Publications

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

Gertz HJ, Xuereb J, Huppert F, Brayne C, McGee MA, Paykel E, Harrington C, Mukaetova-Ladinska E, Arendt T, Wischik CM

Acta Neuropathol (Berl) 1998 Feb;95(2):154-8

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.

Presence of the apolipoprotein E type epsilon 4 allele is not associated with neurofibrillary pathology or biochemical changes to tau protein.

Mukaetova-Ladinska EB, Harrington CR, Roth M, Wischik CM

Dement Geriatr Cogn Disord 1997 Sep-Oct;8(5):288-95

Alzheimer's disease (AD) represents a heterogeneous disorder, and several factors have been associated with its development. The presence of the apolipoprotein E type (APOE) epsilon 4 allele has been proposed as a risk factor for AD, but how it influences the development of the characteristic hallmarks of the disease remains unknown. In the present study, the neuropathological changes and levels of both core PHF-tau and normal tau protein in 4 neocortical areas, cerebellum and medial temporal cortex were determined in 18 AD cases. The extent of these changes was compared between 10 cases possessing an epsilon 4 allele and 8 cases without. These two groups were indistinguishable in terms of neurofibrillary pathology, whereas cases with an epsilon 4 allele had more diffuse plaques, particularly in the temporal neocortex. Biochemically, there was no difference in the levels of PHF-tau protein between the two groups. These data indicate that APOE epsilon 4 allele may influence deposition of diffuse amyloid, but altered tau protein processing, which underlies the development of the neurofibrillary pathology in AD, is not influenced by this allele.

Microsatellite polymorphism of the alpha 1-antichymotrypsin gene locus associated with sporadic Alzheimer's disease.

Morgan K, Morgan L, Carpenter K, Lowe J, Lam L, Cave S, Xuereb J, Wischik C, Harrington C, Kalsheker NA

Hum Genet 1997 Jan;99(1):27-31

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 alpha 1-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.

Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines.

Wischik CM, Edwards PC, Lai RY, Roth M, Harrington CR

Proc Natl Acad Sci U S A 1996 Oct 1;93(20):11213-8

Full reprint

In Alzheimer disease (AD) the microtubule-associated protein tau is redistributed exponentially into paired helical filaments (PHFs) forming neurofibrillary tangles, which correlate with pyramidal cell destruction and dementia. Amorphous neuronal deposits and PHFs in AD are characterized by aggregation through the repeat domain and C-terminal truncation at Glu-391 by endogenous proteases. We show that a similar proteolytically stable complex can be generated in vitro following the self-aggregation of tau protein through a high-affinity binding site in the repeat domain. Once started, tau capture can be propagated by seeding the further accumulation of truncated tau in the presence of proteases. We have identified a nonneuroleptic phenothiazine previously used in man (methylene blue, MB), which reverses the proteolytic stability of protease-resistant PHFs by blocking the tau-tau binding interaction through the repeat domain. Although MB is inhibitory at a higher concentration than may be achieved clinically, the tau-tau binding assay was used to identify desmethyl derivatives of MB that have Ki values in the nanomolar range. Neuroleptic phenothiazines are inactive. Tau aggregation inhibitors do not affect the tau-tubulin interaction, which also occurs through the repeat domain. Our findings demonstrate that biologically selective pharmaceutical agents could be developed to facilitate the proteolytic degradation of tau aggregates and prevent the further propagation of tau capture in AD.

Apolipoprotein E genotype in the prediction of cognitive decline and dementia in a prospectively studied elderly population.

Brayne C, Harrington CR, Wischik CM, Huppert FA, Chi LY, Xuereb JH, O'Connor DW, Paykel ES

Dementia 1996 May-Jun;7(3):169-74

An increased apolipoprotein E (ApoE) type epsilon 4 allele frequency is associated with both sporadic and familial late-onset Alzheimer's 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 individual's susceptibility to either dementia or cognitive decline.

