Biomedical subjects

D R Howlett

Publications and source records attributed to D R Howlett.

At least 19 recordsLinked to original sources

PubMed2003

Ultrastructural and behavioural changes precede amyloid deposition in a transgenic model of Alzheimer's disease.

We describe the thorough characterisation of a new transgenic mouse line overexpressing the 695-amino acid isoform of human amyloid precursor protein harbouring the Swedish double familial Alzheimer's disease mutation. This line, referred to as TAS10, exhibits neuropathological features and cognitive deficits that are closely correlated to the accumulation of Abeta in their brain and that are reminiscent of those observed in AD. Data on the TAS10 line are presented at five time points: 2, 6, 12, 18 and 24 months in a longitudinal study. The TAS10 line is characterised by the following changes: i) significant age-related increases in the levels of total and individual species (1-40, 1-42) of beta-amyloid in the brains of transgenics compared with non-transgenic littermates; ii) transgenic mice showed pronounced spatial learning deficits in the Morris water maze at 6 months and working memory deficits by 12 months; iii) amyloid plaque and associated pathologies were observed by the 12-month time point and the burden increased substantially, particularly in the cortex, by 18 months; iv) electron microscopy of the hippocampus of transgenic mice showed evidence of abnormal ultrastructural features such as dystrophic neurites and lipid deposits that developed from 6 months and increased in number and severity with age. Morphometric studies demonstrate that the synapse to neuron ratio is higher in transgenics than in control mice at 12 months, but this ratio decreases as they age and synapse size increases. Thus, this mouse model exhibits a close correlation of amyloid burden with behavioural deficits and ultrastructural abnormalities and so represents an ideal system to study the mechanisms underlying the impact of amyloid pathology on CNS function.

Alzheimer Disease↗

PubMed2003

Hyperphosphorylated tau and paired helical filament-like structures in the brains of mice carrying mutant amyloid precursor protein and mutant presenilin-1 transgenes.

Senile plaques composed mainly of beta-amyloid (Abeta) and neurofibrillary tangles principally composed of hyperphosphorylated tau are the major pathological features of Alzheimer's disease (AD). Despite the fact that increased expression of amyloid precursor protein (APP) and presenilin-1 (PS1) transgenes in mice lead to increased Abeta deposition in plaquelike structures in the brain, little is known about the nature and distribution of tau in these mice. Therefore the relationship between Abeta and hyperphosphorylated tau was investigated in mice carrying mutant APP and mutant PS1 transgenes using both light (LM) and electron microscopy (EM) with immunocytochemistry. LM immunocytochemistry revealed cerebral Abeta deposits to be present from 8 weeks of age, whereas hyperphosphorylated tau was not detected until 24 weeks of age, when it appeared as punctate deposits in close association with the Abeta deposits in the cortex and hippocampus. However, dystrophic neurites were not as heavily immunolabeled as they are in AD brain. EM revealed that aggregations of straight filaments (10-12 nm wide) were present in some cellular processes at the periphery of Abeta plaques in 8-month-old APP/PS1 mice. In one such mouse, single filaments and paired filaments showing a helical configuration (50-55 nm half-period, 25 nm max. width) were present in a dark, atrophic hippocampal neuron. Immunogold labeling of APP/PS1 mouse brain revealed hyperphosphorylated tau epitopes in some dystrophic neurites from 24 weeks of age that were similar to those present in AD. These results suggest that hyperphosphorylated tau appears in APP/PS1 mouse brain after the onset of Abeta deposition and although it is associated with Abeta deposits, its distribution is not identical to that in AD.

Adult↗

PubMed2002

Production of beta-amyloid by primary human foetal mixed brain cell cultures and its modulation by exogenous soluble beta-amyloid.

