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C M Wischik

Publications and source records attributed to C M Wischik.

At least 37 records · Page 2Linked to original sources
PubMed1993

Molecular characterization of the minimal protease resistant tau unit of the Alzheimer's disease paired helical filament.

The Alzheimer's disease paired helical filament (PHF), after digestion with Pronase, retains its characteristic morphological features. We term this the protease resistant core PHF. A 12 kDa tau fragment can be released from the core as an essentially pure preparation. Sequence analysis of this fragment revealed six distinct N-termini beginning in the repeat region of tau. The precise C-terminus is unknown, but the fragment is approximately 100 residues long. A monoclonal antibody, mAb 423, which recognizes the core PHF and the 12 kDa tau fragment, does not recognize normal full-length tau. We describe cDNA synthesis and expression of candidate 12 kDa tau analogues which permit the mapping of the mAb 423 epitope. mAb 423 recognizes all and only those analogues which terminate at Glu391, which lies beyond the homology repeat region. Addition or removal of a single residue at the C-terminus abolishes immunoreactivity. Therefore, mAb 423, together with knowledge of the N-terminus, can be used to measure the precise extent of 12 kDa PHF core tau fragment which we term the minimal protease resistant tau unit of the core PHF. This unit is 93-95 residues long, which is equivalent to three repeats, but is 14-16 residues out of phase with respect to the maximum homology organization of the repeat region. mAb 423 labels isolated PHFs prior to Pronase digestion and intracellular granular and neurofibrillary degeneration in Alzheimer's disease tissues. The constraints which determine endogenous truncation at Glu391 appear to be characteristic of an assembled configuration of tau, either within the PHF or its precursor.

Alzheimer Disease↗
PubMed1993

Biochemical and anatomical redistribution of tau protein in Alzheimer's disease.

We have developed assays that distinguish tau protein incorporated into the core structure of the paired helical filament (PHF) from non-PHF tau protein in brain tissue, whether soluble or insoluble. The PHF content was 19-fold greater in Alzheimer's disease cases compared with cognitively intact controls, and in temporal cortex the difference was 40-fold. There was a threefold decrease in soluble tau protein in Alzheimer's disease cases compared with normal age-matched controls, the decrease being greatest in frontal cortex. The PHF content was closely correlated with the number of tau-immunoreactive dystrophic neurites in plaques and throughout the neuropil, whereas counts of neurofibrillary tangles were poorer predictors of PHF content. beta-Amyloid deposits correlated neither with PHF content nor with neurofibrillary pathology. These findings suggest that Alzheimer's disease is characterized by a substantial redistribution of available tau protein from free to PHF-incorporated fractions and that PHF accumulation may be important in neurites as well as tangles in predicting the extent of cognitive impairment in Alzheimer's disease.

Adult↗
PubMed1992

Differentiation of neuroblastoma cells correlates with an altered splicing pattern of tau RNA.

Morphological differentiation of N2A neuroblastoma cells is associated with an altered splicing of the gene of the microtubule-associated protein, tau. Two populations of RNA (coding for tau proteins containing three or four tubulin-binding motifs) are present in a similar proportion in undifferentiated neuroblastoma cells while in differentiated cells the proportion is changed in favour of that population coding for tau protein containing four tubulin-binding motifs. An increase in a high molecular weight tau isoforms correlates with the increase in the RNA population coding for four tubulin-binding motifs. A possible consequence of expressing a higher proportion of the tau protein containing four tubulin-binding motifs could be an increase in microtubule stability of differentiated neuroblastoma cells.

Animals↗
PubMed1992

Molecular characterization and measurement of Alzheimer's disease pathology: implications for genetic and environmental aetiology.