Alterations in tau protein metabolism during normal aging.

Mukaetova-Ladinska EB, Harrington CR, Roth M, Wischik CM

Dementia 1996 Mar-Apr;7(2):95-103

It is unknown whether aging and Alzheimer's disease (AD) are on the same continuum, or whether they are qualitatively distinct. Tau protein has been identified as a major constituent of paired helical filaments (PHFs) and AD is characterised by a major redistribution of the normal tau protein pool into PHFs. Little is known about the changes in tau protein distribution that occur in the course of normal aging. We have examined PHF-bound and normal tau fractions in frontal, temporal, parietal and occipital neocortex, cerebellum, hippocampus and entorhinal cortex in 15 cognitively unimpaired individuals aged 19-88 years at death. Insoluble tau protein in the PHF fraction did not increase with aging in any brain region, despite the appearance of neurofibrillary pathology at low density in the more elderly cases. By contrast, normal tau protein decreased with aging (r = 0.32, p < 0.001), with an average loss of 14% of soluble tau per decade after the age of 20 years. This was unrelated either to neurofibrillary or beta-amyloid pathology. Frontal grey matter and hippocampus were most vulnerable to age-related tau loss, decreasing by as much as 90% in the older subgroup. These findings contrast with those we have previously reported in AD, where the redistribution of tau protein into the PHF-bound fraction was highly correlated with the extent of neurofibrillary pathology, and suggest that the mechanisms of tau loss in aging and AD are distinct. Age-related tau loss may underlie the neuropsychological impairments seen in the non-demented elderly.

Staging the pathological assembly of truncated tau protein into paired helical filaments in Alzheimer's disease.

Mena R, Edwards PC, Harrington CR, Mukaetova-Ladinska EB, Wischik CM

Acta Neuropathol (Berl) 1996;91(6):633-41

Tau protein, which is incorporated into the core of paired helical filaments (PHFs) in Alzheimer's disease (AD), can be characterised immunochemically by C-terminal truncation at Glu-391 recognised by monoclonal antibody (mAb) 423, and acid-reversible occlusion of a generic tau epitope in the tandem repeat region recognised by mAb 7.51. PHFs are also characterised by the presence of binding sites for a fluorescent dye (thiazin red) which can be used to differentiate between amorphous and fibrillar states of tau and beta-amyloid proteins in AD. We have used double labelling confocal microscopy to investigate that state of aggregation of the tau antigens associated with the core structure of the PHF at early stages of neurofibrillary pathology. We report that the early abnormal tau deposits in cells vulnerable to neurofibrillary degeneration are characterised by C-terminal truncation at Glu-391, acid-reversible occlusion of the mAb 7.51 epitope, and the absence of binding sites for thiazin red, consistent with the amorphous non-fibrillar structure demonstrated by immunoelectron microscopy. Transition to the fibrillar state in the PHF is associated with acid-reversible occlusion of both mAb 7.51 and 423 epitopes, and acquisition of binding sites for thiazin red. In neurites, the transition between the two states of aggregation shows distal to proximal polarity, with the fibrillar state found nearest the cell body. These findings demonstrate that the assembly of tau protein into the PHF occurs in at least two stages, an amorphous stage characterised by C-terminal truncation and occlusion of sites within the tandem repeat region, and a fibrillar stage characterised by acid-reversible occlusion of both epitopes via addition of intact tau molecules in the fuzzy coat of the PHF.

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

Gertz HJ, Xuereb JH, Huppert FA, Brayne C, Kruger H, McGee MA, Paykel ES, Harrington CR, Mukaetova-Ladinska EB, O'Connor DW, Wischik CM

Eur Arch Psychiatry Clin Neurosci 1996;246(3):132-6

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.

Apolipoprotein E type epsilon 4 allele frequency is increased in patients with schizophrenia.