Previous studies on beta-amyloid production have been carried out using transfected cells and cell lines. We measured the 40 and 42 amino acid forms of beta-amyloid released into the culture medium by primary human foetal mixed brain cell aggregate culture over 3 months. In this model, neurones and supporting cells are maintained in serum-free defined medium. The secretion of significant amounts of beta-amyloid 40 and 42 was observed throughout culture for three separate cultures. Levels of beta-amyloid 40 and 42 closely followed the neuronal content of the cultures as estimated by cellular neurone-specific enolase. Addition of synthetic beta-amyloid 1-40 to the cultures for 1 week at 35 days in vitro resulted in a dose-related reduction in cellular neurone-specific enolase levels. Primary human aggregate brain cell cultures produced multimeric beta-amyloid, as determined by immunoassay. beta-Amyloid-treated cultures released diminishing amounts of multimeric beta-amyloid and contained increasing amounts of intracellular multimeric beta-amyloid with increasing exogenous beta-amyloid. These results suggest that release of multimeric beta-amyloid into the extracellular environment by human primary neurones can be affected by the presence of extracellular beta-amyloid. This has implications for Alzheimer's disease in that beta-amyloid released into the extracellular environment by dead/dying neurones could modulate beta-amyloid release by surrounding neurones, potentially causing amplification of toxicity. Moreover, intracellular beta-amyloid oligomer-dependent neurotoxicity may be a component of neurodegeneration in Alzheimer's disease, and other conditions with increased beta-amyloid synthesis, suggesting anti-amyloid therapies for Alzheimer's disease may have to target intracellular beta-amyloid.

Amyloid beta-Peptides↗

PubMed2001

Neurodegenerative changes associated with beta-amyloid deposition in the brains of mice carrying mutant amyloid precursor protein and mutant presenilin-1 transgenes.

Mutations of amyloid precursor protein (APP) and presenilin-1 (PS1) lead to an increase in beta-amyloid (Abeta) production. Despite the fact that a number of transgenic mice develop cerebral Abeta plaques, few have been subjected to ultrastructural investigation and the sequence of events leading to Abeta plaque formation is unclear. We therefore investigated the doubly transgenic (mutant APP(K670N,M671L)-mutant PS1(M146L)) mouse, which develops Abeta deposits much earlier than singly transgenic littermates. Widespread Abeta plaques with or without a distinct core were found in gray matter. Abeta plaques were also present in white matter. Astrocytosis was greater around gray matter plaques than around white matter plaques. In some plaques, Abeta cores were associated with cell profiles containing prominent endoplasmic reticulum and a homogeneous cytoplasm that appeared to be neuronal. The morphology and location of other profiles indicated them to be microglia or oligodendrocytes. Some Abeta fibrils appeared to lie within these profiles, but they may have been simply surrounded by the cell profile since the profile membrane was not always visible. Dark atrophic neurons, whose morphology suggested that they were apoptotic, were present around gray matter plaques. Cerebrovascular Abeta deposition was also observed in the brains of APP/PS1 transgenic mice. Thus, the amyloid deposition and neuropathology observed in APP/PS1 mouse brain are similar to those in Alzheimer's disease and they appear to develop earlier and become more severe than in the other transgenic models currently available.

Aging↗

PubMed2000

Fractionation and characterization of oligomeric, protofibrillar and fibrillar forms of beta-amyloid peptide.

The beta-amyloid (Abeta) peptide, a major component of senile plaques in Alzheimer's disease brain, has been shown previously to undergo a process of polymerization to produce neurotoxic forms of amyloid. Recent literature has attempted to define precisely the form of Abeta responsible for its neurodegenerative properties. In the present study we describe a novel density-gradient centrifugation method for the isolation and characterization of structurally distinct polymerized forms of Abeta peptide. Fractions containing protofibrils, fibrils, sheet structures and low molecular mass oligomers were prepared. The fractionated forms of Abeta were characterized structurally by transmission electron microscopy. The effects on cell viability of these fractions was determined in the B12 neuronal cell line and hippocampal neurons. Marked effects on cell viability in the cells were found to correspond to the presence of protofibrillar and fibrillar structures, but not to monomeric peptide or sheet-like structures of polymerized Abeta. Biological activity correlated with a positive reaction in an immunoassay that specifically detects protofibrillar and fibrillar Abeta; those fractions that were immunoassay negative had no effect on cell viability. These data suggest that the effect of Abeta on cell viability is not confined to a single conformational form but that both fibrillar and protofibrillar species have the potential to be active in this assay.

Amyloid beta-Peptides↗

PubMed2000

ASP1 (BACE2) cleaves the amyloid precursor protein at the beta-secretase site.