The neuropathological changes seen in Alzheimer's disease represent an interaction between the ageing process in which normal intellectual function is retained, and changes which are specifically associated with severe cognitive deterioration. Molecular analysis of these changes has tended to emphasize the distinction between neurofibrillary pathology, which is intracellular and highly correlated with cognitive deterioration, and the changes associated with the deposition of extracellular amyloid, which appears to be widespread in normal ageing. Extracellular amyloid deposits consist of fibrils composed of a short 42 amino acid peptide (beta/A4) derived by abnormal proteolysis from a much larger precursor molecule (APP). The recent demonstration of a mutation associated with APP in rare cases with familial dementia, neurofibrillary pathology in the hippocampus and atypical cortical Lewy body pathology raises the possibility that abnormal processing of APP could be linked directly with neurofibrillary pathology. Neurofibrillary tangles and neuritic plaques are sites of dense accumulation of pathological paired helical filaments (PHFs) which are composed in part of an antigenically modified form of the microtubule-associated protein tau. The average brain tissue content of PHFs measured biochemically does not increase in the course of normal ageing but increases 10-fold relative to age-matched controls in patients with Alzheimer's disease. There is also a substantial (three-fold) disease-related decline in normal soluble tau protein relative to age-matched controls. This intracellular redistribution of a protein essential for microtubule stability in cortico-cortical association circuits may play an important part in the molecular pathogenesis of dementia in Alzheimer's disease. The role of abnormal proteolysis of APP in this process remains to be elucidated. Immunohistochemical studies on renal dialysis cases have failed to detect evidence of neurofibrillary pathology related to aluminium accumulation in brain tissue. Nevertheless it needs to be seen whether more sensitive biochemical assays of neurofibrillary pathology can demonstrate evidence of an association with aluminium.

Alzheimer Disease↗
PubMed1991

Angiotensin converting enzyme density is increased in temporal cortex from patients with Alzheimer's disease.

The present study assesses the binding density of the selective angiotensin converting enzyme (ACE) radioligand [3H]ceranapril in brain tissue homogenates derived from patients with Alzheimer's disease and those from age-, sex- and post-mortem delay-matched neurologically normal patients. Saturation studies with [3H]ceranapril identified that the specific binding (defined by captopril, 10 microM) was homogenous and of high affinity. ACE inhibitor recognition site density was higher by some 70% in the temporal cortex (Brodmann area 22) from Alzheimer's patients whereas densities were similar in frontal cortex and cerebellum when compared to control tissue. It is unknown whether this apparently selective alteration in ACE density is directly related to, or a compensatory effect of the disease, but it provides additional support for the development of compounds which interact with the central angiotensin system as novel therapies for cognitive dysfunction.

Aged↗
PubMed1991

Difference between the tau protein of Alzheimer paired helical filament core and normal tau revealed by epitope analysis of monoclonal antibodies 423 and 7.51.

The microtubule-associated protein tau that is incorporated into paired helical filaments (PHFs) undergoes some form of aberrant posttranslational processing in Alzheimer disease. Difficulties in deciding which changes are critical for PHF formation stem in part from the lack of immunochemical markers specific for PHF tau. The only monoclonal antibody (mAb) that is known to react with PHF tau but not with the predominant normal adult tau species is mAb 423. Another mAb (7.51, described in this paper) recognizes a segment of tau that is included in the minimal recognition unit required by mAb 423. Unlike 423, which is PHF tau-specific, mAb 7.51 recognizes all PHF core-derived tau as well as native soluble tau and recombinant tau expressed in bacteria and so serves as a generic tau marker. Both epitopes are in the 12-kDa fragment released from the Pronase-resistant core of the PHF (which encompasses the tandem repeat region). The mAb 7.51 epitope requires segments located in the last two repeats, which are common to all tau isoforms. The mAb 423 epitope requires sequences located near both the N and the C terminus of the 12-kDa fragment common to three- and four-repeat tau isoforms. Fragments denatured by concentrated formic acid and SDS regain 423 reactivity when denaturing agents are removed. Since the primary amino acid sequences of PHF tau and normal tau are identical in the repeat region, we conclude that 423 reactivity also requires a modification(s) occurring within an approximately 90-residue segment that are not present in tau proteins so far described in the human brain.

