Amyloid-β aggregates induced by β-cholesteryl glucose-embedded liposomes

Senile plaques that is characterized as an amyloid deposition found in Alzheimer's disease are composed primarily of fibrils of an aggregated peptide, amyloid β (Aβ). The ability to monitor senile plaque formation on a neuronal membrane under physiological conditions provides an attractive model. In this study, the growth behavior of amyloid Aβ fibrils in the presence of liposomes incorporating β-cholesteryl-D-glucose (β-CG) was examined using total internal reflection fluorescence microscopy, transmittance electron microscopy, and other spectroscopic methods. We found that β-CG on the liposome membrane induced the spontaneous formation of spherulitic Aβ fibrillar aggregates. The β-CG cluster formed on liposome membranes appeared to induce the accumulation of Aβ, followed by the growth of the spherulitic Aβ aggregates. In contrast, DMPC and DMPC incorporated cholesterol-induced fibrils that are laterally associated with each other. A comparison study using three types of liposomes implied that the induction of glucose contributed to the agglomeration of Aβ fibrils and liposomes. This agglomeration required the spontaneous formation of spherulitic Aβ fibrillary aggregates. This action can be regarded as a counterbalance to the growth of fibrils and their toxicity, which has great potential in the study of amyloidopathies.     

The relationship between senile plaques and cerebral blood vessels in Alzheimer's disease and senile dementia. Morphological mechanism of senile plaque production

Several kinds of senile plaque found in 6 brains (4 from patients with Alzheimer's disease and 2 from patients with senile dementia) were examined in serial sections by light electron microscopy. The results obtained were as follows. All the senile plaques contained at least some amyloid fibrils, and these seemed to be produced at the basement membranes of capillary endothelial cells and projected into the surrounding parenchyma. Even when the senile plaques themselves appeared to lack amyloid fibrils by light microscopy, at least one degenerable capillary containing amyloid fibrils was demonstrable when serial sections were examined ultrastructurally. The findings described above suggest that the amyloid fibrils which form the cores of the several kinds of senile plaque, seem to be produced at the basement membrane of the endothelial cell. It is speculated that the capillary degeneration with the formation of amyloid fibrils may be primary change in the genesis of senile plaques.     

Visualization and classification of amyloid beta supramolecular assemblies

Deposition of amyloid beta (Abeta) fibrils has been suggested to play a central role in Alzheimer's disease. In clarifying the mechanism by which fibrils form and moreover in developing new treatments for amyloidosis, direct observation is important. Focusing on the interactions with surfaces at the early stages, we studied the spontaneous formation of Abeta(1-40) fibrils on quartz slides, monitored by total internal reflection fluorescence microscopy combined with thioflavin T, an amyloid-specific fluorescence dye. Self-assembly of Abeta(1-40), accelerated by a low concentration of sodium dodecyl sulfate, produced various remarkable amyloid assemblies. Densely packed spherulitic structures with radial fibril growth were typically observed. When the packing of fibrils was coarse, extremely long fibrils often protruded from the spherulitic cores. In other cases, a large number of wormlike fibrils were formed. Transmission electron microscopy and atomic force microscopy revealed relatively short and straight fibrillar blocks associated laterally without tight interaction, leading to random-walk-like fibril growth. These results suggest that, during spontaneous fibrillation, the nucleation occurring in contact with surfaces is easily affected by environmental factors, creating various types of nuclei, and hence variations in amyloid morphology. A taxonomy of amyloid supramolecular assemblies will be useful in clarifying the structure-function relationship of amyloid fibrils.     

