Claude Michel Wischik

Senile dementia of Lewy body type and Alzheimer type are biochemically distinct in terms of paired helical filaments and hyperphosphorylated tau protein.

We have used biochemical assays to examine cingulate and occipital cortices from age-matched cases of Alzheimers disease (AD; n = 12), senile dementia of the Lewy body type (SDLT; n = 13), Parkinsons disease (PD; 5 non-demented cases and 7 cognitively impaired cases) and controls (n = 11) for paired helical filaments (PHFs), phosphorylated and normal tau protein and beta/A4-protein. Whereas cingulate cortex is characterised by relatively high densities of cortical Lewy bodies in the SDLT cases and lower numbers in PD, these inclusion bodies were absent in the cingulate cortex from AD and control cases. Protease-resistant PHFs and hyperphosphorylated tau protein were found in AD and, at low levels, in a minority of SDLT cases. Qualitatively, both of these preparations were indistinguishable in SDLT from those found in AD but levels of both parameters in SDLT were less than 5% of those in AD. SDLT, PD and control groups did not differ from each other in terms of the quantity of protease-resistant PHFs or the level of hyperphosphorylated tau. Furthermore, PHF accumulation did not distinguish between PD cases with or without dementia. The levels of normal tau protein did not differ between the four groups. beta/A4 protein levels did not distinguish between PD and control groups, between AD and SDLT groups, or between SDLT and control groups for either cingulate or occipital cortices. Thus extensive accumulation of PHFs in either neurofibrillary tangles or dystrophic neurites is not a feature of either SDLT or PD. Our findings provide molecular support for the neuropathological and clinical separation of SDLT as a form of dementia that is distinct from AD.

Examination of the validity of the hierarchical model of neuropathological staging in normal aging and Alzheimer's disease.

Abstract The neuropathological staging model of Alzheimer’s disease proposed by Braak and Braak [Acta Neuropathol (1991) 82 : 259] requires that the evolution of neurofibrillary pathology follows a predictable pattern that can be ordered in a regular regional hierarchy. We have operationalized the neuropathological staging system to permit testing of its validity. Forty-two cases were derived from an epidemiological study of cognitive function in an elderly population for which post-mortem brain tissue was collected. Cases with neuropathological diagnoses other than Alzheimer’s disease and normal aging were excluded. Neurofibrillary tangle counts were determined in all cortical laminae and regions used for staging. There was a significant correlation between the overall extent of neurofibrillary pathology and the number of regions affected. There were frequent order violations in the proposed hierarchy: 19 instances (45%) involving entorhinal and transentorhinal cortices, and 16 instances (38%) involving CA1 of hippocampus and entorhinal cortex. Only 6 out of 42 cases conformed in all regions to the expected hierarchy. Nevertheless, 90% of the cases had 2 order violations or less, supporting the approximate validity of the hierarchy.

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 Alzheimers 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.

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 Alzheimers 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 Alzheimers 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.

The relationship between clinical dementia and neuropathological staging (Braak) in a very elderly community sample

The neuropathological staging model proposed by Braak and Braak (1991) implies that the evolution of neurofibrillary pathology follows a predictable sequence and can be ordered in a regular regional hierarchy. A total of 42 cases of an elderly population sample, which had been prospectively clinically assessed, were examined. Clinical diagnosis was made according to the CAMDEX criteria, and the sample reported here did not include cases were vascular dementia according to the criteria proposed by Chui et al. (1991). The neuropathological staging procedure was applied as originally proposed by Braak and Braak (1991). In addition, in all cortical laminae and regions which are essential for the staging model neurofibrillary tangles were quantified. Demented cases had significantly more areas involved and more advanced neuropathological stages. Cases with stages 1–3 tended to be non-demented, and cases with stages 4–6 tended to be demented. However, there was a considerable degree of overlap and no clear-cut threshold could be established. This brings into question the diagnostic value of the staging model.

