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D Allsop

Publications and source records attributed to D Allsop.

At least 19 recordsLinked to original sources
PubMed2005

The production of hydrogen peroxide during early-stage protein aggregation: a common pathological mechanism in different neurodegenerative diseases?

By means of an ESR spin-trapping method, we have shown that Abeta (amyloid beta), alpha-synuclein and various toxic forms of the prion protein all appear to generate H2O2 in vitro. A fundamental molecular mechanism underlying the pathogenesis of cell death in several different neurodegenerative diseases could be the direct production of H2O2 during the early stages of protein aggregation.

Amyloid↗
PubMed2005

Protein aggregation, metals and oxidative stress in neurodegenerative diseases.

There is clear evidence implicating oxidative stress in the pathology of many different neurodegenerative diseases. ROS (reactive oxygen species) are the primary mediators of oxidative stress and many of the aggregating proteins and peptides associated with neurodegenerative disease can generate hydrogen peroxide, a key ROS, apparently through interactions with redox-active metal ions. Our recent results suggest that ROS are generated during the very early stages of protein aggregation, when protofibrils or soluble oligomers are present, but in the absence of mature amyloid fibrils. The generation of ROS during early-stage protein aggregation may be a common, fundamental molecular mechanism underlying the pathogenesis of oxidative damage, neurodegeneration and cell death in several different neurodegenerative diseases. Drugs that specifically target this process could be useful in the future therapy of these diseases.

Humans↗
PubMed2002

Inherited frontotemporal dementia in nine British families associated with intronic mutations in the tau gene.

Genetic screening of 171 patients with frontotemporal lobar degeneration disclosed 14 patients, across nine pedigrees, with mutations in the intron to exon 10 in the tau gene, a region regulating the splicing of exon 10 via a stem loop mechanism. Thirteen of these patients had the +16 splice site mutation and one had the +13 splice site mutation. Affected members of all nine families presented with changes in behaviour and social conduct that were prototypical of frontotemporal dementia (FTD). In all patients with the +16 splice site mutation, the behavioural profile was characterized by disinhibition, restless overactivity, a fatuous affect, puerile behaviour and verbal and motor stereotypies. The single patient with the +13 mutation presented a contrasting picture of apathy and inertia. In addition, all patients had evidence of semantic loss. Pathologically, five of the six patients so far autopsied shared frontotemporal atrophy with involvement of the substantia nigra. The underlying histology was that of microvacuolar-type cortical degeneration with a few swollen cells. Tau pathology was widespread throughout the brain and present in neurones and glial cells, mostly in the frontal and temporal cortical regions. This was in the form of neurofibrillary tangles and amorphous tau deposits (pre-tangles); Pick bodies were not observed. Ultrastructurally, the tau filaments had a twisted, ribbon-like morphology distinct from the paired helical filaments of Alzheimer's disease. One patient died from an unrelated illness whilst in the early clinical stages of FTD. In this patient, cortical microvacuolar and astrocytic changes were absent, though there were scattered neurones and glial cells, immunoreactive to tau, throughout the cortical and subcortical regions. The disease process underlying the neurodegeneration within these inherited forms of FTD may therefore stem directly from early, primary alterations in the function of tau. All eight families with the +16 mutation seem to be part of a common extended pedigree, possibly originating from a founder member residing within the North Wales region of Great Britain.

Age of Onset↗
PubMed2001

Fluorescence anisotropy: a method for early detection of Alzheimer beta-peptide (Abeta) aggregation.

Time-resolved anisotropy measurements (TRAMS) have been used to study the aggregation of the beta-amyloid (Abeta) peptide which is suspected of playing a central role in the pathogenesis of Alzheimer's Disease (AD). The experiments, which employ small quantities of fluorescently-labelled Abeta, in addition to the untagged peptide, have shown that the sensitive TRAMS technique detects the presence of preformed "seed" particles in freshly prepared solutions of Abeta. More importantly, as 100 microM solutions of Abeta containing tagged Abeta at a concentration level of either 0.5 or 1 microM are incubated, the TRAMS prove capable of detection of the peptide aggregation process through the appearance of a continuously increasing "residual anisotropy" within the time-resolved fluorescence data. The method detects Abeta aggregation in its earliest stages, well before complexation becomes apparent in more conventional methods such as the thioflavin T fluorescence assay. The TRAMS approach promises to provide a most attractive route for establishment of a high-throughput procedure for the early detection of the presence of amyloid aggregates in the screening of biological samples.

Alzheimer Disease↗
PubMed2001

New evidence that the Alzheimer beta-amyloid peptide does not spontaneously form free radicals: an ESR study using a series of spin-traps.

