Biomedical subjects

B H Anderton

Publications and source records attributed to B H Anderton.

At least 19 recordsLinked to original sources

PubMed2004

Differential involvement and heterogeneous phosphorylation of tau isoforms in progressive supranuclear palsy.

We found previously that aggregated insoluble tau protein in progressive supranuclear palsy (PSP) brains exhibits a heterogeneous pattern that is not segregated by the type of clinical presentation. Here we have investigated tau isoform composition from 20 PSP cases and found marked variation between different brains. Cases were classified into three groups, each comprising essentially of (1) 1N4R; (2) 1N4R and 1N3R; or (3) 1N4R, 1N3R and 0N4R tau isoforms. There was also an absence of a simple relationship between isoform composition and the pattern of insoluble tau before dephosphorylation. We conclude that there is distinct molecular heterogeneity in the involvement of tau isoforms in the tau pathology in PSP.

Aged↗

PubMed2003

Mutant presenilin 1 proteins induce cell death and reduce tau-dependent processes outgrowth.

The expression of familial Alzheimer's disease mutants of presenilin-1 (PS1) proteins has been observed to induce cell death in cellular systems. To investigate how this phenomenon might be associated to alterations of the microtubule network, we have studied the effect of wild-type and mutant (C263R, P264L and delta9) PS1 proteins expression on the formation of microtubule-dependent processes outgrowth and the association of PS1 to the insoluble cytoskeletal fraction in a cell line expressing the tau microtubule-associated protein. Expression of wild-type and mutant PS1 was associated with increased cell death, most marked for the P264L and delta9 mutants. The three PS1 mutants induced a significant reduction of the length of cell processes. These effects were not associated to a change in tau phosphorylation. However, the mutant PS1 proteins increased the proportion of insoluble tau in the cytoskeletal fraction and they were concentrated in the same fraction. These results suggest that PS1 proteins interact with the microtubule network, affect its organization and that this phenomenon, more marked for the PS1 mutants, might play a role in the cell dysfunction induced by mutant PS1 proteins.

Animals↗

PubMed2003

Apolipoprotein E (apoE) uptake and distribution in mammalian cell lines is dependent upon source of apoE and can be monitored in living cells.

As part of investigations of the cellular uptake of apolipoprotein E (apoE) relevant to Alzheimer's disease we have found that different preparations of apoE are handled differently by cells expressing the LDL-receptor. Comparing recombinant, cellular and native apoE, complexed with different preparations of lipid we find that only cellular and native apoE enter a vesicular compartment. Some, but not all of these apoE containing vesicles are lysosomes. In order to further examine the intracellular fate of apoE we demonstrate that apoE-Enhanced green fluorescent protein chimeric protein can be taken up from medium by recipient cells and tracked within these cells for extended periods.

Animals↗

PubMed2002

The complex relationship between soluble and insoluble tau in tauopathies revealed by efficient dephosphorylation and specific antibodies.

Phosphorylated tau is deposited as insoluble inclusion bodies in the tauopathies. We have used a new efficient method to dephosphorylate tau extracted from control and tauopathy brain. In some tauopathies, including Alzheimer's disease and progressive supranuclear palsy, the pattern of insoluble tau isoforms reflected that of soluble tau. In contrast, in corticobasal degeneration, Pick's disease, and some forms of fronto-temporal dementia, specific tau isoforms were selectively sequestered into insoluble inclusion-forming tau. Therefore the overall expression of individual tau isoforms does not predict which tau isoforms are deposited in all tauopathies and different mechanisms must operate that result in the deposition of specific tau isoforms.

Amino Acid Sequence↗

PubMed2002

Pathological, clinical and genetic heterogeneity in progressive supranuclear palsy.

We have identified two groups of patients with clinically typical and atypical, pathologically diagnosed progressive supranuclear palsy (PSP), and investigated their genetic and molecular pathological characteristics. Those with clinically typical PSP are more likely to have the PSP susceptibility genotype and to have the deposition of PSP-type hyperphosphorylated tau protein. The clinically atypical PSP group contains a number of different clinical syndromes, including an L-dopa unresponsive bradykinetic syndrome and a clinical syndrome closely resembling idiopathic Parkinson's disease. The clinically atypical PSP group are less likely to have the PSP susceptibility genotype and often have the deposition of Alzheimer's disease paired helical filament type hyperphosphorylated tau. This study suggests that the tau PSP susceptibility genotype is most strongly associated with clinically typical PSP. Neurofibrillary tangle parkinsonian disorders, which pathologically resemble PSP but involve the deposition of Alzheimer's disease-type tau often without involvement of the tau susceptibility genotype, need to be distinguished for diagnostic and research purposes.