Harrington CR, Roth M, Xuereb JH, McKenna PJ, Wischik CM

Neurosci Lett 1995 Dec 29;202(1-2):101-4

Apolipoprotein E type epsilon 4 alleles are increased in both Alzheimer's disease (AD) and cortical Lewy body dementia, while the epsilon 2 allele has been associated as a protective factor against the development of dementia in AD. We have determined APOE genotype in schizophrenic patients coming to autopsy (age range 19-95 years). The allele frequencies in this group were: epsilon 2, 6.0%; epsilon 3, 67.9%; and epsilon 4, 26.2%. Three patients (14%) were homozygous for the epsilon 4 allele. Thus, schizophrenia is associated with an increased epsilon 4 allele frequency, as compared with controls (P = 0.01), that was indistinguishable from that found in either AD or Lewy body dementia. The increased epsilon 4 allele frequency was not associated with increased age of schizophrenia patient, indicating that it was not due to the co-existence of AD.

Changes in a CSF antigen associated with dementia.

Carretero MT, Harrington CR, Wischik CM

Dementia 1995 Sep-Oct;6(5):281-5

An 85-kDa antigen was detected in cerebrospinal fluid (CSF) using a monoclonal antibody, 11.57, raised against a fraction of paired helical filaments extracted from Alzheimer's disease (AD) brain tissues. The antigen is a thermostable protein that is unrelated either to tau or amyloid beta-protein precursor. Its level was measured in lumbar and post-mortem CSF samples and found to be decreased significantly in patients with dementia as compared with non-demented controls. There was no difference in its level between patients with AD and those with non-Alzheimer type dementia. In AD, however, there was a significant relationship between disease progression and the amount of the 85-kDa antigen.

Absence of abnormal hyperphosphorylation of tau in intracellular tangles in Alzheimer's disease.

Bondareff W, Harrington CR, Wischik CM, Hauser DL, Roth M

J Neuropathol Exp Neurol 1995 Sep;54(5):657-63

Adult human nerve cells contain tau protein, a phosphorylated microtubule-associated protein, that is hyperphosphorylated in the fetus and in patients with Alzheimer's disease. Hyperphosphorylation, which diminishes the microtubule-binding capacity of tau, destabilizes microtubules and may enhance the formation of paired helical filaments that constitute neurofibrillary tangles in Alzheimer's disease. Here, we use phosphorylation-dependent anti-tau antibodies to detect specific epitopes that characterize hyperphosphorylated tau. Our demonstration of intracellular tangles containing full-length tau that are not immunolabeled by these antibodies suggests that hyperphosphorylation of tau is not obligatory in the formation of neurofibrillary tangles in Alzheimer's disease.

Quantitative analysis of tau protein in paired helical filament preparations: implications for the role of tau protein phosphorylation in PHF assembly in Alzheimer's disease.

Wischik CM, Edwards PC, Lai RY, Gertz HN, Xuereb JH, Paykel ES, Brayne C, Huppert FA, Mukaetova-Ladinska EB, Mena R, et al

Neurobiol Aging 1995 May-Jun;16(3):409-17; discussion 418-31

In Alzheimer's disease, there is a major redistribution of the tau protein pool from soluble to PHF-bound forms. PHF-bound tau can be distinguished from normal tau by acid reversible occlusion of a generic tau epitope in the tandem repeat region and characteristic sedimentation in the if-II protocol developed in this laboratory. We show that 85% of tau bound in the PHF-like configuration can be recovered in the if-II PHF-fraction. Less than 1% of this material was phosphorylated at the mAb AT8 site in aged clinical controls or in cases with minimal or mild dementia. Of tau phosphorylated at the mAb AT8 site, only 12% was found to co-sediment with PHFs. These low levels could not be explained by postmortem dephosphorylation. As more than 95% of PHF-tau is not phosphorylated, even at early stages of pathology, it is misleading to use the terms "PHF-tau" and "phosphorylated tau" as though they were synonymous, particularly as this implies a pathogenetic role which phosphorylation need not have.