Alzheimer Disease↗
PubMed1991

Measurement of distinct immunochemical presentations of tau protein in Alzheimer disease.

The tau protein is a microtubule-associated protein that is normally located in nerve axons. In Alzheimer disease, it is a constituent of paired helical filaments (PHFs), which are the principal fibrous component of the characteristic neurofibrillary tangles. The tau protein, therefore, is abnormally sequestered in an insoluble form in PHFs in the cell body and dendrites in Alzheimer disease. We have used two monoclonal antibodies (mAbs) to selectively measure the levels of normal, soluble tau protein and of PHF-associated tau protein in the brain. mAb 423 binds to PHFs and recognizes a 12-kDa fragment of tau protein released by formic acid treatment of PHFs, but it does not recognize normal tau protein. In contrast, mAb 7.51 recognizes normal tau protein as well as the PHF core-derived tau fragment, but its epitope is concealed in the PHF-bound form. The differential binding properties for these two mAbs have enabled us in this study to quantify insoluble PHF-associated tau protein in the somatodendritic compartment as well as normal soluble tau protein in its predominantly axonal location. Our findings demonstrate that a distinct immunochemical presentation of tau protein recognized by mAb 423, a PHF-specific marker, can be used to quantify neurofibrillary pathology in Alzheimer disease independently of the presence of normal tau proteins.

Alzheimer Disease↗
PubMed1991

Identification of 3- and 4-repeat tau isoforms within the PHF in Alzheimer's disease.

The microtubule associated protein tau is incorporated into the pronase resistant core of the paired helical filament (PHF) in such a way that the repeat region is protected from proteases, but can be released as a major 12 kDa species from the PHF core by formic acid treatment and by boiling in SDS. This fragment retains the ability to aggregate in the presence of SDS. Detailed sequence analysis of the 12 kDa species shows that it consists of a mixture of peptides derived from the repeat region of 3- and 4-repeat tau isoforms comigrating as a single electrophoretic band. However, the 4-repeat isoforms released from the core lack either the first or the last repeat. The pronase-protected region of tau within the PHF core is therefore restricted to three repeats, regardless of isoform. The alignment of cleavage sites at homologous positions within tandem repeats after protease treatment indicates that the tau-core association is precisely constrained by the tandem repeat structure of the tau molecule.

Alzheimer Disease↗
PubMed1991

Transplants of mouse trisomy 16 hippocampus provide a model of Alzheimer's disease neuropathology.

Alzheimer's disease, which is characterized by amyloid plaques and neurofibrillary tangles, may be attributed to the abnormal expression of gene(s) located on human chromosome 21. Genetic linkage studies have narrowed the region of candidate genes to 21q11.2-21q22 of the long arm of this chromosome. Several single copy sequences within this region, including the amyloid precursor protein (APP), have been mapped to mouse chromosome 16. Reliable strategies exist for breeding Trisomy 16 mice. However, the consequences of developmental overexpression of genes on chromosome 16 have not been previously investigated, because of the lethal effects of this aneuploidy during gestation. In the present report, we employ neural transplantation to study long-term survival and pathogenesis in Trisomy 16 central nervous system tissues. Immunocytochemical staining with antiserum raised against the synthetic APP, beta-A4 and alpha 1-antichymotrypsin revealed numerous densely stained cells within hippocampal grafts of Trisomy 16 mice. Similarly, a population of grafted cells were positively stained following incubation with an antiserum raised against components of the pathological neurofibrillary tangle and with the monoclonal antibodies Tau 6.423 and ubiquitin.

Alzheimer Disease↗
PubMed1991

A progressive deposition of paired helical filaments (PHF) in the brain characterizes the evolution of dementia in Alzheimer's disease. An immunocytochemical study with a monoclonal antibody against the PHF core.