Aged↗

PubMed2001

Presenilin 1 independently regulates beta-catenin stability and transcriptional activity.

Presenilin 1 (PS1) regulates beta-catenin stability; however, published data regarding the direction of the effect are contradictory. We examined the effects of wild-type and mutant forms of PS1 on the membrane, cytoplasmic, nuclear, and signaling pools of endogenous and exogenous beta-catenin by immunofluorescence microscopy, subcellular fractionation, and in a transcription assay. We found that PS1 destabilizes the cytoplasmic and nuclear pools of beta-catenin when stabilized by Wnt or Dvl but not when stabilized at lower levels of the Wnt pathway. The PS1 mutants examined were less able to reduce the stability of beta-catenin. PS1 also inhibited the transcriptional activity of endogenous beta-catenin, and the PS1 mutants were again less inhibitory at the level of Dvl but showed a different pattern of inhibition toward transcription below Dvl. The transcriptional activity of exogenously expressed wild-type beta-catenin and two mutants, DeltaN89beta-catenin and DeltaSTbeta-catenin, were also inhibited by wild-type and mutant PS1. We conclude that PS1 negatively regulates the stability and transcriptional activity of beta-catenin at different levels in the Wnt pathway, that the effect on transcriptional activity appears to be independent of the GSK-3beta mediated degradation of beta-catenin, and that mutations in PS1 differentially affect the stability and transcriptional activity of beta-catenin.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Dishevelled regulates the metabolism of amyloid precursor protein via protein kinase C/mitogen-activated protein kinase and c-Jun terminal kinase.

Alzheimer's disease (AD) is a disorder of two pathologies: amyloid plaques, the core of which is a peptide derived from the amyloid precursor protein (APP), and neurofibrillary tangles composed of highly phosphorylated tau. Protein kinase C (PKC) is known to increase non-amyloidogenic alpha-secretase cleavage of APP, producing secreted APP (sAPPalpha), and glycogen synthase kinase (GSK)-3beta is known to increase tau phosphorylation. Both PKC and GSK-3beta are components of the wnt signaling cascade. Here we demonstrate that overexpression of another member of this pathway, dishevelled (dvl-1), increases sAPPalpha production. The dishevelled action on APP is mediated via both c-jun terminal kinase (JNK) and protein kinase C (PKC)/mitogen-activated protein (MAP) kinase but not via p38 MAP kinase. These data position dvl-1 upstream of both PKC and JNK, thereby explaining the previously observed dual signaling action of dvl-1. Furthermore, we show that human dvl-1 and wnt-1 also reduce the phosphorylation of tau by GSK-3beta. Therefore, both APP metabolism and tau phosphorylation are potentially linked through wnt signaling.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Functional differences of tau isoforms containing 3 or 4 C-terminal repeat regions and the influence of oxidative stress.

We report functional differences between tau isoforms with 3 or 4 C-terminal repeats and a difference in susceptibility to oxidative conditions, with respect to the regulation of microtubule dynamics in vitro and tau-microtubule binding in cultured cells. In the presence of dithiothreitol in vitro, a 3-repeat tau isoform promotes microtubule nucleation, reduces the tubulin critical concentration for microtubule assembly, and suppresses dynamic instability. Under non-reducing conditions, threshold concentrations of 3-repeat tau and tubulin exist below which this isoform still promotes microtubule nucleation and assembly but fails to reduce the tubulin critical concentration or suppress dynamic instability; above these threshold concentrations, amorphous aggregates of 3-repeat tau and tubulin can be produced at the expense of microtubule formation. A 4-repeat tau isoform is less sensitive to the oxidative potential of the environment, behaving under oxidative conditions similarly to the 3-repeat isoform under reducing conditions. Under conditions of oxidative stress, in Chinese hamster ovary cells stably expressing either 3- or 4-repeat tau, 3-repeat tau disassociates from microtubules more readily than the 4-repeat isoform, and tau-containing high molecular weight aggregates are preferentially observed in lysates from the Chinese hamster ovary cells expressing 3-repeat tau, indicating greater susceptibility of 3-repeat tau to oxidative conditions, compared with 4-repeat tau in vivo.