Monitoring pathological assembly of tau and beta-amyloid proteins in Alzheimer's disease.

Mena R, Edwards P, Perez-Olvera O, Wischik CM

Acta Neuropathol (Berl) 1995;89(1):50-6

This double-labelling confocal microscopy study of the neuropathology of Alzheimer's disease (AD) reports the use of a fluorescent dye, thiazin red, which has staining properties similar to thioflavin-S. Thiazin red fluorescence can be visualised selectively in the red channel, and we have used this property to compare it with the labelling seen using monoclonal antibody (mAb) 423, which detects tau protein C-terminally truncated at Glu-391, and mAb 4G8, which detects beta-amyloid protein. Thiazin red is shown to recognized the typical histopathological deposits associated with both proteins. However, not all deposits containing these proteins are stained. Specifically, diffuse beta-amyloid plaques and severely degraded extracellular tangles are unlabelled. Likewise a characteristic mAb 423-reactive granular plaque-like structure, typically present in cases with abundant extracellular tangels, is unlabelled by thiazin red. Such plaques can be shown to be continuous with the basal dendrites of degraded tangle-bearing pyramidal cells. These findings suggest that paired helical filaments (PHFs) continue to undergo degradation in the extracellular space, which is associated with loss of thiazin red binding sites, but preservation of mAb 423 immunoreactivity. This epitope appears to be characteristic of a stable core element of the PHF which is highly resistant to proteolysis. Compounds such as thiazin red with high affinity for beta-pleated protein structures can be used to monitor the state of pathological assembly of amyloidogenic protein species found in AD.

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

Harrington CR, Perry RH, Perry EK, Hurt J, McKeith IG, Roth M, Wischik CM

Dementia 1994 Sep-Oct;5(5):215-28

We have used biochemical assays to examine cingulate and occipital cortices from age-matched cases of Alzheimer's disease (AD; n = 12), senile dementia of the Lewy body type (SDLT; n = 13), Parkinson's 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.

Presence of axonal paired helical filament-tau in Alzheimer's disease: submicroscopic localization.

Bondareff W, Harrington CR, McDaniel SW, Wischik CM, Roth M

J Neurosci Res 1994 Aug 15;38(6):664-9

While normal adult tau protein is found typically in axons, paired helical filament (PHF) tau has been shown immunohistochemically to be in the somatodendritic compartment in Alzheimer's disease (AD). The small amount of PHF-tau (8-11%) found in white matter with immunochemical methods is shown here by immunoelectron microscopy to be located in axons. It is localized to straight filament variants of PHFs. The small amount of PHF-tau in white matter, therefore, appears not to result from the contamination of immunochemical preparations but to be an integral constituent of affected axons in AD.

Alzheimer's-disease-like changes in tau protein processing: association with aluminium accumulation in brains of renal dialysis patients.

Harrington CR, Wischik CM, McArthur FK, Taylor GA, Edwardson JA, Candy JM

Lancet 1994 Apr 23;343(8904):993-7

Tau protein is a major structural protein of the paired helical filaments (PHFs) found in both neuritic senile plaques and neurofibrillary tangles in Alzheimer's disease (AD). Senile plaques also contain amyloid beta protein (A beta). We did an immunochemical analysis of frontal cortex from 15 dialysis cases, 5 Alzheimer's disease patients, and 6 control cases to see whether AD-like changes in A beta deposition and tau protein were linked to aluminium accumulation. Dialysis patients were used because they are frequently exposed to increased levels of aluminium. 8 of the 15 dialysis cases had insoluble A beta, but there was no association between its presence and the accumulation of aluminium. However, we found AD-like changes in the processing of tau protein. In white matter, truncated tau protein in the PHF-core fraction and endogenously truncated tau in the supernatant fraction were both increased in association with aluminium accumulation in the brain. In grey matter, normal tau protein was depleted and insoluble hyperphosphorylated tau increased in association with aluminium concentration. Protease-resistant PHFs were present in grey matter in 2 dialysis cases, a frequency above that expected for AD in this age group. PHF-core tau in both grey and white matter correlated with decreased levels of normal tau protein in white matter. These findings are consistent with a role for aluminium in the development of AD-like pathology in patients subjected to prolonged aluminium exposure.

