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Biomedical subjects

R A Crowther

Publications and source records attributed to R A Crowther.

At least 19 recordsLinked to original sources
PubMed1999

Effects of frontotemporal dementia FTDP-17 mutations on heparin-induced assembly of tau filaments.

Missense mutations and intronic mutations in the gene for microtubule-associated protein tau cause frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Most missense mutations have as likely primary effect a reduced ability of tau to interact with microtubules. We report here an additional effect of several missense mutations, namely the stimulation of heparin-induced filament assembly of recombinant tau, despite the absence of any change in structure indicated by circular dichroism. These findings indicate that missense mutations in tau lead to frontotemporal dementia through potentially multiple mechanisms.

Actin Cytoskeleton↗
PubMed1999

Tau gene mutation in familial progressive subcortical gliosis.

Familial forms of frontotemporal dementias are associated with mutations in the tau gene. A kindred affected by progressive subcortical gliosis (PSG), a rare form of presenile dementia, has genetic linkage to chromosome 17q21-22. This kindred (PSG-1) is included in the 'frontotemporal dementias and Parkinsonism linked to chromosome 17' group along with kindreds affected by apparently different forms of atypical dementias. Some of these kindreds have mutations in the tau gene. We report here that PSG-1 has a tau mutation at position +16 of the intron after exon 10. The mutation destabilizes a predicted stem-loop structure and leads to an over-representation of the soluble four-repeat tau isoforms, which assemble into wide, twisted, ribbon-like filaments and ultimately result in abundant neuronal and glial tau pathology. The mutations associated with PSG and other atypical dementias can be subdivided into three groups according to their tau gene locations and effects on tau. The existence of tau mutations with distinct pathogenetic mechanisms may explain the phenotypic heterogeneity of atypical dementias that previously led to their classification into separate disease entities.

Cerebral Cortex↗
PubMed1999

Tau pathology in a family with dementia and a P301L mutation in tau.

Familial forms of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) have recently been associated with coding region and intronic mutations in the tau gene. Here we report our findings on 2 affected siblings from a family with early-onset dementia, characterized by extensive tau pathology and a Pro to Leu mutation at codon 301 of tau. The proband, a 55-year-old woman, and her 63-year-old brother died after a progressive dementing illness clinically diagnosed as Alzheimer disease. Their mother, 2 sisters, maternal aunt and uncle, and several cousins were also affected. Autopsy in both cases revealed frontotemporal atrophy and degeneration of basal ganglia and substantia nigra. Sequencing of exon 10 of the tau gene revealed a C to T transition at codon 301, resulting in a Pro to Leu substitution. Widespread neuronal and glial inclusions, neuropil threads, and astrocytic plaques similar to those seen in corticobasal degeneration were labeled with a battery of antibodies to phosphorylation-dependent and phosphorylation-independent epitopes spanning the entire tau sequence. Isolated tau filaments had the morphology of narrow twisted ribbons. Sarkosyl-insoluble tau exhibited 2 major bands of 64 and 68 kDa and a minor 72 kDa band, similar to the pattern seen in a familial tauopathy associated with an intronic tau mutation. These pathological tau bands predominantly contained the subset of tau isoforms with 4 microtubule-binding repeats selectively affected by the P301L missense mutation. Our findings emphasize the phenotypic and genetic heterogeneity of tauopathies and highlight intriguing links between FTDP-17 and other neurodegenerative diseases.

Atrophy↗
PubMed1999

Crystallization of hepatitis B virus core protein shells: determination of cryoprotectant conditions and preliminary X-ray characterization.

Hepatitis B virus causes liver cirrhosis and hepatocellular cancer and is a major cause of death, particularly in Asia and sub-Saharan Africa. The virus consists of an inner core or nucleocapsid, which encloses the viral nucleic acid, with an outer lipid envelope containing surface-antigen proteins. The core protein, when expressed in E. coli, assembles into spherical shells containing 180 or 240 subunits, arranged with T = 3 or T = 4 icosahedral symmetry. The C-terminal region of the protein is involved in nucleic acid binding, and deletion of this region does not prevent capsid formation. C-terminally deleted hepatitis B core shells containing 240 subunits have been crystallized and data has been collected to 3. 6 A resolution from frozen crystals, using butanediol as a cryoprotectant. The crystals have C2 symmetry, with unit-cell parameters a = 538.0, b = 353.0, c = 369.6 A, beta = 132.3 degrees.