Amyloid fibril formation by a synthetic peptide from a region of human acetylcholinesterase that is homologous to the Alzheimer's amyloid-beta peptide

A region near the C-terminus of human acetylcholinesterase (AChE) is weakly homologous with the N-terminus of the Alzheimer's disease amyloid-beta peptide. We report that a 14-amino acid synthetic polypeptide whose sequence corresponds to residues 586-599 of the human synaptic or T form of AChE assembles into amyloid fibrils under physiological conditions. The fibrils have all the classical characteristics of amyloid: they have a diameter of 6-7 nm and bind both Congo red and thioflavin-T. Furthermore, the kinetics of assembly indicate that fibril formation proceeds via a two-step nucleation-dependent polymerization pathway, and a transition in the peptide conformation from random coil to beta-sheet is observed during fibril formation using far-UV circular dichroism spectroscopy. We also show that the peptide in aggregated fibrillar form has a toxic effect upon PC-12 cells in vitro. AChE normally resides mainly on cholinergic neuronal membranes, but is abnormally localized to senile plaques in Alzheimer's disease. Recently, an in vitro interaction between AChE and A beta, the principal constituent of the amyloid fibrils in senile plaques, has been documented. The presence of a fibrillogenic region within AChE may be relevant to the interaction of AChE with amyloid fibrils formed by Abeta.     

Laminin affects polymerization,depolymerization and neurotoxicity of Abeta peptide

Amyloid deposition in Alzheimer fibrils forms neurotoxic senile plaques in a process that may be modulated by associated proteins. In this work we demonstrate the ability of laminin-1 and laminin-2 to inhibit fibril formation and toxicity on cultured rat hippocampal neurons. We confirm that the laminin-1-derived peptide YFQRYLI inhibits efficiently both fibril formation and neurotoxicity and show that the IKVAV peptide inhibits amyloid neurotoxicity despite its slight inhibition of fibril formation. On other hand, laminin-1 induces disaggregation of preformed fibrils in vitro, characterized as a progressive disassembly of fibrils into protofibrils and further clearance of these latter species, leading to a continual inhibition of amyloid neurotoxicity.     

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.     

Flow-induced alignment of amyloid protofilaments revealed by linear dichroism

Amyloid fibrils underlying various serious amyloidoses including Alzheimer and prion diseases form characteristic deposits in which linear fibrils with an unbranched and rigid morphology associate laterally or radially, e.g. radial senile amyloid plaques of amyloid beta. To clarify the formation of these high order amyloid deposits, studying the rheology is important. A 22-residue K3 peptide fragment of beta2-microglobulin, a protein responsible for dialysis-related amyloidosis, forms long and homogeneous protofilament-like fibrils in 20% (v/v) 2,2,2-trifluoroethanol and 10 mM HCl (pH approximately 2). Here, using circular dichroism and linear dichroism, we observed the flow-induced alignment of fibrils. Analysis of far- and near-UV linear dichroism spectra suggested that both the net pi-pi* transition moment of the backbone carbonyl group and L(b) transition moment of the Tyr(26) side chain are oriented in parallel to the fibril axis, revealing the structural details of amyloid protofilaments. Moreover, the intensities of flow-induced circular dichroism or linear dichroism signals depended critically on the length and type of fibrils, suggesting that they are useful for detecting and characterizing amyloid fibrils.     

Mechanisms of the Effects of Crocin on Aggregation and Deposition of Aβ1–40 Fibrils in Alzheimer’s Disease

Alzheimer??s disease (AD) is among the most important health-care problems in the world. The two pathological hallmarks of AD are extracellular neuritic amyloid plaques and intracellular neurofibrillary tangles. The aggregation of A?? and ??-sheet formation are considered to be the critical events which render these peptides neurotoxic. AD is affecting a large percentage of the elderly around the world. Many studies have been done on drugs to cure or at least slow Alzheimer??s disease. Most drugs produced for this disease aim at compensating for the performance of specific cell groups affected by the disease or restoring the function of these cells.This study examined the interaction of crocin, the main pigment of saffron, with the amyloid-?? peptides 1?+?40 (A?? 40) to determine the effects on peptide conformation and fibril formation using fluorescence spectroscopy, CD spectroscopy and electron microscopy. ThT data demonstrated the appearance of well-defined amyloid fibrils indicating an enhanced nucleation of A??40. Incubation of pre-formed A??40 fibrils with crocin resulted in extensive lateral aggregation and precipitation of the fibrils. Consistent with this, electron microscopy data indicated that crocin decreased the number of fibrils formed and significantly reduced the average fibril length of A??40 as assessed by low levels of thioflavin T binding data. The mechanism by which, crocin prevented fibril formation was demonstrated by ANS binding assay and CD spectroscopy. In summary, crocin interacts with A?? peptides and prevents amyloid formation. This means that it has the potential to be an important therapeutic drug against AD.     