See also comment in Lancet 1994 Apr 23;343(8904):989-90, Lancet 1994 Jul 16;344(8916):204-5, Lancet 1994 Aug 13;344(8920):486

Immunohistochemical staging of neurofibrillary degeneration in Alzheimer's disease.

Bondareff W, Harrington C, Wischik CM, Hauser DL, Roth M

J Neuropathol Exp Neurol 1994 Mar;53(2):158-64

Antibodies to different phosphorylated and non-phosphorylated tau epitopes have been used to identify three histologically distinct types of neurofibrillary tangles in Alzheimer's disease. Intracellular tangles (Type 1) were identified by antibodies recognizing epitopes throughout the tau molecule, including the NH2-terminus. Compact extracellular tangles (Type 2) were characterized by the loss of NH2-terminal immunoreactivity and retention of other tau epitopes. Dispersed extracellular tangles (Type 3) were characterized by the presence of epitopes associated with the microtubule binding region and the COOH-terminus. These three types of tangles, found in situ in hippocampus, could be created experimentally by proteolytic treatment of brain sections. These findings suggest that three stages of neurofibrillary degeneration can be understood as a sequential stripping of paired helical filaments in which the loss of amino-terminus epitopes, followed by loss of phosphorylated epitopes, results in the appearance of dispersed extracellular tangles containing PHF-core epitopes.

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 beta/A4-protein.

Harrington CR, Quinn GB, Hurt J, Day IN, Wischik CM

Biochim Biophys Acta 1993 Oct 3;1158(2):120-8

Antibodies to synthetic peptides corresponding to different regions of beta/A4-protein recognize deposits of amyloid in the brains of patients with Alzheimer's disease. Down's 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 Alzheimer's 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.

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

Mukaetova-Ladinska EB, Harrington CR, Roth M, Wischik CM

Am J Pathol 1993 Aug;143(2):565-78

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.

Evidence of subtypes of Alzheimer's disease and implications for etiology.

Bondareff W, Mountjoy CQ, Wischik CM, Hauser DL, LaBree LD, Roth M

Arch Gen Psychiatry 1993 May;50(5):350-6

OBJECTIVE: Because age of onset does not reliably define two subtypes of Alzheimer's disease, classification based on the severity of neuronal degeneration was tested. DESIGN: Numbers of extracellular tangles and pyramidal neurons in the hippocampus were used to group patients. PATIENTS: The study population consisted of 46 elderly patients satisfying DSM-III criteria for dementia and NINCDS-ADRDA criteria for definite Alzheimer's disease after death. RESULTS: Univariate logistic regression analysis showed the numbers of neurofibrillary tangles and pyramidal neurons and the duration of dementia were significantly associated with grouping based on the presence of abundant extracellular tangles. Ninety-one percent of patients were correctly classified as compared with 85% correctly classified by age of onset data. Odds ratios showed that increasing numbers of neurofibrillary tangles predicted greater severity of neuronal loss. CONCLUSION: The results of the study indicate the importance of neurofibrillary degeneration, not the deposition of amyloid, in the pathogenesis of Alzheimer's disease. They support a classification of Alzheimer's disease related more closely to the severity of neurofibrillary degeneration than to age at onset.

Mitochondrial function in Alzheimer's disease.

Cooper JM, Wischik C, Schapira AH

Lancet 1993 Apr 10;341(8850):969-70

Comment on Lancet 1992 Sep 12;340(8820):671

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

Novak M, Kabat J, Wischik CM

EMBO J 1993 Jan;12(1):365-70

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.