Cold Temperature↗
PubMed1998

Peptides that block hepatitis B virus assembly: analysis by cryomicroscopy, mutagenesis and transfection.

Peptides selected to bind to hepatitis B virus (HBV) core protein block interaction with the long viral surface antigen (L-HBsAg) in vitro. High resolution electron cryomicroscopy showed that one such peptide binds at the tips of the spikes of the core protein shell. The peptides contain two basic residues; changing either of two acidic residues at the spike tip to an alanine greatly reduced the binding affinity. Transfection of hepatoma cells with a replication-competent HBV plasmid gave significantly reduced production of virus in the presence of peptide, in a dose-dependent manner. These experiments show that the interaction of L-HBsAg with core particles is critical for HBV assembly, and give proof of principle for its disruption in vivo by small molecules.

Antiviral Agents↗
PubMed1998

Synthetic filaments assembled from C-terminally truncated alpha-synuclein.

Recently two point mutations in the alpha-synuclein gene have been found in familial Parkinson's disease. The characteristic fibrous neuropathological lesions of Parkinson's and other neurodegenerative diseases have been shown to stain strongly with antibodies against alpha-synuclein and extracted filaments have been labelled with anti-alpha-synuclein antibodies. In view of the close involvement of alpha-synuclein filaments with pathology, it was important to establish an in vitro assembly system. We report here that C-terminally truncated recombinant alpha-synuclein readily assembles into filaments resembling those isolated from diseased brain and suggest that truncation by proteolysis may play a role in the pathological process.

Alanine↗
PubMed1998

Filamentous alpha-synuclein inclusions link multiple system atrophy with Parkinson's disease and dementia with Lewy bodies.

Alpha-synuclein forms the major component of Lewy bodies and Lewy neurites, the defining neuropathological characteristics of Parkinson's disease and dementia with Lewy bodies. Here we show that alpha-synuclein is also the major component of the filamentous inclusions of multiple system atrophy which comprises several neurodegenerative diseases with a shared filamentous pathology in nerve cells and glial cells. These findings provide an unexpected link between multiple system atrophy and Lewy body disorders and establish that alpha-synucleinopathies constitute a major class of human neurodegenerative disorder.

Adult↗
PubMed1998

alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with lewy bodies.

Lewy bodies and Lewy neurites are the defining neuropathological characteristics of Parkinson's disease and dementia with Lewy bodies. They are made of abnormal filamentous assemblies of unknown composition. We show here that Lewy bodies and Lewy neurites from Parkinson's disease and dementia with Lewy bodies are stained strongly by antibodies directed against amino-terminal and carboxyl-terminal sequences of alpha-synuclein, showing the presence of full-length or close to full-length alpha-synuclein. The number of alpha-synuclein-stained structures exceeded that immunoreactive for ubiquitin, which is currently the most sensitive marker of Lewy bodies and Lewy neurites. Staining for alpha-synuclein thus will replace staining for ubiquitin as the preferred method for detecting Lewy bodies and Lewy neurites. We have isolated Lewy body filaments by a method used for the extraction of paired helical filaments from Alzheimer's disease brain. By immunoelectron microscopy, extracted filaments were labeled strongly by anti-alpha-synuclein antibodies. The morphologies of the 5- to 10-nm filaments and their staining characteristics suggest that extended alpha-synuclein molecules run parallel to the filament axis and that the filaments are polar structures. These findings indicate that alpha-synuclein forms the major filamentous component of Lewy bodies and Lewy neurites.

Autopsy↗
PubMed1998

Tau pathology in two Dutch families with mutations in the microtubule-binding region of tau.

Different mutations in the microtubule-associated tau protein gene have recently been identified in several families with hereditary frontotemporal dementia and Parkinsonism (FTDP-17) linked to chromosome 17q21-22. Some families show neuronal and glial deposits containing hyperphosphorylated tau in several brain regions. We have investigated the presence of tau deposits by using a panel of anti-tau antibodies in three brains of a family with the P301L mutation (HFTD1) and in another family with the G272V mutation (HFTD2) of the tau gene. Numerous intracytoplasmic tau deposits in neurons, glial cells, and neurites were found in hippocampal formation, neocortex, and substantia nigra. These deposits in three patients from HFTD1 consisted of slender twisted filaments 15 nm wide with variable periodicity and a few straight filaments. Tau extracted from these filaments appeared as two major bands of 64 and 68 kd and a minor band of 72 kd that, after alkaline phosphatase treatment, proved to consist mainly of 4-repeat tau isoforms and one of the 3-repeat isoforms. In three patients from HFTD2 numerous Pick-like bodies were present. The conclusion is that the type and distribution of tau deposits in HFTD1 and HFTD2, the physical structure of filaments, and tau isoform composition in HFTD1 differ from Alzheimer's disease and an FTDP-17 family with a V337M mutation in the tau gene.