Examination of phosphorylated tau protein as a PHF-precursor at early stage alzheimer's disease

Hyperphosphorylated tau protein which can be isolated on the basis of insolubility in 1% sarkosyl (A68-tau fraction) is thought to represent a precursor pool for PHF assembly, associated histologically with neuritic pathology, which feeds into a more resistant tangle-associated PHF pool via cross-linking and proteolysis. We examined these predictions at the earliest detectable stages of neurofibrillary pathology. We report that there is no evidence that neuritic pathology represents an early pathologic stage, no evidence of an association between neuritic pathology and phosphorylated tau, no evidence of selective accumulation of phosphorylated tau at early stages of pathology, and no evidence for a precursor/product relationship between phosphorylated tau and PHFs during progression of pathology. We conclude that altered phosphorylation is a secondary process affecting 5% of PHFs and does not explain PHF assembly in Alzheimers disease.

Apolipoprotein E Genotype in the Prediction of Cognitive Decline and Dementia in a Prospectively Studied Elderly Population

An increased apolipoprotein E (ApoE) type epsilon 4 allele frequency is associated with both sporadic and familial late-onset Alzheimers disease (AD). The age of onset of disease in patients homozygous for the epsilon 4 allele appears to be decreased by approximately 15 years compared with E2/3 individuals. In order to assess the influence of this allele on both dementia and cognitive decline in the elderly we have determined the ApoE genotype of 150 individuals over the age of 75 years who have taken part in a longitudinal study. Homozygosity for the epsilon 4 allele was rare. Of the 2 homozygotes, 1 was severely demented but the other did not receive a clinical diagnosis of dementia. The latter individual did demonstrate marked cognitive decline over a 28-month period. There was a consistent association between the presence of an epsilon 4 allele and both the clinical diagnosis of dementia and cognitive decline. These findings confirm a genetic heterogeneity in late-onset sporadic AD and prompt caution in the use of ApoE genotype to predict an elderly individuals susceptibility to either dementia or cognitive decline.

Characterisation of an epitope specific to the neuron-specific isoform of human enolase recognised by a monoclonal antibody raised against a synthetic peptide corresponding to the C-terminus of β/A4-protein

Antibodies to synthetic peptides corresponding to different regions of beta/A4-protein recognize deposits of amyloid in the brains of patients with Alzheimers disease. Downs syndrome cases and in the normal ageing brain. We have prepared a monoclonal antibody, mAb 22.212, raised against a synthetic C-terminal peptide of beta/A4 protein (residues 28-40) which labelled senile plaques in Alzheimers disease after proteolytic treatment of tissue sections. In addition to recognising synthetic beta/A4-peptides that include the C-terminal residues 28-42, the mAb 22.212 was found to cross-react with a soluble, 47 kDa protein found in brain homogenates. This protein was shown, by amino acid sequence analysis and immunoassay, to be neuron-specific enolase (NSE). The mAb 22.212 did not recognize the non-neuronal enolase (NNE) or muscle-specific enolase (MSE) isoforms and its epitope was mapped to a short stretch of amino-acids unique to NSE, near the C-terminus. The cross-reactive NSE epitope is sited between residues 402-423 in NSE and shows no common sequence with beta/A4, perhaps suggesting that it is a conformational epitope. The significance and applications of these findings are discussed.

Microsatellite polymorphism of the α1-antichymotrypsin gene locus associated with sporadic Alzheimer's disease

Abstract A variant of the apolipoprotein E gene, APOE*4, is associated with both sporadic Alzheimer’s disease (AD) and a subset of familial AD and this association is stronger with early as opposed to late onset AD. Both APOE*4 and α1-antichymotrypsin (ACT) will accelerate the rate of amyloid filament formation and are major constituents of the plaques associated with AD. We now show that a dinucleotide microsatellite allele in the 5′-flanking sequence of the ACT gene, designated A10, in association with APOE*4 significantly increases the risk of developing sporadic AD, which accounts for the majority of AD cases.

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 Alzheimers 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.