Molecular dissection of the neurofibrillary lesions of Alzheimer's disease.
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Alzheimer Disease↗
Biomedical subjects
R A Crowther
Publications and source records attributed to R A Crowther.
Steps towards a mouse model of Alzheimer's disease.
Alzheimer's disease, a form of senile dementia, is characterised by two kinds of pathological deposits in the brain, called plaques and tangles. The molecular nature of the deposits has been identified but there is as yet little understanding of the underlying biochemistry and cell biology that lead to their formation. Progress in this area would be greatly aided by a realistic animal model. Two recent papers describe the production of transgenic mice that develop significant aspects of the Alzheimer-like pathology. The mice produced by Games et al.(1) develop plaques while those produced by Götz et al.(2) display the early stages of tangle pathology.
Alzheimer Disease↗
Tau protein in Alzheimer's disease.
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Alzheimer Disease↗
Hepatitis B virus core particles as epitope carriers.
HBV core (HBc) particle is one of the most intensively studied particulate carriers for the insertion of foreign peptide sequences. Recombinant HBc protein expressed from the cloned gene undergoes the correct folding in a large variety of bacterial, yeast, insect and mammalian cells. Unique assembly properties and shape of 30/34-nm HBc particles allow substantial insertions into their primary structure without loss of their capsid-forming ability. N- and C-terminal regions, as well as the immunodominant loop in the middle of the molecule are widely accepted as targets for the introduction of foreign epitopes, ensuring retention and even enhancement of the original immunological activity of inserted sequences. Special sets of display vectors have been constructed on the basis of the cloned HBc gene. Epitope sequences of viral (BLV, FeLV, FMDV, HBV, HCV, HIV-1, HRV2, MCMV, PV-1, SIV) and nonviral (human chorionic gonadotropin) origin have been studied as model display moieties.
B-Lymphocytes↗
Epitope mapping of monoclonal antibodies to the paired helical filaments of Alzheimer's disease: identification of phosphorylation sites in tau protein.
Tau is a neuronal phosphoprotein the expression of which is developmentally regulated. A single tau isoform is expressed in fetal human brain but six isoforms are expressed in adult human brain, with the fetal isoform corresponding to the shortest adult isoform. Phosphorylation is also developmentally regulated, as fetal tau is phosphorylated at more sites than adult tau. In Alzheimer's disease, the six adult tau isoforms become hyperphosphorylated and form the paired helical filament (PHF), the major fibrous component of the neurofibrillary lesions. One way to identify phosphorylated sites in tau is to use antibodies that recognize phosphorylated residues within a specific amino acid sequence. We here characterize the two novel phosphorylation-dependent anti-tau antibodies AT270 and AT180 and identify their epitopes as containing phosphorylated Thr-181 and Thr-231 respectively. With these antibodies we show that these two threonine residues are partially phosphorylated in fetal and adult tau and almost fully phosphorylated in PHF tau. This result contrasts with previous studies of Ser-202 and Ser-396 which are partially phosphorylated in fetal tau, unphosphorylated in adult tau but almost fully phosphorylated in PHF tau.
Alzheimer Disease↗
Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy.
Human hepatitis B virus core protein expressed in E. coli assembles into two sizes of particle. We have determined their three-dimensional structures by electron cryomicroscopy and image processing. The large and small particles correspond to triangulation number T = 4 and T = 3 dimer clustered packings, containing 240 and 180 protein subunits, respectively. The local packing of subunits is very similar in the two sizes of particle and shows holes or channels through the shell. The native viral core particle packages RNA and is active in reverse transcription to DNA. The holes we observe may provide access for the necessary small molecules. Shells assembled from the intact core protein contain additional material, probably RNA, which appears as an icosahedrally ordered inner shell in the three-dimensional map.
Cryopreservation↗
Assembly of Alzheimer-like filaments from full-length tau protein.
The principal fibrous component of neurofibrillary pathology in Alzheimer's disease, the paired helical filament, is formed from hyperphosphorylated microtubule-associated protein tau. Here we show that recombinant tau protein either in a non-phosphorylated state or following phosphorylation with brain extract can be assembled in vitro into filaments resembling those seen in Alzheimer's disease.
Alzheimer Disease↗
Crossbridges in the complete unit cell of rigor insect flight muscle imaged by three-dimensional reconstruction from oblique sections.
