Lars O. Tjernberg

ARREST OF BETA -AMYLOID FIBRIL FORMATION BY A PENTAPEPTIDE LIGAND

Polymerization of amyloid β-peptide (Aβ) into amyloid fibrils is a critical step in the pathogenesis of Alzheimers disease. Here, we show that peptides incorporating a short Aβ fragment (KLVFF; Aβ) can bind full-length Aβ and prevent its assembly into amyloid fibrils. Through alanine substitution, it was demonstrated that amino acids Lys, Leu, and Phe are critical for binding to Aβ and inhibition of Aβ fibril formation. A mutant Aβ molecule, in which these residues had been substituted, had a markedly reduced capability of forming amyloid fibrils. The present data suggest that residues Aβ serve as a binding sequence during Aβ polymerization and fibril formation. Moreover, the present KLVFF peptide may serve as a lead compound for the development of peptide and non-peptide agents aimed at inhibiting Aβ amyloidogenesis in vivo.

Defeating Alzheimer's disease and other dementias: a priority for European science and society

Defeating Alzheimers disease and other dementias : a priority for European science and society

A molecular model of Alzheimer amyloid beta-peptide fibril formation.

Polymerization of the amyloid beta (Aβ) peptide into protease-resistant fibrils is a significant step in the pathogenesis of Alzheimer’s disease. It has not been possible to obtain detailed structural information about this process with conventional techniques because the peptide has limited solubility and does not form crystals. In this work, we present experimental results leading to a molecular level model for fibril formation. Systematically selected Aβ-fragments containing the Aβ16–20sequence, previously shown essential for Aβ-Aβ binding, were incubated in a physiological buffer. Electron microscopy revealed that the shortest fibril-forming sequence was Aβ14–23. Substitutions in this decapeptide impaired fibril formation and deletion of the decapeptide from Aβ1–42 inhibited fibril formation completely. All studied peptides that formed fibrils also formed stable dimers and/or tetramers. Molecular modeling of Aβ14–23 oligomers in an antiparallel β-sheet conformation displayed favorable hydrophobic interactions stabilized by salt bridges between all charged residues. We propose that this decapeptide sequence forms the core of Aβ-fibrils, with the hydrophobic C terminus folding over this core. The identification of this fundamental sequence and the implied molecular model could facilitate the design of potential inhibitors of amyloidogenesis.

Characterization of stable complexes involving apolipoprotein E and the amyloid β peptide in Alzheimer's disease brain

Genetic evidence suggests a role for apolipoprotein E (apoE) in Alzheimers disease (AD) amyloidogenesis. Here, amyloid-associated apoE from 32 AD patients was purified and characterized. We found that brain amyloid-associated apoE apparently exists not as free molecules but as complexes with polymers of the amyloid beta peptide (A beta). Brain A beta-apoE complexes were detected irrespective of the apoE genotype, and similar complexes could be mimicked in vitro. The fine structure of purified A beta-apoE complexes was fibrillar, and immunogold labeling revealed apoE immunoreactivity along the fibrils. Thus, we conclude that A beta-apoE complexes are principal components of AD-associated brain amyloid and that the data presented here support a role for apoE in the pathogenesis of AD.

Degradation of the amyloid beta-protein by the novel mitochondrial peptidasome, PreP.

