeta) in the brain by mechanisms which remain unclear. One hypothesis suggests that oxidative stress and Abeta aggregation are interrelated processes. Protein kinase C, a major neuronal regulatory protein is activated after oxidative stress and is also altered in the Alzheimer's disease brain. Therefore, we examined the effects of Abeta(1-40) peptide on the protein kinase C cascade and cell death in primary neuronal cultures following anoxic conditions. Treatment with Abeta(1-40) for 48 h caused a significant increase in the content and activity of Ca2+ dependent and Ca2+ independent protein kinase C isoforms. By 72 h various protein kinase C isoforms were down-regulated. Following 90 min anoxia and 6 h normoxia, a decrease in protein kinase C isoforms was noticed, independent of Abeta(1-40) treatment. A combination of Abeta(1-40) and 30-min anoxia enhanced cytotoxicity as noticed by a marked loss in the mitochondrial ability to convert 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and by enhanced 4',6-diamidino-2-phenylindole nuclear staining. Phosphorylation of two downstream protein kinase C substrates of apparent molecular mass 80 and 43 kDa, tentatively identified as the myristoyl alanine-rich C-kinase substrate (MARCKS), were gradually elevated up to 72 h upon incubation with Abeta(1-40). Anoxia followed by 30 min normoxia enhanced MARCKS phosphorylation in the membrane but not in the cytosolic fraction. In the presence of Abeta(1-40), phosphorylation of MARCKS was reduced. After 6 h normoxia, MARCKS phosphorylatability was diminished possibly because of protein kinase C down-regulation. The data suggest that a biphasic modulation of protein kinase C and MARCKS by Abeta(1-40) combined with anoxic stress may play a role in Alzheimer's disease pathology.     

Promoting α‐secretase cleavage of beta‐amyloid with engineered proteolytic antibody fragments

Deposition of beta‐amyloid (Aβ) is considered as an important early event in the pathogenesis of Alzheimer's Disease (AD), and reduction of Aβ levels by various therapeutic approaches is actively being pursued. A potentially non‐inflammatory approach to facilitate clearance and reduce toxicity is to hydrolyze Aβ at its α‐secretase site. We have previously identified a light chain fragment, mk18, with α‐secretase‐like catalytic activity, producing the 1–16 and 17–40 amino acid fragments of Aβ40 as primary products, although hydrolysis is also observed following other lysine and arginine residues. To improve the specific activity of the recombinant antibody by affinity maturation, we constructed a single chain variable fragment (scFv) library containing a randomized CDR3 heavy chain region. A biotinylated covalently reactive analog mimicking α‐secretase site cleavage was synthesized, immobilized on streptavidin beads, and used to select yeast surface expressed scFvs with increased specificity for Aβ. After two rounds of selection against the analog, yeast cells were individually screened for proteolytic activity towards an internally quenched fluorogenic substrate that contains the α‐secretase site of Aβ. From 750 clones screened, the two clones with the highest increase in proteolytic activity compared to the parent mk18 were selected for further study. Kinetic analyses using purified soluble scFvs showed a 3‐ and 6‐fold increase in catalytic activity (k_cat/K_M) toward the synthetic Aβ substrate compared to the original scFv primarily due to an expected decrease in K_M rather than an increase in k_cat. This affinity maturation strategy can be used to select for scFvs with increased catalytic specificity for Aβ. These proteolytic scFvs have potential therapeutic applications for AD by decreasing soluble Aβ levels in vivo. © 2009 American Institute of Chemical Engineers. Biotechnol. Prog., 2009     

Review: Alzheimer's Amyloid β-Peptide-Associated Free Radical Oxidative Stress and Neurotoxicity

Alzheimer's disease, the major dementing disorder of the elderly that affects over 4 million Americans, is related to amyloid β-peptide, the principal component of senile plaques in Alzheimer's disease brain. Oxidative stress, manifested by protein oxidation and lipid peroxidation, among other alterations, is a characteristic of Alzheimer's disease brain. Our laboratory united these two observations in a model to account for neurodegeneration in Alzheimer's disease brain, the amyloid β-peptide-associated oxidative stress model for neurotoxicity in Alzheimer's disease. Under this model, the aggregated peptide, perhaps in concert with bound redox metal ions, initiates free radical processes resulting in protein oxidation, lipid peroxidation, reactive oxygen species formation, cellular dysfunction leading to calcium ion accumulation, and subsequent neuronal death. Free radical antioxidants abrogate these findings. This review outlines the substantial evidence from multiidisciplinary approaches for amyloid β-peptide-associated free radical oxidative stress and neurotoxicity and protection against these oxidative processes and cell death by free radical scavengers. In addition, we review the strong evidence supporting the notion that the single methionine residue of amyloid β-peptide is vital to the oxidative stress and neurotoxicological properties of this peptide. Further, we discuss studies that support the hypothesis that aggregated soluble amyloid β-peptide and not fibrils per se are necessary for oxidative stress and neurotoxicity associated with amyloid β-peptide.     

