Astrocytes are important mediators of Aß-induced neurotoxicity and tau phosphorylation in primary culture.

Alzheimer's disease (AD) is pathologically characterised by the age-dependent deposition of ß-amyloid (Aß) in senile plaques, intraneuronal accumulation of tau as neurofibrillary tangles, synaptic dysfunction and neuronal death. Neuroinflammation, typified by the accumulation of activated microglia and reactive astrocytes, is believed to modulate the development and/or progression of AD. We have used primary rat neuronal, astrocytic and mixed cortical cultures to investigate the contribution of astrocyte-mediated inflammatory responses during Aß-induced neuronal loss. We report that the presence of small numbers of astrocytes exacerbate Aß-induced neuronal death, caspase-3 activation and the production of caspase-3-cleaved tau. Furthermore, we show that astrocytes are essential for the Aß-induced tau phosphorylation observed in primary neurons. The release of soluble inflammatory factor(s) from astrocytes accompanies these events, and inhibition of astrocyte activation with the anti-inflammatory agent, minocycline, reduces astrocytic inflammatory responses and the associated neuronal loss. Aß-induced increases in caspase-3 activation and the production of caspase-3-truncated tau species in neurons were reduced when the astrocytic response was attenuated with minocycline. Taken together, these results show that astrocytes are important mediators of the neurotoxic events downstream of elevated Aß in models of AD, and suggest that mechanisms underlying pro-inflammatory cytokine release might be an important target for therapy.

Transgenic mouse models of tauopathy in drug discovery.

Tauopathies, including Alzheimer's disease, are neurodegenerative diseases characterized by the deposition of hyperphosphorylated tau protein in the central nervous system, and are the major cause of dementia in later life. Considerable advances have been made in developing mouse models that recapitulate, to varying extents, the development of human tau pathology, and the learning and memory deficits characteristic of some tauopathies. Furthermore, such models have been used to show promising disease-modifying effects in pre-clinical testing of new therapeutics. Various strategies have been utilised to generate mouse models of tauopathies. Some of the most enlightening models developed to date either constitutively or inducibly express pathogenic tau mutations. These animals have been instrumental in defining critical disease-related mechanisms, including the observation that tangles are not the toxic form of tau in disease. Here, we discuss the strengths and weaknesses of well characterised transgenic models that emulate human tauopathy, and include a comprehensive listing of the main phenotypic characteristics of all reported tau transgenic rodents. We summarise the use of tau mice for the development and evaluation of new therapeutic approaches, and their utility in identifying novel drug targets. In addition, we review the parameters to be considered in the development of the next generation of mouse models of tauopathy, aimed at further increasing our understanding of disease aetiology and in evaluating novel treatments.

Quantitative analysis of tau isoform transcripts in sporadic tauopathies.

A number of neurodegenerative diseases, including Alzheimer's disease (AD), are characterized by intraneuronal accumulation of the tau protein. Some forms of FTDP-17 are caused by mutations in the tau gene affecting exon 10 splicing. Therefore, dysregulation of tau pre-mRNA splicing may be a contributing factor to sporadic tauopathies. To address this question, we devised a real-time RT-PCR strategy based on the use of a single fluorogenic probe to evaluate the ratio between tau isoforms containing or lacking exon 10 (4R/3R ratio) in post-mortem brain samples. We found a two- to six-fold increase in the 4R/3R ratio in cases of FTDP-17 linked to a splice site mutation, hence confirming the validity of the strategy. The difference in the 4R/3R ratio in the superior temporal and superior frontal gyri between AD and control brains was not statistically significant. Similarly, there was no significant difference in the 4R/3R ratio between Pick's disease cases and controls, indicating that the predominance of tau3R protein in PiD reflects post-translational modifications of specific isoforms. This study indicates that post-translational events are likely to be the main factors controlling tau isoform composition in sporadic tauopathies and highlights the benefit of quantitative RT-PCR in the assessment of splicing abnormalities in tauopathies.

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.

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.

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.

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.

Autopsy-confirmed familial early-onset Alzheimer disease caused by the l153V presenilin 1 mutation.

BACKGROUND: Three affected individuals are described from a small English kindred with early-onset autosomal dominant familial Alzheimer disease (FAD) caused by a leucine-to-valine change at codon 153 (L153V) of the presenilin 1 (PSEN1) gene. METHODS: Clinical information on the pedigree was collected directly from family members and from hospital records. Samples of DNA were screened by means of direct sequencing of all coding exons of PSEN1. One patient underwent neuropathological examination. RESULTS: Mean age at onset of symptoms was 35.3 years (95% confidence interval [CI], 34.6-36.0 years); at death, 44.0 years (95% CI, 39.1-48.9 years). Mean duration of illness was 8.3 years (95% CI, 4.7-11.9 years). Myoclonus was a late feature in 1 patient; seizures were not reported in any subjects. Spastic paraparesis and extrapyramidal signs were absent. The neuropsychometric profile of 1 patient showed relatively preserved naming skills in the setting of global cognitive deficits. Results of neuropathological examination demonstrated the signature lesions of Alzheimer disease and the presence of occasional cortical Lewy bodies. CONCLUSIONS: The PSEN1 L153V mutation lies in the main mutation cluster of PSEN1 in the second transmembrane domain. It causes early-onset FAD with clinical features similar to those of other reported FAD pedigrees.