We have computed two types of 3-D reconstructions from single images of oblique transverse sections through rigor insect flight muscle (IFM) that permit simultaneous examination of all myosin crossbridges within the unit cell. One type, crystallographic serial section reconstruction (CSSR), utilizes primarily real space image manipulations of the periodic crossbridge lattice to obtain a 3-D reconstruction from a single image. The CSSRs, which do not average successive unit cells along the filament axis, reveal variations in the rigor double chevrons within the 116 nm long axial repeat and in particular show that specific crossbridges are absent. CSSRs establish that in rigor, the 116 nm period contains nine 12.9 nm repeats of attached crossbridges rather than the eight 14.5 nm repeats of myosin head origins observed in the relaxed state. This indicates that dominance of the actin repeat on myosin head form enforces axial and azimuthal changes on the crossbridge origins on the thick filament. The second type, superlattice reconstruction (SLR), is carried out entirely in Fourier space and produces an averaged reconstruction with the symmetry of the unit cell enforced. SLRs measure the 3-D transform of the complete unit cell, permitting direct comparison with X-ray diagrams from native muscle. Averaging several SLRs together has produced the highest resolution reconstruction of IFM to date. Oblique section reconstructions made by both methods confirm in greater detail the presence of two-headed lead crossbridges and single-headed rear crossbridges implying heads with differing angles and conformation. Reduced twist in the thin filament coincident with the lead crossbridge is also apparent. We have modeled several interpretations of this reduced twist in terms of structural changes in both myosin and actin at the lead bridge. In addition, these 3-D images resolve a feature just Z-ward of the rear crossbridge where antibody labeling has identified part of the large troponin complex of IFM.
Actins↗
The abnormal phosphorylation of tau protein at Ser-202 in Alzheimer disease recapitulates phosphorylation during development.
Tau is a neuronal phosphoprotein whose expression is developmentally regulated. A single tau isoform is expressed in fetal human brain but six isoforms are expressed in adult brain, with the fetal isoform corresponding to the shortest of the adult isoforms. Phosphorylation of tau is also developmentally regulated, as fetal tau is phosphorylated at more sites than adult tau. In Alzheimer disease, the six adult tau isoforms become abnormally phosphorylated and form the paired helical filament, the major fibrous component of the characteristic neurofibrillary lesions. We show here that Ser-202 (in the numbering of the longest human brain tau isoform) is a phosphorylation site that distinguishes fetal from adult tau and we identify it as one of the abnormal phosphorylation sites in Alzheimer disease. The abnormal phosphorylation of tau at Ser-202 in Alzheimer disease thus recapitulates normal phosphorylation during development.
Adult↗
The microtubule binding repeats of tau protein assemble into filaments like those found in Alzheimer's disease.
The paired helical filament, which comprises the major fibrous element of the neurofibrillary tangle in Alzheimer's disease, contains abnormally phosphorylated microtubule-associated protein tau as its principal constituent. The repeat region of tau protein, which represents the microtubule binding domain, forms the core of the filament. Here we show that an expressed fragment of tau protein spanning the repeat region can assemble in vitro into filaments like those found in Alzheimer's disease.
Alzheimer Disease↗
Cloning of a big tau microtubule-associated protein characteristic of the peripheral nervous system.
Microtubule-associated protein tau consists in brain of a series of isoforms of 48- to 67-kDa apparent molecular mass that are encoded by mRNAs of approximately 6 kilobases (kb) and that are generated from a single gene by alternative splicing. Previously, a tau-like protein of 110-kDa apparent molecular mass was described in peripheral ganglia and in peripheral neuronlike cell lines. We now report the cloning and sequencing of a rat cDNA encoding this big tau. The corresponding protein contains sequence identical to the longest of the previously cloned small tau isoforms but with an additional 254 amino acid insert in the amino-terminal half. Big tau is produced from an 8-kb mRNA generated by alternative splicing from the same gene that encodes small tau. Production of big tau from the cloned sequence gives a protein of 110-kDa apparent molecular mass that aligns on SDS/PAGE with big tau protein extracted from peripheral ganglia. RNA blots show that in peripheral ganglia from adult rats only the 8-kb mRNA band corresponding to big tau is found, whereas in ganglia from newborn rats both 6- and 8-kb tau mRNA bands are found. In tissues from the central nervous system only the 6-kb mRNA band can be detected. Big tau protein is therefore produced specifically in the peripheral nervous system, and it will be interesting to see whether further molecular differences between the two major divisions of the vertebrate nervous system will be discovered.
Amino Acid Sequence↗
Tau proteins of Alzheimer paired helical filaments: abnormal phosphorylation of all six brain isoforms.
Preparations of dispersed paired helical filaments (PHFs) from the brains of Alzheimer's disease and Down's syndrome patients display on gels three principal bands corresponding to abnormally modified forms of the microtubule-associated protein tau. Interpretation of the pattern is difficult because there are six tau isoforms in normal brain and phosphorylation changes their mobility. By enzymatic dephosphorylation at high temperature, we have shifted the three abnormal bands obtained from dispersed PHFs to align with the six nonphosphorylated tau isoforms. By using antibodies specific for some of the inserts that distinguish the various isoforms and label PHFs, we have established a correspondence between PHFs, abnormal bands, and isoforms. This identification of isoforms is a necessary step in unravelling the molecular pathogenesis of PHFs.
Aged↗
Straight and paired helical filaments in Alzheimer disease have a common structural unit.