The direct formation of free radicals from Abeta has been suggested to be a key neurotoxic mechanism in Alzheimer's disease (AD). We have explored the possibility of the spontaneous formation of peptide-derived free radicals during the incubation of Abeta 1-40 by ESR spectroscopy using N-tert-butyl-alpha-phenylnitrone (PBN), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), and 3,5-dibromo-4-nitrosobenzenesulfonic acid sodium salt (DBNBS) as spin traps. Employing PBN, we observed spectra during the incubation of beta-amyloid peptide, at 37 degrees C, which included adducts of 2-methyl-2-nitrosopropane (MNP), despite rigorous purification of the PBN before incubation. The formation of some of these adducts was found to be enhanced by ambient laboratory light. Our experiments have led us to propose a hypothesis that PBN undergoes hydrolysis and decomposition in the presence of oxidants, which explains the origin of all of the PBN and MNP adducts observed (even when the PBN is highly purified). Hydrogen peroxide, formed during incubation, could play a major role as an oxidant in these experiments. Of the other three spin traps, only DMPO gave (very weak) spectra, but these could be assigned to its hydroxyl radical adduct, formed as an artifact by the nucleophilic addition of water to DMPO, catalyzed by trace levels of iron ions. Thus, while spectra are observed during our experiments, none of them can be assigned to adducts of radicals derived from the peptide and, therefore, our data do not support the suggestion that radicals are spontaneously formed from beta-amyloid peptide.

Alzheimer Disease↗
PubMed2001

alpha-Synuclein implicated in Parkinson's disease catalyses the formation of hydrogen peroxide in vitro.

Some rare inherited forms of Parkinson's disease (PD) are due to mutations in the gene encoding a 140-amino acid presynaptic protein called alpha-synuclein. In PD, and some other related disorders such as dementia with Lewy bodies, alpha-synuclein accumulates in the brain in the form of fibrillar aggregates, which are found inside the neuronal cytoplasmic inclusions known as Lewy bodies. By means of an electron spin resonance (ESR) spin trapping method, we show here that solutions of full-length alpha-synuclein, and a synthetic peptide fragment of alpha-synuclein corresponding to residues 61-95 (the so-called non-Abeta component or NAC), both liberate hydroxyl radicals upon incubation in vitro followed by the addition of Fe(II). We did not observe this property for the related beta- and gamma-synucleins, which are not found in Lewy bodies, and are not linked genetically to any neurodegenerative disorder. There is abundant evidence for the involvement of free radicals and oxidative stress in the pathogenesis of nigral damage in PD. Our new data suggest that the fundamental molecular mechanism underlying this pathological process could be the production of hydrogen peroxide by alpha-synuclein.

Cell Death↗
PubMed2001

3-p-Toluoyl-2-[4'-(3-diethylaminopropoxy)-phenyl]-benzofuran and 2-[4'-(3-diethylaminopropoxy)-phenyl]-benzofuran do not act as surfactants or micelles when inhibiting the aggregation of beta-amyloid peptide.

The cmc and IC50 values of the beta-amyloid (Abeta) aggregation inhibitors, 3-p-toluoyl-2-[4'-(3-diethylaminopropoxy)-phenyl]-benzofuran 1, and 2-[4'-(3-diethylaminopropoxy)-phenyl]-benzofuran 2 have been determined. After comparison of the cmc data and biological data (IC50 values), we conclude that these active benzofurans do not act as surfactants or micelles at the concentration required to inhibit beta-amyloid-peptide aggregation.

Amyloid beta-Peptides↗
PubMed2001

Modulation of beta-amyloid production and fibrillization.

Alzheimer's disease (AD) is the most common cause of dementia in old age and presently affects an estimated 4 million people in the U.S.A. and 0.75 million people in the U.K. It is a relentless, degenerative brain disease, characterized by progressive cognitive impairment. In the final stages of the disease, patients are often bedridden, doubly incontinent and unable to speak or to recognize close relatives. Pathological changes of Alzheimer's disease include extensive neuronal loss and the presence of numerous neurofibrillary tangles and senile plaques in the brain. The senile plaques contain amyloid fibrils derived from a 39-43-amino-acid peptide referred to as beta-amyloid or A beta. The basic theory of the so-called 'amyloid hypothesis' is that the deposition of aggregated forms of A beta in the brain parenchyma triggers a pathological cascade of events that leads to neurofibrillary tangle formation, neuronal loss and the associated dementia [1]. Here we discuss progress towards the identification of inhibitors of A beta production and fibrillization.

Alzheimer Disease↗
PubMed2001

Production of reactive oxygen species from aggregating proteins implicated in Alzheimer's disease, Parkinson's disease and other neurodegenerative diseases.