Chicken synucleins: cloning and expression in the developing embryo.

Synucleins comprise a family of small intracellular proteins that have recently attracted considerable attention because of their involvement in human diseases. Mutations of alpha-synuclein has been found in several families with hereditary early-onset Parkinson's disease and accumulation of this protein in characteristic cytoplasmic inclusions is a pathohistological hallmark of several neurodegenerative diseases that have been recently classified as 'alpha;-synucleinopathies' (reviewed in Brain Res. Bull. 50 (1999) 465; J. Neurosci. Res. 58 (1999) 120; Philos. Trans. R. Soc. Lond. Biol. Sci. 354 (1999) 1101; Brain Pathol. 9 (1999) 733). Aggregates of beta-synuclein and persyn (gamma-synuclein) also have been found in dystrophic neurites associated with Parkinson's and other neurodegenerative diseases (Proc. Natl. Acad. Sci. USA 96 (1999) 13450; and our unpublished observations). Moreover, persyn has been implicated in malignization of breast tumours (Cancer Res. 57 (1997) 759; Cancer Res. 59 (1999) 742; Hum. Mol. Genet. 7 (1998) 1417). All synucleins have distinct, although overlapping, patterns of expression in the embryonic, postnatal and adult mammalian nervous systems, suggesting important, although still not clear, biological functions in neuronal developing. Chicken embryo is a unique object for developmental studies that allows in vivo manipulations not always possible for mammalian embryos. Studies of synucleins expression in this model system could shed light on their functions in the developing nervous system. We cloned three chicken synucleins from the embryonic neural cDNA libraries and studied their expression in normal chicken embryonic tissues by Northern and in situ hybridization with specific probes. Our results demonstrate that primary structures and expression patterns of synucleins are similar in birds and mammals, suggesting that conserved function of synucleins is important for embryonic development of vertebrates.

Induction of neuronal death by alpha-synuclein.

The molecular and cellular mechanisms underlying neuronal loss in neurodegenerative diseases are unclear. It is generally thought that aggregation of mutated, abnormally modified or abnormally folded proteins leads to the accumulation of extracellular, intracellular or intranuclear deposits that severely compromise cell physiology, leading to the death of the affected neurons. However, there is growing evidence that neuronal apoptosis in the absence of obvious pathological deposits could have a serious impact on the pathogenesis of neurodegenerative diseases. alpha-Synuclein has been implicated in aetiology and pathogenesis of certain neurodegenerative diseases, although the precise role of this protein in neurodegeneration is uncertain. The normal functions of alpha-synuclein and other members of the synuclein family in the development and function of the nervous system also remain elusive. Here we show that overexpression of wild-type and mutant forms of alpha-synuclein in cultured neurons, but not the closely related persyn (gamma-synuclein), causes apoptosis. These findings suggest that abnormalities of alpha-synuclein metabolism could lead to the neuronal loss occurring in certain forms of neurodegeneration before the formation of characteristic pathological lesions.

New phosphorylation sites identified in hyperphosphorylated tau (paired helical filament-tau) from Alzheimer's disease brain using nanoelectrospray mass spectrometry.

Paired helical filaments (PHFs) are the structural constituents of neurofibrillary tangles in Alzheimer's disease and are composed of hyperphosphorylated forms of the microtubule-associated protein tau (PHF-tau). Pathological hyperphosphorylation of tau is believed to be an important contributor to the destabilisation of microtubules and their subsequent disappearance from tangle-bearing neurons in Alzheimer's disease, making elucidation of the mechanisms that regulate tau phosphorylation an important research goal. Thus, it is essential to identify, preferably by direct sequencing, all of the sites in PHF-tau that are phosphorylated, a task that is incomplete because of the difficulty to date of purifying insoluble PHF-tau to homogeneity and in sufficient quantities for structural analysis. Here we describe the solubilisation of PHF-tau followed by its purification by Mono Q chromatography and reversed-phase HPLC. Phosphopeptides from proteolytically digested PHF-tau were sequenced by nanoelectrospray mass spectrometry. We identified 22 phosphorylation sites in PHF-tau, including five sites not previously identified. The combination of our new data with previous reports shows that PHF-tau can be phosphorylated on at least 25 different sites.