Recently we have identified the novel mitochondrial peptidase responsible for degrading presequences and other short unstructured peptides in mitochondria, the presequence peptidase, which we named PreP peptidasome. In the present study we have identified and characterized the human PreP homologue, hPreP, in brain mitochondria, and we show its capacity to degrade the amyloid β-protein (Aβ). PreP belongs to the pitrilysin oligopeptidase family M16C containing an inverted zinc-binding motif. We show that hPreP is localized to the mitochondrial matrix. In situ immuno-inactivation studies in human brain mitochondria using anti-hPreP antibodies showed complete inhibition of proteolytic activity against Aβ. We have cloned, overexpressed, and purified recombinant hPreP and its mutant with catalytic base Glu78 in the inverted zinc-binding motif replaced by Gln. In vitro studies using recombinant hPreP and liquid chromatography nanospray tandem mass spectrometry revealed novel cleavage specificities against Aβ-(1-42), Aβ-(1-40), and Aβ Arctic, a protein that causes increased protofibril formation an early onset familial variant of Alzheimer disease. In contrast to insulin degrading enzyme, which is a functional analogue of hPreP, hPreP does not degrade insulin but does degrade insulin B-chain. Molecular modeling of hPreP based on the crystal structure at 2.1 Å resolution of AtPreP allowed us to identify Cys90 and Cys527 that form disulfide bridges under oxidized conditions and might be involved in redox regulation of the enzyme. Degradation of the mitochondrial Aβ by hPreP may potentially be of importance in the pathology of Alzheimer disease.

Amyloid β-peptide polymerization studied using fluorescence correlation spectroscopy

Background The accumulation of fibrillar deposits of amyloid β-peptide (Aβ) in brain parenchyma and cerebromeningeal blood vessels is a key step in the pathogenesis of Alzheimers disease. In this report, polymerization of Aβ was studied using fluorescence correlation spectroscopy (FCS), a technique capable of detecting small molecules and large aggregates simultaneously in solution. Results The polymerization of Aβ dissolved in Tris-buffered saline, pH 7.4, occurred above a critical concentration of 50 μM and proceeded from monomers/dimers into two discrete populations of large aggregates, without any detectable amount of oligomers. The aggregation showed very high cooperativity and reached a maximum after 40 min, followed by an increase in the amount of monomers/dimers and a decrease in the size of the large aggregates. Electron micrographs of samples prepared at the time for maximum aggregation showed a mixture of an amorphous network and short diffuse fibrils, whereas only mature amyloid fibrils were detected after one day of incubation. The aggregation was reduced when Aβ was incubated in the presence of Aβ ligands, oligopeptides previously shown to inhibit fibril formation, and aggregates were partly dissociated after the addition of the ligands. Conclusions The polymerization of Aβ is a highly cooperative process in which the formation of very large aggregates precedes the formation of fibrils. The entire process can be inhibited and, at least in early stages, partly reversed by Aβ ligands.

The role of protein glycosylation in Alzheimer disease

Glycosylation is one of the most common, and the most complex, forms of post‐translational modification of proteins. This review serves to highlight the role of protein glycosylation in Alzheimer disease (AD), a topic that has not been thoroughly investigated, although glycosylation defects have been observed in AD patients. The major pathological hallmarks in AD are neurofibrillary tangles and amyloid plaques. Neurofibrillary tangles are composed of phosphorylated tau, and the plaques are composed of amyloid β‐peptide (Aβ), which is generated from amyloid precursor protein (APP). Defects in glycosylation of APP, tau and other proteins have been reported in AD. Another interesting observation is that the two proteases required for the generation of amyloid β‐peptide (Aβ), i.e. γ‐secretase and β‐secretase, also have roles in protein glycosylation. For instance, γ‐secretase and β‐secretase affect the extent of complex N‐glycosylation and sialylation of APP, respectively. These processes may be important in AD pathogenesis, as proper intracellular sorting, processing and export of APP are affected by how it is glycosylated. Furthermore, lack of one of the key components of γ‐secretase, presenilin, leads to defective glycosylation of many additional proteins that are related to AD pathogenesis and/or neuronal function, including nicastrin, reelin, butyrylcholinesterase, cholinesterase, neural cell adhesion molecule, v‐ATPase, and tyrosine‐related kinase B. Improved understanding of the effects of AD on protein glycosylation, and vice versa, may therefore be important for improving the diagnosis and treatment of AD patients.