β‐Hairpin stabilization in a 28‐residue peptide derived from the β‐subunit sequence of human chorionic gonadotropin hormone

The β‐subunit of the human chorionic gonadotropin (hCG) hormone, which is believed to be related to certain types of cancer, contains three hairpin‐like fragments. To investigate the role of β‐hairpin formation in the early stages of the hCGβ folding, a 28‐residue peptide with the sequence RDVRFESIRLPGSPRGVNPVVSYAVALS, corresponding to the H3‐β hairpin fragment (residues 60–87) of the hCGβ subunit, was studied under various conditions using three optical spectroscopic methods: Fourier transform ir spectroscopy, electronic CD, and vibrational CD. Environmental conditions are critical factors for formation of secondary structure in this peptide. TFE : H₂O mixed solvents induced helical formation. Formation of β‐structure in this peptide, which may be related to the native β‐hairpin formation in the intact hormone, was found to be induced only under conditions such as high concentration, high temperature, and the presence of nonmicellar sodium dodecyl sulfate concentrations. These findings support a protein folding mechanism for the hCGβ subunit in which an initial hydrophobic collapse, which increases intermolecular interactions in hCGβ, is needed to induce the H3‐β hairpin formation. © 1999 John Wiley & Sons, Inc. Biopoly 50: 413–423, 1999     

Huntingtin associated protein 1 regulates trafficking of the amyloid precursor protein and modulates amyloid beta levels in neurons

J. Neurochem. (2012) 122, 1010-1022. ABSTRACT: Amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease. It is axonally transported, endocytosed and sorted to different cellular compartments where amyloid beta (Aβ) is produced. However, the mechanism of APP trafficking remains unclear. We present evidence that huntingtin associated protein 1 (HAP1) may reduce Aβ production by regulating APP trafficking to the non-amyloidogenic pathway. HAP1 and APP are highly colocalized in a number of brain regions, with similar distribution patterns in both mouse and human brains. They are associated with each other, the interacting site is the 371-599 of HAP1. APP is more retained in cis-Golgi, trans-Golgi complex, early endosome and ER-Golgi intermediate compartment in HAP1-/- neurons. HAP1 deletion significantly alters APP endocytosis and reduces the re-insertion of APP into the cytoplasmic membrane. Amyloid precursor protein-YFP(APP-YFP) vesicles in HAP1-/- neurons reveal a decreased trafficking rate and an increased number of motionless vesicles. Knock-down of HAP1 protein in cultured cortical neurons of Alzheimer's disease mouse model increases Aβ levels. Our data suggest that HAP1 regulates APP subcellular trafficking to the non-amyloidogenic pathway and may negatively regulate Aβ production in neurons.     

Kinetics of the competitive reactions of isomerization and peptide bond cleavage at l‐α‐ and d‐β‐aspartyl residues in an αA‐crystallin fragment

d ‐β‐aspartyl (Asp) residue has been found in a living body such as aged lens crystallin, although l ‐α‐amino acids are constituents in natural proteins. Isomerization from l ‐α‐ to d ‐β‐Asp probably modulates structures to affect biochemical reactions. At Asp residue, isomerization and peptide bond cleavage compete with each other. To gain insight into how fast each reaction proceeds, the analysis requires the consideration of both pathways simultaneously and independently. No information has been provided, however, about these competitive processes because each reaction has been studied separately. The contribution of Asp isomers to the respective pathways has still been veiled. In this work, the two competitive reactions, isomerization and spontaneous peptide bond cleavage at Asp residue, were simultaneously observed and compared in an αA‐crystallin fragment, S⁵¹LFRTVLD⁵⁸SG⁶⁰ containing l ‐α‐ and d ‐β‐Asp58 isomers. The kinetics showed that the formation of l ‐ and d ‐succinimide (Suc) intermediate, as a first step of isomerization, was comparable at l ‐α‐ and d ‐β‐Asp. Although l ‐Suc was converted to l ‐β‐Asp, d ‐Suc was liable to return to the original d ‐β‐Asp, the reverse reaction marked enough to consider d ‐β‐Asp as apparently stable. d ‐β‐Asp was also resistant to the peptide bond cleavage. Such apparent less reactivity is probably the reason for gradual and abnormal accumulation of d ‐β‐Asp in a living body under physiological conditions. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Mitotic spindle association of TACC3 requires Aurora‐A‐dependent stabilization of a cryptic α‐helix

Aurora‐A regulates the recruitment of TACC3 to the mitotic spindle through a phospho‐dependent interaction with clathrin heavy chain (CHC). Here, we describe the structural basis of these interactions, mediated by three motifs in a disordered region of TACC3. A hydrophobic docking motif binds to a previously uncharacterized pocket on Aurora‐A that is blocked in most kinases. Abrogation of the docking motif causes a delay in late mitosis, consistent with the cellular distribution of Aurora‐A complexes. Phosphorylation of Ser558 engages a conformational switch in a second motif from a disordered state, needed to bind the kinase active site, into a helical conformation. The helix extends into a third, adjacent motif that is recognized by a helical‐repeat region of CHC, not a recognized phospho‐reader domain. This potentially widespread mechanism of phospho‐recognition provides greater flexibility to tune the molecular details of the interaction than canonical recognition motifs that are dominated by phosphate binding.