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

Montejo de Garcini E, Corrochano L, Wischik CM, Diaz Nido J, Correas I, Avila J

FEBS Lett 1992 Mar 24;299(1):10-4

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.

Immunological characterization of the region of tau protein that is bound to Alzheimer paired helical filaments.

Caputo CB, Wischik C, Novak M, Scott CW, Brunner WF, De Garcini EM, Lo MM, Norris TE, Salama AI

Neurobiol Aging 1992 Mar-Apr;13(2):267-74

Tau protein is known to be present in the paired helical filaments (PHFs) of Alzheimer brains. This study investigated the fragments of tau protein that remain bound to pronase-treated PHFs and conditions that lead to the release of these tau fragments from the core structure of the PHF. Antibody 423 reacted with PHFs and with fetal rat tau but not with adult rat tau, pig tau, or recombinant human tau. Three other antibodies that react with the tubulin binding region of tau only reacted with PHFs after they were disrupted with formic acid or guanidine. Other antibodies that recognize tau sequences C terminal to the tubulin binding region also recognized pronase-treated PHFs. Antibodies SMI34 and T3P that recognize phosphorylated epitopes were reactive with pronase-treated PHFs. Tau fragments from the PHF were solubilized by acid or guanidine treatment. These findings suggest that the fragments of tau that are bound to PHFs and protected from pronase digestion include sequences from the tubulin binding region to the C terminus of tau. In addition, some of these sequences appear to be conformationally or post-translationally modified.

Therapeutic approaches targeted at the amyloid proteins in Alzheimer's disease.

Caputo CB, Scott CW, Sobel IR, Sygowski LA, Wischik CM, Brunner WF

Clin Neuropharmacol 1992;15 Suppl 1 Pt A:414A-415A

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

Wischik CM, Harrington CR, Mukaetova-Ladinska EB, Novak M, Edwards PC, McArthur FK

Ciba Found Symp 1992;169:268-93; discussion 293-302

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.

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

Jakes R, Novak M, Davison M, Wischik CM

EMBO J 1991 Oct;10(10):2725-9

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.

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

Bondareff W, Wischik CM, Novak M, Roth M

Am J Pathol 1991 Sep;139(3):641-7

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.

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

Barnes NM, Cheng CH, Costall B, Naylor RJ, Williams TJ, Wischik CM

Eur J Pharmacol 1991 Aug 6;200(2-3):289-92

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.

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.

Mena R, Wischik CM, Novak M, Milstein C, Cuello AC

J Neuropathol Exp Neurol 1991 Jul;50(4):474-90

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.

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

Harrington CR, Mukaetova-Ladinska EB, Hills R, Edwards PC, Montejo de Garcini E, Novak M, Wischik CM

Proc Natl Acad Sci U S A 1991 Jul 1;88(13):5842-6

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.

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.

Novak M, Jakes R, Edwards PC, Milstein C, Wischik CM

Proc Natl Acad Sci U S A 1991 Jul 1;88(13):5837-41

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.

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

Richards SJ, Waters JJ, Beyreuther K, Masters CL, Wischik CM, Sparkman DR, White CL III, Abraham CR, Dunnett SB

EMBO J 1991 Feb;10(2):297-303

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.

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

Harrington CR, Edwards PC, Wischik CM

J Immunol Methods 1990 Dec 5;134(2):261-71

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.

Normal densities of 5-HT3 receptor recognition sites in Alzheimer's disease.