Using the monoclonal antibody (mAb) 6.423 which recognizes epitopes of the pronase-resistant core of paired helical filaments (PHF), we studied postmortem frontal cortex from Alzheimer's disease (AD) patients with short (Group II) and long (Group III) histories of clinical dementia. Four cases with clinically unconfirmed dementia and a postmortem diagnosis of AD (Group I) were also studied. In Group I, the 6,423 mAb was negative whereas in Group II, the antibody recognized primarily neurofibrillary tangles (NFT). In contrast, brains in Group III contained a dense network of 6,423-immunoreactive (IR) thread-like structures ("ghost" neurites) and plaque-like structures with granular appearance, in addition to NFT. The number of 6,423-IR structures appeared to be related to the duration of clinical dementia and the age of onset. Furthermore, "ghost" neurites were more abundant in young AD cases. The possible significance of the 6,423-IR pattern in the pathogenesis of AD is discussed.

Adult↗
PubMed1991

Sequestration of tau by granulovacuolar degeneration in Alzheimer's disease.

Antibodies directed against three regions of tau have been used in a histologic study of granulovacuolar degeneration (GVD) in Alzheimer's disease (AD). Granulovascular degeneration complexes, consisting of a dense granule in a less-dense vacuole, were found in hippocampal pyramidal neurons in all patients studied. Anti-tau antibodies directed against the N-and C-termini, and the repeat region of tau, were found to immunolabel the granule of the GVD complex. Intracellular neurofibrillary tangles also were labeled by these antibodies. In particular, MAb6.423, which recognizes tau protein sequestered in paired helical filaments (PHF) in AD, but not the normal tau proteins so far described in human brain, labeled GVD granules. Contrarily, a generic tau marker (MAb7.51), which immunolabels all known isoforms of isolated and expressed tau protein, including PHF-tau, did not label the GVD granule. These findings demonstrate that the entire tau molecule is sequestered within the GVD granule, and that the tau protein found in GVD complexes is antigenically related to that found in PHFs. There is, however, a difference in the way in which the repeat region of tau is incorporated into the two structures, making the MAb7.51 epitope unavailable in the GVD complex. These findings suggest that the formation of GVD complexes in hippocampal pyramidal neurons vulnerable to neurofibrillary degeneration may represent an alternative pathway for dealing with an aberrant molecular complex, which contributes to the formation of GVD granules and neurofibrillary tangles in AD.

Adult↗
PubMed1990

Competitive ELISA for the measurement of tau protein in Alzheimer's disease.

Tau protein is a major component of paired helical filaments (PHFs) which constitute the characteristic neurofibrillary tangle lesions observed in Alzheimer's disease. Two tau mAbs have been produced which show distinct patterns of immunoreactivity with intact human tau and with tau incorporated in PHFs. The mAb 423 recognises PHFs but not human tau on immunoblots whereas mAb 7/51 reacts with human tau but its epitope is buried within the PHF and is only exposed after formic acid treatment. A competitive ELISA has been developed for both of these mAbs and these have been used to quantify the two distinct tau epitopes in PHFs. Samples containing antigen are incubated with horseradish peroxidase-conjugated mAb at 4 degrees C for 16 h and non-adsorbed antibody then measured by binding, at 37 degrees C for 1 h, to a fragment of tau coated on microtitre plates. Bound enzyme-labelled antibody is measured kinetically using a spectrophotometer capable of automatically mixing the samples throughout a 2-min incubation with substrate and chromogen. The interfacing of the plate reader with a computer permits competitive curves to be plotted automatically using Softmax. Curves are fitted using a 4-parameter logistic algorithm which allows one to determine the relative immunoreactivity for different samples. The application of these assays to monitoring biochemical fractions and quantifying distinct immunochemical presentations of tau protein with these two mAbs is described.

Alzheimer Disease↗
PubMed1990

Molecular analysis of neurofibrillary degeneration in Alzheimer's disease. An immunohistochemical study.