Sequential proteolytic processing of the Amyloid Precursor Protein (APP) by beta- and gamma-secretases generates the 4-kDa amyloid (A beta) peptide, a key component of the amyloid plaques seen in Alzheimer's disease (AD). We and others have recently reported the identification and characterisation of an aspartic proteinase, Asp2 (BACE), as beta-secretase. Here we describe the characterization of a second highly related aspartic proteinase, Asp1 as a second beta-secretase candidate. Asp1 is expressed in brain as detected at the mRNA level and at the protein level. Transient expression of Asp1 in APP-expressing cells results in an increase in the level of beta-secretase-derived soluble APP and the corresponding carboxy-terminal fragment. Paradoxically there is a decrease in the level of soluble A beta secreted from the cells. Asp1 colocalizes with APP in the Golgi/endoplasmic reticulum compartments of cultured cells. Asp1, when expressed as an Fc fusion protein (Asp1-Fc), has the N-terminal sequence ALEP..., indicating that it has lost the prodomain. Asp1-Fc exhibits beta-secretase activity by cleaving both wild-type and Swedish variant (KM/NL) APP peptides at the beta-secretase site.

Amyloid Precursor Protein Secretases↗

PubMed2000

In search of an enzyme: the beta-secretase of Alzheimer's disease is an aspartic proteinase.

The deposition of beta-amyloid (Abeta) in the brain is a neuropathological feature of Alzheimer's disease. Abeta is cleaved from its precursor protein (APP) by processing at its N and C termini by enzymes known as beta- and gamma-secretases,respectively. The identity of these enzymes has been elusive but the search for the N-terminal secretase might have ended recently with the almost simultaneous publication by five major laboratories claiming a transmembrane aspartic proteinase to be the long sought after beta-secretase. Even at this early stage of its characterization, this aspartic proteinase fulfils many of the key criteria necessary for beta-secretase. The race is now on to develop inhibitors that could prove effective in halting the progression of Alzheimer's disease.

Alzheimer Disease↗

PubMed1999

Common structural features determine the effectiveness of carvedilol, daunomycin and rolitetracycline as inhibitors of Alzheimer beta-amyloid fibril formation.

One of the major pathological features of Alzheimer's disease is the deposition of beta-amyloid peptide (Abeta). Cellular toxicity has been shown to be associated with fibrillar forms of Abeta; preventing this fibril formation is therefore viewed as a possible method of slowing disease progression in Alzheimer's disease. With the use of a series of tetracyclic and carbazole-type compounds as inhibitors of Abeta fibril formation, we here describe a number of common structural features that seem to be associated with the inhibitory properties of these agents. Compounds such as carvedilol, rolitetracycline and daunomycin, which are shown to inhibit Abeta fibril formation, also prevent the formation of species of peptide that demonstrate biological activity in a human neuroblastoma cell line. Molecular modelling data suggest that these compounds have in common the ability to adopt a specific three-dimensional pharmacophore conformation that might be essential for binding to Abeta and preventing it from forming fibrils. Understanding such drug-peptide interactions might aid the development of disease-modifying agents.

Alzheimer Disease↗

PubMed1999

Inhibition of fibril formation in beta-amyloid peptide by a novel series of benzofurans.

A series of benzofuran derivatives have been identified as inhibitors of fibril formation in the beta-amyloid peptide. The activity of these compounds has been assessed by a novel fibril-formation-specific immunoassay and for their effects on the production of a biologically active fibril product. The inhibition afforded by the compounds seems to be associated with their binding to beta-amyloid, as identified by scintillation proximity binding assay. Binding assays and NMR studies also indicate that the inhibition is associated with self-aggregation of the compounds. There is a close correlation between the activity of the benzofurans as inhibitors of fibril formation and their ability to bind to beta-amyloid. Non-benzofuran inhibitors of the fibril formation process do not seem to bind to the same site on the beta-amyloid molecule as the benzofurans. Thus a specific recognition site might exist for benzofurans on beta-amyloid, binding to which seems to interfere with the ability of the peptide to form fibrils.

Amyloid beta-Peptides↗

PubMed1999

Computationally derived structural models of the beta-amyloid found in Alzheimer's disease plaques and the interaction with possible aggregation inhibitors.