Interactions of Alzheimer amyloid peptides with cultured cells and brain tissue, and their biological consequences

The Alzheimer amyloid peptides are the main constituent of the diagnostic hallmark of Alzheimer disease, the senile plaque. A halo of neurodegeneration surrounds the senile plaques observed in the brains of Alzheimer patients. Significant efforts are under way to determine whether the Alzheimer peptides are the causal agents of this neurodegeneration. We review the developments in identifying the putative interaction sites of Alzheimer amyloid peptides on cells and intact brain tissue. We focus on the specificity of this interaction and on the molecular nature of potential receptors. These studies form the bases for developing therapeutics that target potential interaction sites and inhibit Alzheimer amyloid peptide deposition.     

Congo Red Inhibits Proteoglycan and Serum Amyloid P Binding to Amyloid β Fibrils

Abstract: Various data suggest that Alzheimer's disease results from the accumulation of amyloid β (Aβ) peptide fibrils and the consequent formation of senile plaques in the cognitive regions of the brain. One approach to lowering senile plaque burden in Alzheimer's disease brain is to identify compounds that will increase the degradation of existing amyloid fibrils. Previous studies have shown that proteoglycans and serum amyloid P (SAP), molecules that localize to senile plaques, bind to Aβ fibrils and protect the amyloid peptide from proteolytic breakdown. Therefore, molecules that prevent the binding of SAP and/or proteoglycans to fibrillar Aβ might increase plaque degradation and prove useful in the treatment of Alzheimer's disease. The nature of SAP and proteoglycan binding to Aβ is defined further in the present study. SAP binds to both fibrillar and nonfibrillar forms of Aβ. However, only the former is rendered resistant to proteolysis after SAP association. It is interesting that both SAP and proteoglycan binding to Aβ fibrils can be inhibited by glycosaminoglycans and Congo red. Unexpectedly, Congo red protects fibrillar Aβ from breakdown, suggesting that this compound and other structurally related molecules are unlikely to be suitable for use in the treatment of Alzheimer's disease.     

High density lipoprotein inhibits assembly of amyloid beta-peptides into fibrils

The extracellular deposition of amyloid beta (Abeta) in senile plaques constitutes one of the defining hallmarks of Alzheimer's disease. Abeta peptides can aggregate spontaneously to highly insoluble amyloid fibrils, but several components are likely to influence the kinetics of fibrillogenesis in vivo. We report here that high density lipoprotein (HDL), the predominant lipoprotein in the human brain, reduces amyloid formation in vitro as determined by thioflavin T fluorescence and high speed sedimentation assays. The inhibition occurred in a dose dependent manner, and with concentrations of HDL above 1% resulting in more than 70% inhibition. We also examined the combined effect of apolipoprotein E (apoE) and HDL on Abeta fibrillogenesis. We found that HDL particles enriched with any of the three apoE isoforms inhibited Abeta fibrillogenesis as their native counterparts. Taken together, these findings suggest that HDL-like particles in the brain may prevent the formation of Abeta fibrils.     

CLAC binds to amyloid beta peptides through the positively charged amino acid cluster within the collagenous domain 1 and inhibits formation of amyloid fibrils

CLAC (collagenous Alzheimer amyloid plaque component) is a proteolytic fragment derived from a novel membrane-bound collagen, CLAC-P/collagen type XXV, that deposits in senile plaques associated with amyloid beta peptides (Abeta) in the brains of patients with Alzheimer's disease. We previously showed that CLAC binds to the fibrillized form of Abeta in vitro, although the mechanism and the subdomains that mediate interaction of CLAC with Abeta as well as the effect of binding of CLAC on amyloid fibril formation remain unknown. Here we show that the collagenous domain 1 of CLAC, which is rich in positively charged amino acid residues, mediates its interaction with Abeta and that this binding is mediated by an electrostatic interaction and requires formation of the triple helix structure of CLAC. The soluble form of CLAC purified from the media of cells transfected with CLAC-P inhibited fibrillization of Abeta in vitro, especially in its elongation phase. These results suggest the anti-amyloidogenic roles of CLAC in the pathophysiology of Alzheimer's disease.     