The deposition of abnormal protein fibrils is a prominent pathological feature of many different 'protein conformational' diseases, including some important neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), motor neurone disease and the 'prion' dementias. Some of the fibril-forming proteins or peptides associated with these diseases have been shown to be toxic to cells in culture. A clear understanding of the molecular mechanisms responsible for this toxicity should shed light on the probable link between protein deposition and cell loss in these diseases. In the case of the beta-amyloid (Abeta), which accumulates in the brain in AD, there is good evidence that the toxic mechanism involves the production of reactive oxygen species (ROS). By means of an electron spin resonance (ESR) spin-trapping method, we have shown recently that solutions of Abeta liberate readily detectable amounts of hydroxyl radicals upon incubation in vitro followed by the addition of small amounts of Fe(II). We have also obtained similar results with alpha-synuclein, which accumulates in Lewy bodies in PD. Our data suggest that hydrogen peroxide accumulates during Abeta or alpha-synuclein incubation and that this is subsequently converted to hydroxyl radicals, on addition of Fe (II), by Fenton's reaction. Consequently, we now support the idea that one of the fundamental molecular mechanisms underlying the pathogenesis of cell death in AD, PD, and possibly some other protein conformational diseases, could be the direct production of ROS during formation of the abnormal protein aggregates. This hypothesis suggests a novel approach to the therapy of this group of diseases.

Alzheimer Disease↗
PubMed2000

Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells.

Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The K(i) values for the best inhibitors were in the range 10-100 nM with suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3xK(i)). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFkappaB activation with IC(50) values comparable to their K(i) values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells.

Animals↗
PubMed2000

Pick's disease is associated with mutations in the tau gene.

Recently, mutations within the tau gene have been associated with some familial forms of frontotemporal dementia. To investigate whether tau gene mutations are also associated with Pick's disease, we analyzed the tau gene in 30 cases of pathologically confirmed Pick's disease. Two coding mutations were identified in separate cases of Pick's disease. A glycine-to-arginine mutation at codon 389 was detected in 1 case and a lysine-to-threonine mutation at codon 257 was identified in another. Analysis of dephosphorylated tau from the brain of the patient with the codon 389 mutation revealed a prominent band representing tau, with four microtubule-binding domains and no amino terminal inserts. This is in contrast to Pick's disease without any tau gene mutations, which consist of tau with mainly three microtubule-binding domains and only a trace of tau, with four microtubule-binding domains. Functional analysis of tau with these two mutations demonstrated a reduced ability of tau to promote microtubule assembly. Surprisingly, these mutations increased tau's susceptibility to calpain I digestion, suggesting that this feature may be related to the formation of a Pick type of histology. Moreover, these data suggest that Pick's disease is not a separate entity but part of the frontotemporal dementia disease spectrum.

Adult↗
PubMed1999

Alzheimer's disease: correlation of the suppression of beta-amyloid peptide secretion from cultured cells with inhibition of the chymotrypsin-like activity of the proteasome.

Peptide aldehyde inhibitors of the chymotrypsin-like activity of the proteasome (CLIP) such as N-acetyl-Leu-Leu-Nle-H (or ALLN) have been shown previously to inhibit the secretion of beta-amyloid peptide (A beta) from cells. To evaluate more fully the role of the proteasome in this process, we have tested the effects on A beta formation of a much wider range of peptide-based inhibitors of CLIP than published previously. The inhibitors tested included several peptide boronates, some of which proved to be the most potent peptide-based inhibitors of beta-amyloid production reported so far. We found that the ability of the peptide aldehyde and boronate inhibitors to suppress A beta formation from cells correlated extremely well with their potency as CLIP inhibitors. Thus, we conclude that the proteasome may be involved either directly or indirectly in A beta formation.

Aldehydes↗
PubMed1998

Presenilins--in search of functionality.

The discovery of the PS proteins, the complexities of their biochemistry and their potential involvement in signalling pathways and in apoptosis have galvanized research into AD. To date, the aspect of the functionality of the PSs most relevant to the pathology of AD is the effect of PS FAD mutants to increase the proportion of A beta 42 produced from cells. This, coupled to the observation that gamma-secretase cleavage is considerably reduced in neurons derived from PS-1 knockout mice, argues strongly that PS plays a very direct role in the proteolytic processing of APP.

Alzheimer Disease↗
PubMed1997

Distinct sites of intracellular production for Alzheimer's disease A beta40/42 amyloid peptides.