Aged↗
PubMed1997

Alzheimer-like changes in microtubule-associated protein Tau induced by sulfated glycosaminoglycans. Inhibition of microtubule binding, stimulation of phosphorylation, and filament assembly depend on the degree of sulfation.

Hyperphosphorylated microtubule-associated protein tau is the major proteinaceous component of the paired helical and straight filaments which constitute a defining neuropathological characteristic of Alzheimer's disease and a number of other neurodegenerative disorders. We have recently shown that full-length recombinant tau assembles into Alzheimer-like filaments upon incubation with heparin. Heparin also promotes phosphorylation of tau by a number of protein kinases, prevents tau from binding to taxol-stabilized microtubules, and produces rapid disassembly of microtubules assembled from tau and tubulin. Here, we have used the above parameters to study the interactions between tau protein and a number of naturally occurring and synthetic glycosaminoglycans. We show that the magnitude of the glycosaminoglycan effects is proportional to their degree of sulfation. Thus, the strongly sulfated glycosaminoglycans dextran sulfate, pentosan polysulfate, and heparin were the most potent, whereas the non-sulfated dextran and hyaluronic acid were without effect. The moderately sulfated glycosaminoglycans heparan sulfate, chondroitin sulfate, and dermatan sulfate had intermediate effects, whereas keratan sulfate had little or no effect. These in vitro interactions between tau protein and sulfated glycosaminoglycans reproduced the known characteristics of paired helical filament-tau from Alzheimer's disease brain. Sulfated glycosaminoglycans are present in nerve cells in Alzheimer's disease brain in the early stages of neurofibrillary degeneration, suggesting that their interactions with tau may constitute a central event in the development of the neuronal pathology of Alzheimer's disease.

Alzheimer Disease↗
PubMed1997

Familial multiple system tauopathy with presenile dementia: a disease with abundant neuronal and glial tau filaments.

Neurofibrillary lesions made of hyperphosphorylated microtubule-associated protein tau constitute not only one of the defining neuropathological features of Alzheimer disease but also are present in a number of other neurodegenerative diseases with dementia. Here we describe a novel autosomal dominant disease named familial "multiple system tauopathy with presenile dementia," which is characterized by abundant fibrillary deposits of tau protein in both neurons and glial cells. There are no detectable deposits of beta-amyloid. The tau deposits are in the form of twisted filaments that differ in diameter and periodicity from the paired helical filaments of Alzheimer disease. They are stained by both phosphorylation-independent and -dependent anti-tau antibodies. Moreover, tau immunoreactivity coexists with heparan sulfate in affected nerve and glial cells. Tau protein extracted from filaments of familial multiple system tauopathy with presenile dementia shows a minor 72-kDa band and two major bands of 64 and 68 kDa that contain mainly hyperphosphorylated four-repeat tau isoforms of 383 and 412 amino acids.

Aged↗
PubMed1997

Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy.

Hepatitis B virus, a major human pathogen with an estimated 300 million carriers worldwide, can lead to cirrhosis and liver cancer in cases of chronic infection. The virus consists of an inner nucleocapsid or core, surrounded by a lipid envelope containing virally encoded surface proteins. The core protein, when expressed in bacteria, assembles into core shell particles, closely resembling the native core of the virus. Here we use electron cryomicroscopy to solve the structure of the core protein to 7.4 A resolution. Images of about 6,400 individual particles from 34 micrographs at different levels of defocus were combined, imposing icosahedral symmetry. The three-dimensional map reveals the complete fold of the polypeptide chain, which is quite unlike previously solved viral capsid proteins and is largely alpha-helical. The dimer clustering of subunits produces spikes on the surface of the shell, which consist of radial bundles of four long alpha-helices. Our model implies that the sequence corresponding to the immunodominant region of the core protein lies at the tip of the spike and also explains other properties of the core protein.