Tau protein in the glial cytoplasmic inclusions of multiple system atrophy can be distinguished from abnormal tau in Alzheimer's disease.

The glial cytoplasmic inclusion (GCI) is a histological hallmark for multiple system atrophy (MSA). These inclusions are in oligodendrocytes, contain microtubular structures of 20-30 nm diameter, and can be labelled immunohistochemically with antibodies to ubiquitin, alphaB-crystallin, alpha- and beta-tubulin, and the microtubule-associated protein tau. GCIs have been compared with neuronal inclusions in other neurodegenerative disorders including the neurofibrillary tangles (NFTs) found in Alzheimer's disease (AD), which also contain tau protein. In order to determine whether the tau protein of GCIs in MSA is similar to that observed in AD we used a panel of antibodies to phosphorylation-independent (SMI51, TP007, TP70), dephosphorylation-dependent (Tau.1), and phosphorylation-dependent antibodies to tau and neurofilaments (AT8, AT180, AT270, SMI31, SMI34, RT97, BF10, 8D8). Immunohistochemistry was performed on paraffin wax-embedded brain tissue of the cerebellum, brainstem, and frontal lobes (Brodmann areas 4/6) of ten clinically and neuropathologically well-characterised cases of MSA, two cases of AD, and two normal controls. The NFTs of the AD cases were labelled with all the phosphorylation-dependent and phosphorylation-independent antibodies and with Tau.1 only after treatment with alkaline phosphatase. In contrast, GCIs were immunolabelled by the phosphorylation-independent antibodies and Tau.1, but not by the phosphorylation-dependent antibodies. These data demonstrate that the tau in GCIs is different from the abnormally phosphorylated tau found in AD and is similar to normal adult tau. The mechanism causing the abnormal accumulation of tau in GCIs remains to be elucidated.

Oxidative stress induces dephosphorylation of tau in rat brain primary neuronal cultures.

Oxidative stress and free radical damage have been implicated in the neurodegenerative changes characteristic of several neurodegenerative diseases, including Alzheimer's disease. There is experimental evidence that the neurotoxicity of beta-amyloid is mediated via free radicals, and as the deposition of beta-amyloid apparently precedes the formation of paired helical filaments (PHF) in Alzheimer's disease, we have investigated whether subjecting primary neuronal cultures to oxidative stress induces changes in the phosphorylation state of the principal PHF protein tau that resemble those found in PHF-tau. Contrary to causing an increase in tau phosphorylation, treatment of neurones with hydrogen peroxide caused a dephosphorylation of tau and so we conclude that oxidative stress is not the direct cause of tau hyperphosphorylation and hence of PHF formation.

Pathological lesions of Alzheimer's disease and dementia with Lewy bodies brains exhibit immunoreactivity to an ATPase that is a regulatory subunit of the 26S proteasome.

MS73 is one of a family of ATPases that act as regulatory subunits of the 26S proteasome. Localisation of this ATPase in histological sections of hippocampus from Alzheimer's disease (AD) and in cingulate gyrus sections of dementia with Lewy bodies (DLB) brains was examined immunohistochemically. In all cases of AD (n = 10) neurofibrillary tangles (NFT), plaque neurites and neuropil threads were immunoreactive for MS73. In seven out of the nine cases of DLB, distinctive MS73-positive structures were detected within cortical Lewy bodies. The association of MS73 with these neuronal abnormalities provides further evidence that proteolytic processing involving the 26S proteasome occurs in lesions of AD and DLB.

Neurodegenerative changes including altered tau phosphorylation and neurofilament immunoreactivity in mice transgenic for the serine/threonine kinase Mos.

Transgenic mice expressing the oncogenic protein-serine/threonine kinase Mos at high levels in the brain display progressive neuronal degeneration and gliosis. Gliosis developed in parallel with the onset of postnatal transgene expression and led to a dramatic increase in the number of astrocytes positive for GFAP, vimentin, and possibly tau. Interestingly, vimentin is normally expressed only in immature or neoplastic astrocytes, but appears to be induced to high levels in Mos-transgenic, mature astrocytes. Mos can activate mitogen activated protein kinase (MAPK) and MAPK has been implicated in Alzheimer-type tau phosphorylation. In the Mos-transgenic brain we found increased levels of phosphorylation at one epitope on tau containing serines 199 and 202 (numbering according to human tau), a pattern similar but not identical to that found in Alzheimer's disease. In addition, Mos-transgenic mice express a novel neurofilament-related protein that might be a proteolytic neurofilament heavy chain degradation product. These results suggest that activation of protein phosphorylation in neurons can result in changes in cytoskeletal proteins that might contribute to neuronal degeneration.

The phosphorylation state of the microtubule-associated protein tau as affected by glutamate, colchicine and beta-amyloid in primary rat cortical neuronal cultures.