Barnes NM, Costall B, Naylor RJ, Williams TJ, Wischik CM

Neuroreport 1990 Nov-Dec;1(3-4):253-4

The present studies investigate the possible difference in 5-HT3 receptor recognition site density in amygdaloid and hippocampal homogenates from patients with Alzheimer's disease compared with age-matched controls. The 5-HT3 receptor recognition site radioligand [3H]-(S)-zacopride identified a comparable density of specific sites (defined by granisetron 10.0 microM) in tissues obtained from patients with Alzheimer's disease as compared to age-matched controls (amygdala, Bmax 41 +/- 4 and 43 +/- 9; hippocampus, Bmax 59 +/- 9 and 51 +/- 9 fmol mg-3 protein; tissue from Alzheimer's disease patients and age-matched controls, respectively, mean +/- s.e.m., n = 7-8). These results suggest that the density of 5-HT3 receptor recognition sites labelled by [3H]-(S)-zacopride are not altered in the amygdala and hippocampus of patients with Alzheimer's disease.

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

Bondareff W, Wischik CM, Novak M, Amos WB, Klug A, Roth M

Am J Pathol 1990 Sep;137(3):711-23

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.

Cell biology of the Alzheimer tangle.

Wischik CM

Curr Opin Cell Biol 1989 Feb;1(1):115-22

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

Novak M, Wischik CM, Edwards P, Pannell R, Milstein C

Prog Clin Biol Res 1989;317:755-61

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.

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

Crowther T, Goedert M, Wischik CM

Ann Med 1989;21(2):127-32

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.

Structural characterization of the core of the paired helical filament of Alzheimer disease.

Wischik CM, Novak M, Edwards PC, Klug A, Tichelaar W, Crowther RA

Proc Natl Acad Sci U S A 1988 Jul;85(13):4884-8

The paired helical filament, the principal constituent of the neurofibrillary tangles characteristic of Alzheimer 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. Scanning transmission electron microscopy gives an estimate for the mass per unit length as 79 kDa.nm-1 before Pronase treatment and 65 kDa.nm-1 after treatment. The fuzzy region carries all the epitopes recognized by two different antisera against microtubule-associated protein tau. By contrast, a monoclonal antibody (mAb) we have raised to paired helical filament cores (mAb 423) decorates Pronase-treated filaments much more strongly than it does untreated ones. We have shown in previous papers that the epitope recognized by mAb 423 is carried by a central 9.5-kDa fragment of tau protein, which therefore forms part of the Pronase-resistant core structure. The remainder of the tau protein incorporated into the filaments must contribute part, if not all, of the fuzzy region. The mass per unit length measurements imply that the three-domain structural subunit of the core that we visualized previously by image reconstruction has a molecular mass of approximately equal to 100 kDa.

Isolation of a fragment of tau derived from the core of the paired helical filament of Alzheimer disease.

Wischik CM, Novak M, Thogersen HC, Edwards PC, Runswick MJ, Jakes R, Walker JE, Milstein C, Roth M, Klug A

Proc Natl Acad Sci U S A 1988 Jun;85(12):4506-10

A substantially enriched preparation of Alzheimer paired helical filaments (PHFs) has been used as a starting point for biochemical studies. Pronase treatment, which strips off adhering proteins, leaves a resistant core that is structurally intact. This has been used to raise a monoclonal antibody that decorates the filament core. The antibody has been used to follow the extraction of two peptide fragments (9.5 and 12 kDa) by immunoblotting. The link between the PHF as a morphological entity and these peptides has been established independently by photoaffinity labeling with a chemical ligand to the PHF core. Sequence analysis of these peptides was used to design oligonucleotide probes for cloning a cognate cDNA, which leads to its identification as human microtubule-associated tau protein. The sequencing of the 9.5- and 12-kDa peptides shows they are derived from a conserved region of tau containing three repeating segments. Since these fragments have been copurified with the Pronase-resistant core and are only released by subsequent steps, the corresponding part of the tau molecule must be tightly bound in the PHF core.

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.

Goedert M, Wischik CM, Crowther RA, Walker JE, Klug A

Proc Natl Acad Sci U S A 1988 Jun;85(11):4051-5

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.

Subunit structure of the Alzheimer tangle.