Metals accelerate the formation and direct the structure of amyloid fibrils of NAC

Non-beta amyloid component of Alzheimer's disease amyloid or NAC is a highly amyloidogenic peptide consisting of 35 amino acids which was first identified associated with senile plaques in the Alzheimer's disease brain. It is a fragment of the presynaptic protein alpha-synuclein and, as such, it is implicated in the aetiologies of both Alzheimer's (AD) and Parkinson's (PD) disease. Metals are involved in the aggregation of amyloidogenic peptides such as beta amyloid (Abeta), British amyloid peptide (ABri) and alpha-synuclein though nothing is yet known about how they might influence the aggregation of NAC. We show herein that NAC will form beta-pleated conformers at a peptide concentration of only 2.0 microM and that metals, and Zn(II) and Cu(II) in particular, accelerate the formation of these fibrils. Cu(II) and Zn(II) did not influence the diameter or general structure of the fibrils which were formed though many more shorter fibrils were observed in their presence and these shorter fibrils were highly thioflavin T positive and they were efficient catalysts of the redox cycling of added Fe(II). By way of contrast, beta-pleated conformers of NAC which were formed in the presence of Al(III) showed much lower levels of thioflavin T fluorescence and were poorer catalysts of the redox cycling of added Fe(II) and these properties were commensurate with an increased abundance of a novel amyloid morphology which consisted of twisted fibrils with a periodicity of about 100 nm. These spirals of twisted fibrils were especially abundant in the presence of added Al(III) and it is speculated that NAC binding of Al(III) may be important in their formation and subsequent stability.     

Membrane-mediated amyloidogenesis and the promotion of oxidative lipid damage by amyloid beta proteins

Evidence of oxidative stress and the accumulation of fibrillar amyloid beta proteins (Abeta) in senile plaques throughout the cerebral cortex are consistent features in the pathology of Alzheimer disease. To define a mechanistic link between these two processes, various aspects of the relationship between oxidative lipid membrane damage and amyloidogenesis were characterized by chemical and physical techniques. Earlier studies of this relationship demonstrated that oxidatively damaged synthetic lipid membranes promoted amyloidogenesis. The studies reported herein specify that 4-hydroxy-2-nonenal (HNE) is produced in both synthetic lipids and human brain lipid extracts by oxidative lipid damage and that it can account for accelerated amyloidogenesis. Abeta promotes the copper-mediated generation of HNE from polyunsaturated lipids, and in turn, HNE covalently modifies the histidine side chains of Abeta. HNE-modified Abeta have an increased affinity for lipid membranes and an increased tendency to aggregate into amyloid fibrils. Thus, the prooxidant activity of Abeta leads to its own covalent modification and to accelerated amyloidogenesis. These results illustrate how lipid membranes may be involved in templating the pathological misfolding of Abeta, and they suggest a possible chemical mechanism linking oxidative stress with amyloid formation.     

In vitro formation of amyloid fibrils from two synthetic peptides of different lengths homologous to Alzheimer's disease beta-protein

Two synthetic peptides corresponding to the reported 28-residue sequence of Alzheimer's Disease beta-protein (SP28) and to residues 12-28 (SP17) were used to form fibrils in vitro. Synthetic fibrils bound Congo Red and closely resembled amyloid fibrils isolated from leptomeninges and senile plaques of Alzheimer's brain by electron microscopy. A polyclonal antiserum to SP28 specifically decorated both synthetic and native amyloid by colloidal gold immunoelectron microscopy. Amyloid fibrils isolated from tissue were insoluble on SDS-Polyacrylamide gels, and tended to aggregate while synthetic amyloid fibrils were completely solubilized, releasing only monomers of SP28 and SP17. Anti-SP28 immunostained cerebrovascular and plaque core amyloid, but not neurofibrillary tangles, in tissue section. Western blot analysis showed that anti-SP28 reacted with a 4 kDa band released from amyloid core-enriched preparations and leptomeninges. By contrast, a 16 kDa band corresponding to the tetramer of beta-protein was not recognized. These data suggest that as little as a 17 residue sequence of beta-protein may be required to form fibrils and that the complete sequence of the 4 kDa beta-protein may be important in determining insolubility and the formation of intermediate size polymers.     