We report the modeling of and possible interactions within the solid beta-amyloid (ABeta) 1-43 fibril, the most fibrillogenic peptide known. All models proposed are consistent with the known experimental structural data, in terms of both secondary structure and packing motifs. The model containing antiparallel beta-sheets, and a beta-turn at G(25)S(26)N(27)K(28) has the lowest calculated packing energy. As such, it can be considered a reasonable model for solid beta-amyloid in Alzheimer's disease plaques. Interestingly, with the turn located at this position, the 1-43 structure is stabilized by a number of complementary intermolecular interactions between the beta-sheets. These well-defined interactions exist for the side-chain residues of 41, 42, and 43 with adjacent ABeta molecules. These interactions would not be conserved in the 1-40 peptide, and indeed, this enhanced interaction is proposed to give rise to the increased fibrillogenic nature of the ABeta 1-43 species over the 1-40 form. The models are used to explain the increased fibrillogenic nature of the Dutch family mutation of ABeta. These models are also employed to examine possible docking interactions of previously reported antiaggregation inhibitors, such as 4'-deoxy-4'-iododoxorubicin (IDOX) onto the theoretical growing surface. A docked structure of IDOX with the model of the solid fibril is described and a proposal for the mechanism of its antiaggregation properties is presented.

Alzheimer Disease↗

PubMed1999

Identification of a novel aspartic protease (Asp 2) as beta-secretase.

The Alzheimer's disease beta-amyloid peptide (Abeta) is produced by excision from the type 1 integral membrane glycoprotein amyloid precursor protein (APP) by the sequential actions of beta- and then gamma-secretases. Here we report that Asp 2, a novel transmembrane aspartic protease, has the key activities expected of beta-secretase. Transient expression of Asp 2 in cells expressing APP causes an increase in the secretion of the N-terminal fragment of APP and an increase in the cell-associated C-terminal beta-secretase APP fragment. Mutation of either of the putative catalytic aspartyl residues in Asp 2 abrogates the production of the fragments characteristic of cleavage at the beta-secretase site. The enzyme is present in normal and Alzheimer's disease (AD) brain and is also found in cell lines known to produce Abeta. Asp 2 localizes to the Golgi/endoplasmic reticulum in transfected cells and shows clear colocalization with APP in cells stably expressing the 751-amino-acid isoform of APP.

Alzheimer Disease↗

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↗

PubMed1995

Aggregation state and neurotoxic properties of Alzheimer beta-amyloid peptide.

The behaviour of synthetic batches of beta-amyloid (beta A) 1-40 peptide in solution has been studied. The effects of beta A1-40 on a PC12 cell toxicity assay was dependent upon the time of preincubation of an aqueous solution of the peptide before application to the cells. Fibrillization of the beta A1-40, quantitatively assessed by the binding of Congo red to amyloid fibrils, also increased in a time dependent manner over the 168 h incubation period studied. The degree of Congo red binding, in the absence of any preincubation, differed between two synthetically distinct batches of the peptide. The rate of development of fibril formation during subsequent incubation also differed between the two batches and appeared to parallel the effects on cell viability. Infra-red spectroscopic analysis revealed beta-sheet formation for both batches and other more subtle conformational differences between the peptides. Electron microscope examination of the batches of beta A1-40 confirmed the difference in occurrence and development of fibrils. At high magnification, fibrils of both batches exhibited a helical structure. The results suggest that the development of neurotoxicity of beta A1-40 is related to the fibrillar state of the peptide.

Alzheimer Disease↗

PubMed1994

beta-Cyclodextrin interacts with the Alzheimer amyloid beta-A4 peptide.

Electrospray ionisation mass spectrometry has been used to show that the synthetic 40 amino acid beta-amyloid peptide (beta 1-40) interacts with the cyclic oligosaccharide beta-cyclodextrin. This interaction, presumably with the hydrophobic aromatic moieties on the peptide, has been shown to diminish substantially the neurotoxic effects of beta 1-40 in a cell line.

Amino Acid Sequence↗

PubMed1990

Synthesis and antihypertensive activity of 4-(substituted-carbonylamino)-2H-1-benzopyrans.

The synthesis and antihypertensive activity of a series of novel 4-(substituted-carbonylamino)-2H-1-benzopyran-3-ols, administered orally to conscious spontaneously hypertensive rats, are described. Optimum activity was observed for compounds with alkyl, amino, or aryl groups flanking the carbonyl group. Of the alkyl and amino series the most potent compounds contained the methyl and methylamino groups, respectively. Several analogues have been compared with cromakalim (1) for their effects on potassium ion efflux in the rabbit mesenteric artery using rubidium-86 as a marker. The ability of each compound to enhance rubidium-86 efflux is approximately parallelled by its blood pressure lowering activity, and thus these analogues, like compound (1), belong to the series of drugs which have been classified as potassium-channel activators.

