Different pathways of molecular pathophysiology underlie cognitive and motor tauopathy phenotypes in transgenic models for Alzheimer's disease and frontotemporal lobar degeneration.

A poorly understood feature of the tauopathies is their very different clinical presentations. The frontotemporal lobar degeneration (FTLD) spectrum is dominated by motor and emotional/psychiatric abnormalities, whereas cognitive and memory deficits are prominent in the early stages of Alzheimer's disease (AD). We report two novel mouse models overexpressing different human tau protein constructs. One is a full-length tau carrying a double mutation [P301S/G335D; line 66 (L66)] and the second is a truncated 3-repeat tau fragment which constitutes the bulk of the PHF core in AD corresponding to residues 296-390 fused with a signal sequence targeting it to the endoplasmic reticulum membrane (line 1; L1). L66 has abundant tau pathology widely distributed throughout the brain, with particularly high counts of affected neurons in hippocampus and entorhinal cortex. The pathology is neuroanatomically static and declines with age. Behaviourally, the model is devoid of a higher cognitive phenotype but presents with sensorimotor impairments and motor learning phenotypes. L1 displays a much weaker histopathological phenotype, but shows evidence of neuroanatomical spread and amplification with age that resembles the Braak staging of AD. Behaviourally, the model has minimal motor deficits but shows severe cognitive impairments affecting particularly the rodent equivalent of episodic memory which progresses with advancing age. In both models, tau aggregation can be dissociated from abnormal phosphorylation. The two models make possible the demonstration of two distinct but nevertheless convergent pathways of tau molecular pathogenesis. L1 appears to be useful for modelling the cognitive impairment of AD, whereas L66 appears to be more useful for modelling the motor features of the FTLD spectrum. Differences in clinical presentation of AD-like and FTLD syndromes are therefore likely to be inherent to the respective underlying tauopathy, and are not dependent on presence or absence of concomitant APP pathology.

Addictive-like behaviours to ultraviolet light among frequent indoor tanners.

BACKGROUND: Frequent, purposeful exposure to ultraviolet (UV) light may induce a compulsive desire to tan despite the negative consequences being known, suggesting a behavioural complex similar to addictive disorders. AIM: To assess the presence of addictive-like behaviours in subjects using indoor tanning salons. METHODS: Subjects (n = 100) were surveyed by two questionnaires: a modified CAGE questionnaire to assess behaviours consistent with problem tanning and a modified Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) ('substance dependence' criteria) to assess behaviours consistent with a dependence-like disorder. RESULTS: In total, 41% of subjects met criteria consistent with a 'tanning addictive disorder', and an additional 33% met criteria for problematic tanning behaviour based on the modified CAGE criteria or subthreshold criteria on the modified DSM-IV criteria. Female gender and early age of onset were associated with meeting tanning addiction criteria. CONCLUSION: A high percentage of subjects who tan frequently in indoor salons experience behaviours and consequences to their tanning consistent with other identified addictive disorders.

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.

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.

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

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.

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 Alzheimer's disease.

Evolution of a homopurine-homopyrimidine pentanucleotide repeat sequence upstream of the human inducible nitric oxide synthase gene.

We have identified a highly polymorphic pentanucleotide repeat (CCTTT)n within the 5'-putative promoter region of the human inducible nitric oxide synthase gene (iNOS, NOS2). Using a pair of specific primers derived from the human iNOS gene, we have also amplified this iNOS repeat in DNA from the following species: chimpanzee, gorilla, orangutan and macaque. As is found in man, both chimpanzees and gorillas are polymorphic at this locus. In contrast, the locus is monomorphic in macaques and orangutans. While the average number of repeats is similar in gorilla and man, there are considerably fewer repeats in chimpanzees. A comparison of the sequences flanking the (CCTTT)n repeats among these closely related species demonstrates the presence of long stretches of homopurine-homopyrimidine residues. Similar polypurine/polypyrimidine stretches have been identified in the promoter regions of a number of other vertebrate genes where they have been associated with transcriptional regulation, although a role for the (CCTTT)n repeat array in the human iNOS gene has not yet been demonstrated.

Lack of an association of estrogen receptor alpha gene polymorphisms and transcriptional activity with Alzheimer disease.