Wischik CM, Crowther RA

Br Med Bull 1986 Jan;42(1):51-6

Image reconstruction of the Alzheimer paired helical filament.

Crowther RA, Wischik CM

EMBO J 1985 Dec 30;4(13B):3661-5

The paired helical filament (PHF) is the principal constituent of the neurofibrillary tangles that occur in the brain in senile dementias of the Alzheimer type. We have previously shown from fragmentation patterns of isolated PHFs that they consist of a regularly repeating subunit, which is axially compact and not an extended fibrous molecule or protofilament. Here we present diffraction patterns of PHFs and an objective reconstruction of the cross-sectional density of the PHF computed from the diffraction patterns. We demonstrate the presence of an axial periodicity (3 nm) in PHFs. These results establish conclusively that the PHF is made up of a double helical stack of transversely oriented subunits, each of which has three domains, and precludes purely descriptive models based on helical rearrangements of preformed cytoskeletal polymers or protofilaments. The structure is of the type that might arise by the de novo assembly of a single structural subunit, which appears to be produced in considerable abundance in those neurons where tangles form.

Subunit structure of paired helical filaments in Alzheimer's disease.

Wischik CM, Crowther RA, Stewart M, Roth M

J Cell Biol 1985 Jun;100(6):1905-12

The neurofibrillary tangles that occur in the brain in cases of senile dementia of the Alzheimer type contain a distinctive type of filament, the paired helical filament (PHF). We have developed a method for isolating the tangles postmortem in sufficient yield for structural study of PHFs by electron microscopy of negatively stained and shadowed preparations. This material shows the characteristic helical structure seen in sectioned embedded material. In addition, two striking fragmentation patterns are observed. (a) Some filaments show sharp transverse breaks at apparently random positions along the filament. (b) In a few PHFs one strand is missing for a variable length, whereas the other appears to maintain its structural integrity. The shadowed specimens show the PHF to be wound in a left-handed manner. These observations indicate that the PHF consists of subunits of very limited axial extent arranged along two left-handed helical strands. The visualization of the substructure within the PHFs is rather variable and a model building approach has therefore been adopted, which has allowed the main features seen in the images to be interpreted. The subunit appears to have at least two domains in a radial direction and an axial extent of less than 5 nm. The whole structure can best be described as a twisted ribbon and indeed alkali treatment does untwist PHFs to give flat ribbons. The nature of the proposed model makes it most unlikely that the PHF is formed by a simple collapse of normal cytoskeletal elements, such as neurofilaments.

Cell organization in the stroma of the rat uterus. II. Responses to progesterone in the ovariectomised rat.

Rogers AW, Wischik CM

J Anat 1983 Oct;137 (Pt 3):541-53

The organisation and activity of cells in the uterine stroma of ovariectomised rats have been studied over the 24 hours following an injection of progesterone. Autoradiographically, the rate of incorporation of [3H]lysine falls 1 hour after progesterone, rising progressively at 3 and 7 hours to levels higher than in control animals. A gradient of grain density is evident at 1, 3 and 7 hours, higher in regions adjacent to luminal epithelium than near the muscularis. No such gradient appears relative to glandular epithelium. Morphometric studies show that the array of stromal cells 24 hours after progesterone is similar to that in control rats, except that cells near the luminal epithelium are larger in the former group. At intermediate times, changes in cell size and spacing occur throughout the stroma in patterns that vary with distance from luminal epithelium. The organisation and activity of cells in the uterine stroma change in response to progesterone in a manner conditioned by their position in the uterus, in particular by their distance from luminal epithelium.

Cell organization in the stroma of the rat uterus. I. The ovariectomised rat.

Wischik CM, Rogers AW

J Anat 1982 Dec;135(4):707-18

Stromal cell interactions in the rat uterus as influenced by oestrogen and in intrauterine device.

Wischik CM, Rogers AW

Theriogenology 1977 Oct;8(4):144