Animals↗

PubMed1989

Inhibition by glibenclamide of the vasorelaxant action of cromakalim in the rat.

1. In rat isolated thoracic aortic rings pre-contracted with noradrenaline (10(-6) M), cromakalim (3 x 10(-7)-3 x 10(-5) M) produced concentration-related relaxation. This effect was progressively inhibited by increasing concentrations of the anti-diabetic sulphonylurea drug, glibenclamide (10(-6)-10(-5) M). 2. In rat isolated portal veins, cromakalim (3 x 10(-8)-10(-6) M) produced concentration-related inhibition of the spontaneous contractive activity and glibenclamide (3 x 10(-7)-3 x 10(-6) M) prevented this inhibitory action in a concentration-dependent manner. 3. In both rat aortic rings and portal veins, cromakalim (10(-5) M) stimulated 86Rb efflux. Prior exposure to glibenclamide (10(-7)-10(-6) M) produced a concentration-related inhibition of this response. 4. In conscious rats, cromakalim, 0.075 mg kg-1 i.v., produced a rapid and sustained fall in arterial blood pressure which was not influenced by pretreatment (2 h) with a large oral dose of glibenclamide (100 mg kg-1). 5. In conscious rats, the hypotensive action of cromakalim, 0.075 mg kg-1 i.v., was abolished by pretreatment (30 min) with glibenclamide, 20 mg kg-1, given by the intravenous route. 6. The results suggest that the vasorelaxant and hypotensive actions of cromakalim involve a K+ channel which can be inhibited by glibenclamide, but which may be distinct from the ATP-sensitive K+ channel of the pancreatic beta-cell.

Animals↗

PubMed1988

Potassium efflux enhancement by cromakalim (BRL 34915) in rabbit mesenteric artery: an indirect effect independent of calcium?

Experiments have been performed in order to investigate the calcium and exposure time dependency of cromakalim (BRL 34915)-stimulated rubidium efflux in rabbit isolated mesenteric artery. Removal of calcium from the bathing medium prolonged the effects of cromakalim on rubidium efflux. Lanthanum was without effect on cromakalim-induced efflux whilst high concentrations of nifedipine were required to produce a significant inhibitory effect. Decreasing the exposure time to cromakalim, either in the presence or absence of calcium, led to a progressive loss of the response. However, significant increases in rubidium efflux rate were observed after very short exposures (15 sec) to the drug. In normal medium, exposure to cromakalim resulted in an inhibition of a second response when the drug was reapplied. Blockade by tetraethylammonium of the initial rubidium efflux response to cromakalim did not reverse the inhibition of the second response. These results suggest that the stimulation by cromakalim of rubidium efflux in rabbit isolated mesenteric artery is independent of calcium influx and requires only a short initial exposure to the drug in order to develop a response. The development and maintenance of the response after the removal of the drug suggest that cromakalim does not directly interact with the potassium channel through which rubidium efflux enhancement is observed.

Animals↗

PubMed1988

Comparative effects of K+ channel blockade on the vasorelaxant activity of cromakalim, pinacidil and nicorandil.

Three agents with K+ channel blocking activity, procaine, 4-aminopyridine (4-AP) and tetraethylammonium (TEA), were tested for inhibition of vasorelaxation and 86Rb+ efflux induced by cromakalim (BRL 34915), pinacidil and nicorandil in rabbit isolated mesenteric artery. The potency order for inhibition of vasorelaxation was procaine greater than 4-AP greater than TEA and for inhibition of efflux was procaine = 4-AP greater than TEA. The K+ channel blockers did not discriminate between cromakalim, pinacidil or nicorandil on efflux but demonstrated preferential inhibition of vasorelaxation to cromakalim greater than pinacidil greater than nicorandil. In addition, the maximum response to cromakalim was depressed but that to pinacidil and nicorandil was not. The results confirm the role of K+ channel activation in vasorelaxation to cromakalim, pinacidil and nicorandil, but suggest that additional mechanisms may be involved for pinacidil and, in particular, for nicorandil.

4-Aminopyridine↗