The effects of the excitatory amino acid glutamate, the microtubule destabilizing agent colchicine, and beta 25-35-amyloid peptide on the phosphorylation state of tau were studied in rat cortical neurons in primary culture. Using immunocytochemistry and Western-blot analysis, we demonstrated that a proportion of tau in these cultures is normally highly phosphorylated, but most of this tau fraction is dephosphorylated after treatment of the cultures with glutamate or colchicine, but not with beta-amyloid; the glutamate- and colchicine-induced changes in tau phosphorylation commenced before cell death, as assessed by release of lactate dehydrogenase. Dephosphorylation of tau was readily revealed by using the monoclonal antibodies Tau.1 and AT8, which have phosphate-sensitive epitopes that both centre around serine-199 and -202 (numbering of the largest tau isoform). On Western blots and by immunocytochemistry, AT8 labelling strongly decreased after glutamate and colchicine treatments, whereas Tau.1 staining was more intense. Neurofilament monoclonal antibodies, including RT97, 8D8, SMI31 and SMI310, all additionally known to recognize tau in a phosphorylation-dependent manner, also demonstrated that glutamate and colchicine treatments of the cultures induced a dephosphorylation of tau. We also showed immunocytochemically that there is an increase in tau immunoreactivity in neuronal perikarya in response to glutamate and colchicine treatment, and this occurs concomitantly with the dephosphorylation of tau. Treatment of the primary rat cortical neuronal cultures with beta 25-35-amyloid peptide, under conditions which induce neuronal degeneration, did not induce a change in tau phosphorylation, and failed to act synergistically with glutamate to produce an increase in dephosphorylation of tau over that produced by glutamate treatment alone. These findings demonstrate that glutamate and colchicine induce tau dephosphorylation, as opposed to increased tau phosphorylation, which would be more indicative of Alzheimer-type neurodegeneration.

Modulation of PHF-like tau phosphorylation in cultured neurones and transfected cells.

Two cellular systems have been used to investigate the modulation of tau hyperphosphorylation. In the first system, the effects of the excitatory amino acid glutamate, the microtubule destabilising agent colchicine, and beta 25-35-amyloid peptide on tau phosphorylation were studied in rat cortical neurones in primary culture. Using immunocytochemistry and western blot analysis, we demonstrated that tau in these cultures is normally highly phosphorylated, but a proportion becomes rapidly dephosphorylated following treatment of the cultures with glutamate or colchicine. These changes in tau phosphorylation occurred prior to cell death. In the second system, the ability of p42 MAP and p44 MAP kinases, glycogen synthase kinases 3 alpha and 3 beta (GSK-3 alpha and GSK-3 beta) to phosphorylate tau in transfected COS cells was investigated. Both GSK-3 alpha and GSK-3 beta phosphorylated tau to produce a PHF-like state of phosphorylation but the MAP kinases failed to induce such a transformation in tau. These results suggest that aberrant regulation of GSK-3 alpha/beta may be a pathogenic mechanism in Alzheimer's disease.

PHF-tau from Alzheimer's brain comprises four species on SDS-PAGE which can be mimicked by in vitro phosphorylation of human brain tau by glycogen synthase kinase-3 beta.

Extensive in vitro phosphorylation of a purified preparation of control human brain tau consistently produces four rather than, as previously believed, three tau species on SDS-PAGE. The species thus generated are shifted on SDS-PAGE to positions that match those of PHF-tau isolated from Alzheimer's disease brain. A mixture of recombinant human tau isoforms phosphorylated by GSK-3 beta gave similar results to those obtained with control human brain tau. In vitro phosphorylation of the individual recombinant isoforms by GSK-3 beta showed that the four bands of PHF-tau are likely to consist of isoforms 3R,0 alone; 4R,0 with 3R,29; 4R,29 with 3R,58 and 4R,58 alone.

Phosphorylation of tau by cyclic-AMP-dependent protein kinase.

Alzheimer's disease paired helical filaments contain abnormally phosphorylated tau (PHF-tau) which has reduced electrophoretic mobility on sodium dodecyl sulphate polyacrylamide electrophoresis. We have investigated the effects of cyclic-AMP-dependent protein kinase (PKA) on recombinant human tau isoforms and two recombinant tau fragments. PKA phosphorylated tau and reduced its electrophoretic mobility, phosphorylation towards the C-terminus of tau having a major influence on this property. Substitution of serine396 (phosphorylated in PHF-tau) or serine416 (phosphorylated by calcium/calmodulin kinase II) by alanine demonstrated that these are not major sites for PKA phosphorylation. Although the phosphorylated forms of tau generated by PKA are not identical to those of PHF-tau, PKA may be involved in the generation of PHF-tau in Alzheimer's disease via phosphorylation of additional, as yet unidentified, sites on tau.