BACKGROUND: Long-term cognitive decline in postmenopausal women is associated with aging and Alzheimer disease (AD). Estrogen replacement therapy has been reported to reduce the risk of developing AD. The distribution of estrogen receptors (ERs) in neurons overlaps that of the brain neurons known to develop AD. Estrogen increases the secretion and metabolism of amyloid precursor protein, may help synapse formation, and is reported to protect neurons from toxins. Restriction fragment length polymorphisms (RFLPs) of the ERalpha gene at intron 1 and exon 2 were associated with a low bone mineral density in postmenopausal women and also with AD in a Japanese population. OBJECTIVE: To determine whether ERalpha gene polymorphisms are associated with transcriptional activity and AD. METHODS: A luciferase reporter assay analyzed enhancer activity of the ERalpha gene at intron 1 and exon 2. This activity was evaluated according to the RFLPs. The RFLPs of the ERalpha gene were determined in Japanese patients clinically diagnosed as having AD, white patients diagnosed as having AD at autopsy, and corresponding healthy control subjects. The RFLPs were also evaluated for the contribution of the ERalpha gene RFLPs to AD. RESULTS: We found weak (about 2-fold) enhancer activity of the ERalpha gene, which differed among RFLPs. Although there were racial differences in these polymorphisms, we could not confirm the previously reported association between ERalpha gene polymorphisms and AD. CONCLUSION: Regulatory element of the ERalpha gene was found in intron 1, but we found no association between ERalpha gene polymorphisms and AD.

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.

Accumulation of C-terminally truncated tau protein associated with vulnerability of the perforant pathway in early stages of neurofibrillary pathology in Alzheimer's disease.

Neurofibrillary pathology is a characteristic hallmark of Alzheimer's disease that is closely correlated with cognitive decline. We have analysed the density and distribution of neurofibrillary tangles (NFTs) that are immunoreactive with the monoclonal antibody (mAb) 423 in a prospectively analysed population of Alzheimer's disease (AD) cases and age-matched controls. NFTs were examined in allocortical and isocortical areas and correlated with Braak pathological stage and clinical severity of dementia. The mAb 423 was used as it recognises a C-terminally truncated tau fragment that is a major constituent of NFTs. Our results show that extracellular NFTs and, to a lesser extent, intracellular NFTs, correlated significantly with both Braak stages and the clinical index of severity. Furthermore, a differential distribution of the two types of tangles indicates that layer II of the entorhinal cortex and the transentorhinal area are particularly vulnerable to neurofibrillary degeneration. These areas serve as a point of connection between isocortex and hippocampus. Our findings, therefore, suggest that the perforant pathway may be substantially affected by the accumulation of truncated tau protein in AD and that this represents a neuropathological predictor for the clinical severity of dementia. When neurofibrillary pathology was examined by combined labelling with mAbs 423 and Alz-50 and the dye thiazin red, we were able to demonstrate various stages of tau aggregation. The different stages may represent a sequence of conformational changes that tau proteins undergo during tangle formation in the allocortex during the early development of dementia in AD.

Two actin binding proteins, actin depolymerizing factor and cofilin, are associated with Hirano bodies.

Hirano bodies are intracellular, paracrystalline, rod-like structures which contain actin, tropomyosin, vinculin, alpha-actinin, amyloid beta-protein precursor and several microtubule associated proteins (MAPs). These bodies are observed more frequently in the elderly and in a number of neurodegenerative diseases including Alzheimer's disease. Many of the proteins known to be associated with Hirano bodies are actin binding proteins. We present immunohistological evidence that actin depolymerizing factor (ADF) and cofilin, two closely related proteins that bind and sever actin filaments, are also components of Hirano bodies. However, we could detect no difference in the levels of expression of either ADF or cofilin in the hippocampal tissue from normal individuals and Alzheimer's disease patients.

Glycation: a pathological modification in neuropathies?: a hypothesis.

A common feature of a number of neuropathies is the formation of characteristic histopathological lesions of neural amyloid. Although the major components of many of these lesions have been identified, the nature of the modifications of these normal cellular proteins that lead to amyloidogenesis remains elusive. The purpose of this article is to introduce the hypothesis that protein glycation might account for the modifications of normal cellular proteins leading to amyloid formation neuropathogenesis.

The CCTTT polymorphism in the NOS2A gene is associated with dementia with Lewy bodies.

We report the analysis of the allele distribution of a (CCTTT)n pentanucleotide repeat within the promoter region of the NOS2A gene in DNA samples from patients with autopsy confirmed Alzheimer's disease (AD) and dementia with Lewy bodies (DLB) type. A significant difference was observed in the allelic distribution between the control group and the DLB group (chi2 = 15.175, df = 5; p<0.01), with an increased occurrence of the eight and nine repeat alleles, and a marked under representation of the 11 repeat allele. Genotype frequencies in the DLB group also differed significantly from controls (p<0.012). These results suggest that variations in the NOS2A gene may predispose to the development of DLB.

Characterisation of a monoclonal antibody and its use in the immunoaffinity purification of penicillin-binding protein 2' of methicillin-resistant Staphylococcus aureus.