The Alzheimer amyloid precursor protein (APP) is cleaved by several proteases, the most studied, but still unidentified ones, are those involved in the release of a fragment of APP, the amyloidogenic beta-protein A beta. Proteolysis by gamma-secretase is the last processing step resulting in release of A beta. Cleavage occurs after residue 40 of A beta [A beta(1-40)], occasionally after residue 42 [A beta(1-42)]. Even slightly increased amounts of this A beta(1-42) might be sufficient to cause Alzheimer's disease (AD) (reviewed in ref. 1, 2). It is thus generally believed that inhibition of this enzyme could aid in prevention of AD. Unexpectedly we have identified in neurons the endoplasmic reticulum (ER) as the site for generation of A beta(1-42) and the trans-Golgi network (TGN) as the site for A beta(1-40) generation. It is interesting that intracellular generation of A beta seemed to be unique to neurons, because we found that nonneuronal cells produced significant amounts of A beta(1-40) and A beta(1-42) only at the cell surface. The specific production of the critical A beta isoform in the ER of neurons links this compartment with the generation of A beta and explains why primarily ER localized (mutant) proteins such as the presenilins could induce AD. We suggest that the earliest event taking place in AD might be the generation of A beta(1-42) in the ER.

Alzheimer Disease↗
PubMed1996

Effect of aluminium on expression and processing of amyloid precursor protein.

The environmental agent aluminium has been extensively investigated for a potential role in the aetiology of Alzheimer's disease. Despite many investigations there is at present no definite proof for any involvement. If aluminium is involved it is possible that its action is mediated through interaction with the synthesis or processing of amyloid precursor protein (APP). The present study compared aluminium loaded IMR-32 neuroblastoma cells and rat brains with control cells and brains to determine if aluminium affected APP expression and/or processing. In the IMR-32 model system aluminium had no effect on steady-state APP mRNA levels or on the ratio of individual isoforms. It also had no quantitative or qualitative effect on APP-immunoreactive bands detected in protein extracts from conditioned medium of these cells. In total cell extracts, aluminium reduced the intensity of APP-immunoreactive bands between 120-105 kDa but had no effect on a 9 kDa band. In rat brains, aluminium had no effect on APP-immunoreactive bands from soluble or insoluble-membranous extracts. The results, in general, provide no evidence for any effect of aluminium on APP expression or processing.

Aluminum↗
PubMed1996

Aggregation and metal-binding properties of mutant forms of the amyloid A beta peptide of Alzheimer's disease.

The fibrillogenic properties of Alzheimer's A beta peptides corresponding to residues 1-40 of the normal human sequence and to two mutant forms containing the replacement Ala21 to Gly or Glu22 to Gln were compared. At pH 7.4 and 37 degrees C the Gln22 peptide was found to aggregate and precipitate from solution faster than the normal A beta, whereas the Gly21 peptide aggregated much more slowly. Electron microscopy showed that the aggregates all had fibrillar structures. Circular dichroism spectra of these peptides revealed that aggregation of the normal and Gln22 sequences was associated with spectral changes consistent with a transformation from random coil to beta sheet, whereas the spectrum of the Gly21 peptide remained almost unchanged during a period in which little or no aggregation occurred. When immobilised by spotting onto nitrocellulose membranes the peptides bound similar amounts of the radioisotope 65Zn2+. Of several competing metal ions, tested at 20x the concentration of Zn2+, Cu2+ displaced > 95% of the radioactivity from all three peptides and Ni2+ produced >50% displacement in each case. Some other metal ions tested caused lesser displacement, but Fe2+ and Al3+ were without effect. In a saturation binding assay, a value of 3.2 microM was obtained for the binding of Zn2+ to A beta but our data provided no evidence for a reported higher affinity site (107 nM). The results suggest that the neuropathology associated with the Gly21 mutation is not due to enhanced fibrillogenic or different metal-binding properties of the peptide and that the binding of zinc to amyloid peptides is not a specific phenomenon.

Alzheimer Disease↗
PubMed1995

The immunoreactive profile at the N-terminal region of A beta 1-39/40 but not A beta 1-42 changes with transition from monomer/dimer to further peptide aggregates.

Using site-specific antibodies, we assessed the effect of aggregation of various length forms of A beta on the immunoreactive profile of the peptides. All of the antibodies tested reacted with monomeric/dimeric forms of A beta 1-42 and its further aggregates. However, antibodies directed against the 1-24 region of A beta reacted weakly or not at all with A beta 1-39/40 monomers or dimers, but immunoreactivity was enhanced substantially following peptide incubation and aggregation. These results suggest that the conformation of the N-terminal region of monomeric and dimeric A beta 1-39/40 is different from that of aggregated forms, whereas the longer A beta 1-42 does not significantly change its N-terminal conformation during beta-sheet fibril formation. These immunochemical results are consistent with previous structural data, and help to explain the differential effects of A beta 1-39/40 and 1-42 on fibril formation in brain.

Blotting, Western↗