The presence of abundant neurofibrillary tangles in certain areas of the brain constitutes one of the defining pathological characteristics of Alzheimer disease. The predominant component of the tangle is an abnormal fibrous assembly known as the paired helical filament (PHF). The PHF is formed by a twisted double-helical ribbon of subunits that gives rise to an image alternating in width between 8 nm and 20 nm with a cross-over spacing of 80 nm. Also found in tangles is the straight filament (SF), a different kind of abnormal filament, about 15 nm wide, that does not exhibit the marked modulation in width shown by the PHF. It is reported herein that PHFs and SFs form hybrid filaments displaying both morphologies, that PHFs and SFs share surface epitopes, and that computed maps reveal a similar C-shaped morphological unit in PHFs and SFs, though differing in relative arrangement in the two types of filament. The observations imply that the SF is a structural variant of the PHF and establish a common unit of assembly for these two pathological filaments.
Alzheimer Disease↗
Molecular characterization of microtubule-associated proteins tau and MAP2.
Tau and MAP2 are two of the major microtubule-associated proteins in the vertebrate nervous system. They promote microtubule assembly and stability, and might be involved in the establishment and maintenance of neuronal polarity. In nerve cells immunohistochemistry shows complementary distributions, with tau being concentrated in axons and high molecular mass MAP2 being confined to dendrites. Each protein consists of multiple isoforms that contain three or four homologous tandem repeats near the carboxy-terminus, which constitute microtubule-binding domains. In humans, tau consists of at least six isoforms of related amino acid sequences that are produced from a single gene by alternative mRNA splicing and that are expressed in a stage- and cell type-specific manner. Tau is also a component of the paired helical filaments associated with Alzheimer's disease and other disorders of the CNS. Rat MAP2 consists of at least three isoforms produced from a single gene: high molecular mass MAP2a and MAP2b, and low molecular mass MAP2c. MAP2c is expressed only during early development and has so far been seen only in axons; MAP2a appears to replace MAP2c, whereas MAP2b is expressed throughout life. Messenger RNAs for MAP2 of high molecular mass are expressed both in cell bodies and in dendrites, consistent with the dendritic localization of the corresponding protein isoforms.
Amino Acid Sequence↗
A protocol for 3D image reconstruction from a single image of an oblique section.
Oblique section 3D reconstruction can produce a 3D image of a sectioned crystal from a single electron micrograph. We describe here in detail a reconstruction protocol applicable to an electron micrograph of an oblique section through a 3D crystal. The protocol is described in six steps: (1) selection criteria for images, (2) preprocessing steps to correct for image defects, (3) determination of unit cell coordinates, (4) interpolation of strip images with correction for image distortions and crystal disorder, (5) production of a crystallographic serial section reconstruction, (6) correction for skewed sampling to produce an oblique section reconstruction. In addition, we explore Wiener filter deconvolution of the section thickness. We describe a method for determining the section thickness by comparing data from projections of the oblique section reconstruction with corresponding data from a thick longitudinal section. Several schemes for Wiener filter deconvolution are described that differ in the way information on the signal-to-noise ratio is used in the filter.
Animals↗
Tau proteins and neurofibrillary degeneration.
The paired helical filament is the major fibrous component of neurofibrillary pathology in Alzheimer's disease. Over the last three years evidence has accumulated that the microtubule-associated protein tau forms an important, if not the sole, constituent of the paired helical filament. Tau protein in normal brain is bound to axonal microtubules by a tandem repeat region. In Alzheimer's disease a proportion of tau protein becomes abnormally phosphorylated and is no longer associated with axonal microtubules but instead accumulates in paired helical filaments throughout affected nerve cells. The tandem repeat region contributes substantially to the structural core of the paired helical filament, around which the amino-terminal half of the molecule forms a disordered coat.
Alzheimer Disease↗
Structural aspects of pathology in Alzheimer's disease.
The characteristic lesions of Alzheimer's disease, neurofibrillary tangles and neuritic plaques, are the sites of accumulation of abnormal fibrillar material. The structure of the paired helical filament from tangles has been analysed by electron microscopy and biochemical studies have shown that it contains microtubule associated protein tau as a component. Fibrils of beta-amyloid in the neuritic plaque arise by polymerization of a small proteolytic fragment of a much larger precursor protein. It is not yet clear what triggers the events that lead to assembly of the abnormal structures nor why the structures once formed are so resistant to turnover.
Actin Cytoskeleton↗
Computation of a three dimensional image of a periodic specimen from a single view of an oblique section.
We describe here a method for computing a three dimensional map of a periodic specimen from a single electron micrograph of an obliquely cut section. Neighbouring areas of such an image display successively the contents of the unit cell of the structure. The reconstruction procedure can be considered in two steps. The first step involves restacking of successive areas to produce an image akin to that produced by serial section reconstruction. The resolution normal to the section would, at this stage, be limited by the thickness of the section, since the micrograph represents a projection of the density in the section. However, because of the periodic nature of the specimen, the image contains redundant information, which can be used in an attempt to deconvolute the section thickness and thus produce improved resolution normal to the section. The computation can be carried out directly with the densities or more conveniently, particularly for three dimensional crystals, by using Fourier transforms. The approach, which is most powerful when the section is thin, is insensitive to the collapse of the section caused by electron irradiation. Striated muscle provides particularly suitable specimens for such analysis and we present, as examples, computed maps of the M-band of fish muscle and of insect flight muscle in rigor.
Animals↗