Tritium-labeled (E,E)-2,5-bis(4′-hydroxy-3′-carboxystyryl)benzene as a probe for β-amyloid fibrils

Accumulation of Aβ in the brains of Alzheimer disease (AD) patients reflects an imbalance between Aβ production and clearance from their brains. Alternative cleavage of amyloid precursor protein (APP) by processing proteases generates soluble APP fragments including the neurotoxic amyloid Aβ40 and Aβ42 peptides that assemble into fibrils and form plaques. Plaque-buildup occurs over an extended time-frame, and the early detection and modulation of plaque formation are areas of active research. Radiolabeled probes for the detection of amyloid plaques and fibrils in living subjects are important for noninvasive evaluation of AD diagnosis, progression, and differentiation of AD from other neurodegenerative diseases and age-related cognitive decline. Tritium-labeled (E,E)-1-[³H]-2,5-bis(4′-hydroxy-3′-carbomethoxystyryl)benzene possesses an improved level of chemical stability relative to a previously reported radioiodinated analog for radiometric quantification of Aβ plaque and tau pathology in brain tissue and in vitro studies with synthetic Aβ and tau fibrils.     

In vivo detection of amyloid-beta deposits by near-infrared imaging using an oxazine-derivative probe

As Alzheimer's disease pathogenesis is associated with the formation of insoluble aggregates of amyloid beta-peptide, approaches allowing the direct, noninvasive visualization of plaque growth in vivo would be beneficial for biomedical research. Here we describe the synthesis and characterization of the near-infrared fluorescence oxazine dye AOI987, which readily penetrates the intact blood-brain barrier and binds to amyloid plaques. Using near-infrared fluorescence imaging, we demonstrated specific interaction of AOI987 with amyloid plaques in APP23 transgenic mice in vivo, as confirmed by postmortem analysis of brain slices. Quantitative analysis revealed increasing fluorescence signal intensity with increasing plaque load of the animals, and significant binding of AOI987 was observed for APP23 transgenic mice aged 9 months and older. Thus, AOI987 is an attractive probe to noninvasively monitor disease progression in animal models of Alzheimer disease and to evaluate effects of potential Alzheimer disease drugs on the plaque load.     

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.     

Sulfated polysaccharides promote the assembly of amyloid beta(1-42) peptide into stable fibrils of reduced cytotoxicity

The histopathological hallmarks of Alzheimer disease are the self-aggregation of the amyloid beta peptide (Abeta) in extracellular amyloid fibrils and the formation of intraneuronal Tau filaments, but a convincing mechanism connecting both processes has yet to be provided. Here we show that the endogenous polysaccharide chondroitin sulfate B (CSB) promotes the formation of fibrillar structures of the 42-residue fragment, Abeta(1-42). Atomic force microscopy visualization, thioflavin T fluorescence, CD measurements, and cell viability assays indicate that CSB-induced fibrils are highly stable entities with abundant beta-sheet structure that have little toxicity for neuroblastoma cells. We propose a wedged cylinder model for Abeta(1-42) fibrils that is consistent with the majority of available data, it is an energetically favorable assembly that minimizes the exposure of hydrophobic areas, and it explains why fibrils do not grow in thickness. Fluorescence measurements of the effect of different Abeta(1-42) species on Ca(2+) homeostasis show that weakly structured nodular fibrils, but not CSB-induced smooth fibrils, trigger a rise in cytosolic Ca(2+) that depends on the presence of both extracellular and intracellular stocks. In vitro assays indicate that such transient, local Ca(2+) increases can have a direct effect in promoting the formation of Tau filaments similar to those isolated from Alzheimer disease brains.