Animals↗

PubMed2001

Effects of FTDP-17 mutations on the in vitro phosphorylation of tau by glycogen synthase kinase 3beta identified by mass spectrometry demonstrate certain mutations exert long-range conformational changes.

In vitro phosphorylation of recombinant wild-type 2N4R tau and FTDP-17 exonic mutant forms P301L, V337M and R406W by glycogen synthase kinase 3beta (GSK3beta) was examined by two dimensional phosphopeptide mapping analysis on thin layer cellulose plates. Comparison of these peptide maps with those generated from wild-type 1N4R tau isoform from which the phosphopeptide constituents and sites of phosphorylation had been determined previously, enabled us to monitor directly changes in phosphorylation of the individual tau proteins. No differences were found in the phosphorylation of wild-type, P301L or V337M tau by GSK3beta but the R406W mutant showed at least two clear differences from the other three tau proteins. The peptides, identified by mass spectrometry corresponding to phosphorylation at both threonine 231 and serine 235 (spot 3), serines 396, 400 and 404 (spot 6a) and serines 195 and 199 (spot 6b) were absent from the R406W peptide map. The findings imply that the R406W mutation in tau exerts long-range conformational effects on the structure of tau.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed2001

Increase of adenomatous polyposis coli immunoreactivity is a marker of reactive astrocytes in Alzheimer's disease and in other pathological conditions.

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are responsible for colon cancer in familial adenomatous polyposis coli and in many sporadic colorectal tumors. The product of the APC gene is also essential for normal development and is expressed in the adult brain. We have investigated the immunocytochemical localization of APC in the temporal cortex and hippocampus of normal human brain, in Alzheimer's disease (AD) and in several other neuropathological conditions. APC was expressed in neuronal cell bodies and dendrites both in control subjects and in patients with different diseases. In addition, a high APC expression was observed in a proportion of fibrillary and glial fibrillary acidic protein-positive astrocytes in AD. Furthermore, in AD the proportion of APC-positive astrocytes was higher in astrocytes associated with beta-amyloid (Abeta) deposits in senile plaques than in astrocytes not associated to Abeta deposits. APC-positive astrocytes were also observed in control cases, in diffuse Lewy body disease, in Creutzfeldt-Jacob disease, in HIV encephalitis and around cerebral infarcts. Tumoral astrocytes in pilocytic astrocytoma and in glioblastoma were also strongly APC positive. APC was not detected in cultured astroglial cells. These results indicate that APC expression is upregulated in astrocytes following their activation by several types of pathological insults and is a newly identified molecular characteristic of the reactive phenotype of astrocytes, possibly related to the control of cell proliferation. In addition, it also suggests that Abeta, and/or the inflammatory process associated with Abeta deposits, is responsible for a preferential increase of APC expression in astrocytes in AD.

Adenomatous Polyposis Coli Protein↗

PubMed2001

Regional distribution of amyloid-Bri deposition and its association with neurofibrillary degeneration in familial British dementia.

Familial British dementia (FBD), pathologically characterized by cerebral amyloid angiopathy (CAA), amyloid plaques, and neurofibrillary degeneration, is associated with a stop codon mutation in the BRI gene resulting in the production of an amyloidogenic fragment, amyloid-Bri (ABri). The aim of this study was to assess the distribution of ABri fibrillar and nonfibrillar lesions and their relationship to neurofibrillary pathology, astroglial and microglial response using immunohistochemistry, confocal microscopy, and immunoelectron microscopy in five cases of FBD. Abnormal tau was studied with immunoblotting. We present evidence that ABri is deposited throughout the central nervous system in blood vessels and parenchyma where both amyloid (fibrillar) and pre-amyloid (nonfibrillar) lesions are formed. Ultrastructurally amyloid lesions appear as bundles of fibrils recognized by an antibody raised against ABri, whereas Thioflavin S-negative diffuse deposits consist of amorphous electron-dense material with sparse, dispersed fibrils. In contrast to nonfibrillar lesions, fibrillar ABri is associated with a marked astrocytic and microglial response. Neurofibrillary tangles and neuropil threads occurring mainly in limbic structures, are found in areas affected by all types of ABri lesions whereas abnormal neurites are present around amyloid lesions. Immunoblotting for tau revealed a triplet electrophoretic migration pattern. Our observations confirm a close link between ABri deposition and neurodegeneration in FBD.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Tau proteins with frontotemporal dementia-17 mutations have both altered expression levels and phosphorylation profiles in differentiated neuroblastoma cells.