Capsid↗
PubMed1997

Three-dimensional structure of infectious bursal disease virus determined by electron cryomicroscopy.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae group, is a commercially important pathogen of chickens. From electron micrographs of frozen, hydrated, unstained specimens, we have computed a three-dimensional map of IBDV at about 2 nm resolution. The map shows that the structure of the virus is based on a T=13 lattice and that the subunits are predominantly trimer clustered. The subunits close to the fivefold symmetry axes are at a larger radius than those close to the two- or threefold axes, giving the capsid a markedly nonspherical shape. The trimer units on the outer surface protrude from a continuous shell of density. On the inner surface, the trimers appear as Y-shaped units, but the set of units surrounding the fivefold axes appears to be missing. It is likely that the outer trimers correspond to the protein VP2, carrying the dominant neutralizing epitope, and the inner trimers correspond to protein VP3, which has a basic carboxy-terminal tail expected to interact with the packaged RNA.

Animals↗
PubMed1996

Assembly of microtubule-associated protein tau into Alzheimer-like filaments induced by sulphated glycosaminoglycans.

The paired helical filament (PHF) is the major component of the neurofibrillary deposits that form a defining neuropathological characteristic of Alzheimer's disease. PHFs are composed of microtubule-associated protein tau, in a hyperphosphorylated state. Hyperphosphorylation of tau results in its inability to bind to microtubules and is believed to precede PHF assembly. However, it is unclear whether hyperphosphorylation of tau is either necessary or sufficient for PHF formation. Here we show that non-phosphorylated recombinant tau isoforms with three microtubule-binding repeats form paired helical-like filaments under physiological conditions in vitro, when incubated with sulphated glycosaminoglycans such as heparin or heparan sulphate. Furthermore, heparin prevents tau from binding to microtubules and promotes microtubule disassembly. Finally, we show that heparan sulphate and hyperphosphorylated tau coexist in nerve cells of the Alzheimer's disease brain at the earliest known stages of neurofibrillary pathology. These findings, with previous studies which show that heparin stimulates tau phosphorylation by a number of protein kinases, indicate that sulphated glycosaminoglycans may be a key factor in the formation of the neurofibrillary lesions of Alzheimer's disease.

Alzheimer Disease↗
PubMed1996

Difference imaging reveals ordered regions of RNA in turnip yellow mosaic virus.

BACKGROUND: Turnip yellow mosaic virus (TYMV) is a small icosahedral plant virus with a capsid containing 180 subunits arranged with hexamer-pentamer clustering. Cross-linking studies have indicated extensive contacts between RNA and coat protein, suggesting that substantial parts of the RNA might be icosahedrally ordered. RESULTS: Comparison of maps computed to a Fourier cut-off of 1.5 nm from electron micrographs of ice-embedded specimens of TYMV and of empty capsids produced by freeze-thawing reveals strong inner features around the threefold axes in the virus but not in the empty capsid. Internal features of subunit packing indicate that interhexamer contacts are closer than those between pentamers and hexamers and that pentamer density in the empty capsid is reduced relative to that in the virus. CONCLUSIONS: The differences between virus and empty capsid indicate that substantial parts of the RNA are icosahedrally ordered and that the exit of RNA on freeze-thawing is accompanied by the loss of at least one pentamer unit.

Binding Sites↗
PubMed1996

Characterization of mAb AP422, a novel phosphorylation-dependent monoclonal antibody against tau protein.

A monoclonal antibody (AP422) specific for phosphoserine 422 in microtubule-associated protein tau has been produced. It strongly labels paired helical filament (PHF) tau from Alzheimer's disease brain in a phosphorylation-dependent manner. By contrast, AP422 only labels a small fraction of fetal tau and a very small fraction of tau from adult brain. The amount of tau phosphorylated at Ser-422 in normal brain is minor relative to that phosphorylated at sites recognized by other phosphorylation-dependent anti-tau antibodies of known epitope. It follows that AP422 is the most specific anti-tau antibody available for detecting the neurofibrillary lesions of Alzheimer's disease. We also show that Ser-422 in tau is a good in vitro substrate for mitogen-activated protein kinase, but not for glycogen synthase kinase-3 or neuronal cdc2-like kinase.

Alzheimer Disease↗