The additional penicillin-binding protein (PBP) 2' that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been purified by affinity chromatography using monoclonal antibodies. Monoclonal antibody 1/423.10.351 reacted in ELISA with detergent extracts of membranes from resistant organisms, but not in immunoblots with PBP 2' separated by SDS-PAGE. Immunoprecipitation experiments showed that antibody 1/423.10.351 reacted with PBP 2' in detergent extracts. The latter antibody, covalently coupled to protein A-Sepharose through the Fc region, served as an affinity matrix to purify PBP 2'. The PBP was detected in immunoblots using a second monoclonal antibody, 2/401.43. Conjugation of this antibody with alkaline phosphatase afforded more rapid detection of PBP 2' for the immunological detection of PBP 2' both in affinity-purified fractions and in resistant strains.

Immunological detection of penicillin-binding protein 2' in clinical isolates of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis.

The additional penicillin-binding protein (PBP 2') that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been detected immunologically in strains from a variety of world-wide locations. This additional protein has also been definitively identified both immunologically and as a PBP in methicillin-resistant strains of S. epidermidis (MRSE). The assay described is rapid, specific and sensitive and has been used to detect PBP 2' in S. haemolyticus but not in beta-lactam resistant Streptococci.

Apolipoprotein E type epsilon 4 allele frequency is not increased in patients with sporadic inclusion-body myositis.

The apolipoprotein E genotype was determined for 11 patients with sporadic inclusion-body myositis. Seven cases had the genotype epsilon 3/epsilon 3, the other four cases, epsilon 3/epsilon 4. The frequency of the epsilon 4 allele in this group of patients (0.182) was not significantly increased compared with elderly controls (0.147; n = 58), in contrast to Alzheimer's disease in which there was a significant increase (0.328; n = 67). The epsilon 2 allele was not found in any of the 11 sporadic inclusion-body myositis patients and its frequency was decreased in Alzheimer's disease. Despite certain pathological similarities that exist between inclusion body myositis and Alzheimer's disease, their association with particular apolipoprotein E genotypes is distinct.

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

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.

Loss of synaptic but not cytoskeletal proteins in the cerebellum of chronic schizophrenics.

The pathobiology of schizophrenia is poorly understood, and many neuroanatomical domains have been considered to underlie the pathophysiology of the disease. There is considerable clinical and neuroradiological evidence to support cerebellar involvement in the schizophrenic illness. We have analysed the changes in synaptic and cytoskeletal proteins in the cerebellum associated with schizophrenia. The cerebellar expression of tau and MAP2 proteins is similar in schizophrenia to that detected in age-matched controls, whereas the level of SNAP-25 is significantly depleted in the schizophrenic cerebellum. Other synaptic proteins, such as synaptophysin and syntaxin, are not affected. This provides evidence that alterations of the cerebellar synaptic network occur in schizophrenia. These changes may influence cerebellar-forebrain connections, especially those with the frontal lobes, and give rise to the cognitive dysmetria that is characteristic of the clinical phenotype in schizophrenia.

Presenilin-1 intron 8 polymorphism is not associated with autopsy-confirmed late-onset Alzheimer's disease.

Mutations in the presenilin-1 (PS-1) gene may account for the majority of familial early-onset Alzheimer's disease (EOAD) cases. However, there is controversy as to whether the bi-allelic intron 8 PS-1 polymorphism plays a role in late-onset AD (LOAD). As previous association studies with this polymorphism have all investigated clinically diagnosed LOAD cases, we have analysed the frequency of the PS-1 intronic polymorphism in a series of autopsy-confirmed early- (n = 54) and late-onset (n = 199) cases and a large control population of non-demented, aged individuals (n = 215). Our sample size should have had the power to reveal effects of the size previously reported for the PS-1 polymorphism, but we detected no significant increase in the 1/1 risk genotype distribution in EOAD or LOAD cases. Thus, we have been unable to find an association between the PS-1 intronic polymorphism and early- or late-onset AD within this autopsy-confirmed population.

Lowry protein assay containing sodium dodecyl sulfate in microtiter plates for protein determinations on fractions from brain tissue.

A modified Lowry protein assay which contains sodium dodecyl sulfate (Markwell et al., 1978, Anal. Biochem. 87, 207-210) has been adapted for use in 96-well microtiter plates. A spectrophotometer interfaced with a computer is used to plot the standard curve and calculate the protein content of each sample. The method is suitable for measuring 4-40 micrograms of protein/assay in a volume of 50 microliters. There was no need for prior solubilization of proteins which had been extracted from brain tissue. Test samples, up to 21 in triplicate, can be tested on a single microtiter plate plus standards. Alternatively, further samples can be tested separately on extra plates using the standard curve from the original plate for calculations.