The inherited form of frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) has been attributed to mutations in the tau gene. Pathologically, affected FTDP-17 brains share tau aggregates with other tauopathies, the most common being Alzheimer's disease. FTDP-17 mutations may therefore affect tau function leading to tau aggregation and cell loss. Interaction of tau with microtubules is thought to be regulated by phosphorylation. Investigating FTDP-17 mutations transiently expressed as enhanced green fluorescent protein (EGFP)-tagged proteins for the first time in differentiated neuronal cells, we found that two out of three missense mutations showed surprisingly decreased phosphorylation at the pathologically relevant S202/T205 site, mutant EGFP-tau being completely dephosphorylated in most cells. Moreover, phosphorylation at the S396/S404 site was moderately decreased for all mutant isoforms. Although microtubule integrity was not affected, with all mutants tested we demonstrated an increase in cellular tau protein level, some of which is microtubule-bound. Further enhancing this EGFP-tau accumulation by inhibition of tau degradation resulted in the previously less phosphorylated mutant EGFP-tau becoming highly phosphorylated. We conclude that the missense tau mutations primarily result in an excess of neuronal tau, which may interfere with important cellular functions such as axonal transport.

Alzheimer Disease↗

PubMed2001

Sites of phosphorylation in tau and factors affecting their regulation.

The microtubule-associated protein, tau, is the principal component of paired helical filaments (PHFs) in Alzheimer's disease. PHF-tau is highly phosphorylated and a total of 25 sites of phosphorylation have so far been identified. Many of these sites are serine or threonine residues that are immediately followed in the sequence by proline residues, and hence are candidate phosphorylation sites for proline-directed kinases. In vitro, glycogen synthase kinase-3 (GSK-3), extracellular signal-related kinase-1 and -2, and mitogen-activated protein kinases, p38 kinase and c-jun N-terminal kinase, all phosphorylate many of these sites, although with different efficiencies for particular sites. Phosphorylation studies in transfected cells and neurons show that GSK-3 phosphorylates tau more extensively than do these other proline-directed kinases. Mutations in tau have been shown to affect in vitro phosphorylation of tau by GSK-3. The Arg406-->Trp (R406W) tau mutation also affects tau phosphorylation in cells.

Alzheimer Disease↗

PubMed2001

Neurofibrillary tangles and tau phosphorylation.

Neurofibrillary tangles (NFTs) are a characteristic neuropathological lesion of Alzheimer's disease (AD). They are composed of a highly-phosphorylated form of the microtubule-associated protein tau. We are investigating the relationship between NFTs and microtubule stability and how tau phosphorylation and function is affected in transgenic models and by co-expression with beta-amyloid precursor protein and presenilins. In most NFT-bearing neurons, we observed a strong reduction in acetylated alpha-tubulin immunoreactivity (a marker of stable microtubules) and a reduction of the in situ hybridization signal for tubulin mRNA. In transfected cells, mutated tau forms (corresponding to tau mutations identified in familial forms of frontotemporal dementias linked to chromosome 17) were less efficient in their ability to sustain microtubule growth. These observations are consistent with the hypothesis that destabilization of the microtubule network is an important mechanism of cell dysfunction in Alzheimer's disease. The glycogen synthase kinase-3 beta (GSK-3 beta) generates many phosphorylated sites on tau. We performed a neuroanatomical study of GSK-3 beta distribution showing that developmental evolution of GSK-3 beta compartmentalization in neurons paralleled that of phosphorylated tau. Studies on transfected cells and on cultured neurons showed that GSK-3 beta activity controls tau phosphorylation and tau functional interaction with microtubules. Tau phosphorylation was not affected in neurons overexpressing beta-amyloid precursor protein. Transgenic mice expressing a human tau isoform and double transgenic animals for tau and mutated presenilin 1 have been generated; a somatodendritic accumulation of phosphorylated transgenic tau proteins, as observed in the pretangle stage in AD, has been observed but NFTs were not found, suggesting that additional factors might be necessary to induce their formation.