Aβ43 is more frequent than Aβ40 in amyloid plaque cores from Alzheimer disease brains

One hallmark of Alzheimer disease (AD) is the extracellular deposition of the amyloid β‐peptide (Aβ) in senile plaques. Two major forms of Aβ are produced, 40 (Aβ40) and 42 (Aβ42) residues long. The most abundant form of Aβ is Aβ40, while Aβ42 is more hydrophobic and more prone to form toxic oligomers and the species of particular importance in early plaque formation. Thus, the length of the hydrophobic C‐terminal seems to be very important for the oligomerization and neurotoxicity of the Aβ peptide. Here we investigated which Aβ species are deposited in AD brain. We analyzed plaque cores, prepared from occipital and frontal cortex, from sporadic and familial AD cases and performed a quantitative study using Aβ standard peptides. Cyanogen bromide was used to generate C‐terminal Aβ fragments, which were analyzed by HPLC coupled to an electrospray ionisation ion trap mass spectrometer. We found a longer peptide, Aβ43, to be more frequent than Aβ40. No variants longer than Aβ43 could be observed in any of the brains. Immunohistochemistry was performed and was found to be in line with our findings. Aβ1‐43 polymerizes rapidly and we suggest that this variant may be of importance for AD.

Active γ‐secretase is localized to detergent‐resistant membranes in human brain

Several lines of evidence suggest that polymerization of the amyloid β‐peptide (Aβ) into amyloid plaques is a pathogenic event in Alzheimer’s disease (AD). Aβ is produced from the amyloid precursor protein as the result of sequential proteolytic cleavages by β‐secretase and γ‐secretase, and it has been suggested that these enzymes could be targets for treatment of AD. γ‐Secretase is an aspartyl protease complex, containing at least four transmembrane proteins. Studies in cell lines have shown that γ‐secretase is partially localized to lipid rafts, which are detergent‐resistant membrane microdomains enriched in cholesterol and sphingolipids. Here, we studied γ‐secretase in detergent‐resistant membranes (DRMs) prepared from human brain. DRMs prepared in the mild detergent CHAPSO and isolated by sucrose gradient centrifugation were enriched in γ‐secretase components and activity. The DRM fraction was subjected to size‐exclusion chromatography in CHAPSO, and all of the γ‐secretase components and a lipid raft marker were found in the void volume (> 2000 kDa). Co‐immunoprecipitation studies further supported the notion that the γ‐secretase components are associated even at high concentrations of CHAPSO. Preparations from rat brain gave similar results and showed a postmortem time‐dependent decline in γ‐secretase activity, suggesting that DRMs from fresh rat brain may be useful for γ‐secretase activity studies. Finally, confocal microscopy showed co‐localization of γ‐secretase components and a lipid raft marker in thin sections of human brain. We conclude that the active γ‐secretase complex is localized to lipid rafts in human brain.

Binding of amyloid β-peptide to mitochondrial hydroxyacyl-CoA dehydrogenase (ERAB): regulation of an SDR enzyme activity with implications for apoptosis in Alzheimer’s disease

The intracellular amyloid β‐peptide (Aβ) binding protein, ERAB, a member of the short‐chain dehydrogenase/reductase (SDR) family, is known to mediate apoptosis in different cell lines and to be a class II hydroxyacyl‐CoA dehydrogenase. The Aβ peptide inhibits the enzymatic reaction in a mixed type fashion with a K i of 1.2 μmol/l and a K iES of 0.3 μmol/l, using 3‐hydroxybutyryl‐CoA. The peptide region necessary for inhibition comprises residues 12–24 of Aβ1–40, covering the 16–20 fragment, which is the minimum sequence for the blockade of Aβ polymerization, but that minimal fragment is not sufficient for more than marginal inhibition. The localization of ERAB to the endoplasmic reticulum and mitochondria suggests a complex interaction with components of the programmed cell death machinery. The interaction of Aβ with ERAB further links oxidoreductase activity with both apoptosis and amyloid toxicity.