Antibodies directed against three regions of tau, ubiquitin, and B-amyloid were used in a histologic study of neurofibrillary degeneration in Alzheimer's disease to distinguish two populations of neurofibrillary tangles. Intracellular tangles were immunolabeled exclusively by two antibodies raised against antigens in the fuzzy coat of the paired helical filament (PHF). Extracellular tangles were distinguished by selective immunolabeling with a monoclonal antibody raised against antigens in the PHF core. This was associated with removal of the fuzzy coat and exposure of PHF-core epitopes. In the transition from intracellular to extracellular compartments in vivo, tangles appeared to undergo changes similar to protease digestion in vitro. The transition was associated with the appearance of amyloid immunoreactivity. These findings suggest that tangle degradation occurs in a series of distinct stages, including ubiquitination of some unknown molecule, a change in tau immunoreactivity, and partial proteolysis of tangle-bound tau in extracellular tangles.

Aged↗
PubMed1989

The repeat region of microtubule-associated protein tau forms part of the core of the paired helical filament of Alzheimer's disease.

The paired helical filament, the principal component of the neurofibrillary tangles characteristic of Alzheimer's disease, is shown to consist of two structurally distinct parts. An external fuzzy region can be removed by pronase treatment to leave a pronase-resistant morphologically recognizable core. A monoclonal antibody has been raised which both decorates the core and labels peptide fragments extracted from the core. Amino acid sequence derived from such peptides was used to design oligonucleotide probes with which cDNA libraries were screened and clones coding for the corresponding proteins were isolated. The sequences proved to code for two isoforms of human microtubule-associated protein tau, which contained respectively three or four tandem repeats of 31 or 32 amino acids each with a characteristic Pro-Gly-Gly-Gly motif. The patterns of mRNA expression for the two isoforms were found to be stage and cell-type specific but were apparently unaltered in Alzheimer's disease. The repeat region of tau is believed to be the microtubule binding domain and it is this region of the molecule which is tightly and specifically bound in the core of the paired helical filament.

Alzheimer Disease↗
PubMed1989

Characterisation of the first monoclonal antibody against the pronase resistant core of the Alzheimer PHF.

A monoclonal antibody, NOAL 6.423 has been produced which unequivocally identifies a protein which is a constituent of the pronase resistant core of the Alzheimer PHF. Although the site recognised by this antibody is contained somewhere within the highly conserved triple repeat region of the tau molecule, no experiments have succeeded so far in demonstrating NOAL 6.423 reactivity in any brain proteins other than those derived from PHFs. A site which can be recognised by NOAL 6.423 cannot be generated in preparations of normal porcine tau by simple proteolytic cleavage with pronase. Likewise reactivity with the PHF peptide recognised by NOAL 6.423 is not altered by alkaline phosphatase. The unusual properties of NOAL 6.423 are consistent with the possibility that its recognition site is not determined solely by the primary structure of the conserved region of the tau molecule.

Alzheimer Disease↗
PubMed1988

Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer disease: identification as the microtubule-associated protein tau.

Screening of cDNA libraries prepared from the frontal cortex of an Alzheimer disease patient and from fetal human brain has led to isolation of the cDNA for a core protein of the paired helical filament of Alzheimer disease. The partial amino acid sequence of this core protein was used to design synthetic oligonucleotide probes. The cDNA encodes a protein of 352 amino acids that contains a characteristic amino acid repeat in its carboxyl-terminal half. This protein is highly homologous to the sequence of the mouse microtubule-associated protein tau and thus constitutes the human equivalent of mouse tau. RNA blot analysis indicates the presence of two major transcripts, 6 and 2 kilobases lon g, with a wide distribution in normal human brain. Tau protein mRNAs were found in normal amounts in the frontal cortex from patients with Alzheimer disease. The proof that at least part of tau protein forms a component of the paired helical filament core opens the way to understanding the mode of formation of paired helical filaments and thus, ultimately, the pathogenesis of Alzheimer disease.

Alzheimer Disease↗