Alzheimer Disease↗

PubMed2000

Molecular cloning and expression analysis of human glycogen synthase kinase-3 alpha promoter.

Human glycogen synthase kinase-3 alpha (GSK-3 alpha) is a serine/threonine kinase that phosphorylates a variety of cytoplasmic and nuclear proteins. It also phosphorylates components of the neuronal cytoskeleton including tau and neurofilament heavy chain. Hyperphosphorylated tau is found in neurofibrillary tangles, a hallmark of Alzheimer's disease and aberrant phosphorylation of neurofilament heavy chain is observed in motor neuron disease. Alterations in GSK-3 alpha activity may therefore contribute to the disease process in these disorders. As a first step to understand the transcriptional regulation of GSK-3 alpha, a 2-kb (p-1751/+243) DNA fragment upstream of the GSK-3 alpha initiation codon was obtained from a YAC clone and characterised. Using primer extension assays, a putative transcriptional start site was located to a G nucleotide 244 bp upstream of the ATG codon. Several transcription factor-binding sites were identified on the promoter region, but no TATA-like element was located close to the start site. Deletion mutants of the 2-kb DNA fragment were generated and fused to a promoterless chloramphenicol acetyltransferase (CAT) gene. Transfection study in a neuroblastoma cell line revealed the 1-kb (p-719/+243) fragment carried strong promoter activity, while the 2-kb construct that contains an Alu-like sequence was only 50% active.

Alu Elements↗

PubMed2000

Differential effects of apolipoprotein E isoforms on phosphorylation at specific sites on tau by glycogen synthase kinase-3 beta identified by nano-electrospray mass spectrometry.

Previously published data have shown an allele-specific variation in the in vitro binding of apolipoprotein E (apoE) to tau, which prompted the hypothesis that apoE binding may protect tau from phosphorylation, apoE3 being more efficient than apoE4. We have, therefore, investigated the effects of apoE on tau phosphorylation in vitro by the proline-directed kinase, glycogen synthase kinase (GSK)-3 beta. The phosphopeptide maps of tau alone, of tau with apoE3 and of tau with apoE4 were very similar. When apoE2 was present a further four spots were evident. Additionally, of the 15 peptides phosphorylated in the presence or absence of apoE, subtle differences, some isoform-specific, in the relative amounts of phosphorylation were observed.

Apolipoprotein E2↗

PubMed2000

Glutamate slows axonal transport of neurofilaments in transfected neurons.

Neurofilaments are transported through axons by slow axonal transport. Abnormal accumulations of neurofilaments are seen in several neurodegenerative diseases, and this suggests that neurofilament transport is defective. Excitotoxic mechanisms involving glutamate are believed to be part of the pathogenic process in some neurodegenerative diseases, but there is currently little evidence to link glutamate with neurofilament transport. We have used a novel technique involving transfection of the green fluorescent protein-tagged neurofilament middle chain to measure neurofilament transport in cultured neurons. Treatment of the cells with glutamate induces a slowing of neurofilament transport. Phosphorylation of the side-arm domains of neurofilaments has been associated with a slowing of neurofilament transport, and we show that glutamate causes increased phosphorylation of these domains in cell bodies. We also show that glutamate activates members of the mitogen-activated protein kinase family, and that these kinases will phosphorylate neurofilament side-arm domains. These results provide a molecular framework to link glutamate excitotoxicity with neurofilament accumulation seen in some neurodegenerative diseases.

Axonal Transport↗

PubMed2000

The function of the microtubule-associated protein tau is variably modulated by graded changes in glycogen synthase kinase-3beta activity.

The microtubule-associated protein tau favors microtubule nucleation and stabilization and plays a role in the elongation of axons. We have investigated the ability of glycogen synthase kinase-3beta (GSK-3beta) to control tau-induced processes outgrowth. Tau-transfected Chinese hamster ovary (CHO) cells developed processes containing microtubule bundles after cytochalasin treatment, but a significant reduction in the number of cells harboring processes was observed in tau/GSK-3beta-co-transfected cells. Lithium, an inhibitor of GSK-3beta, counteracted in a dose-dependent manner this inhibitory effect of GSK-3beta. These findings suggest that GSK-3beta modulates in a graded manner the ability of tau to control the microtubule-dependent induction of cell processes.

Animals↗