Diane P. Hanger

Glycogen synthase kinase-3 induces Alzheimer's disease-like phosphorylation of tau : generation of paired helical filament epitopes and neuronal localisation of the kinase

Glycogen synthase kinase-3 (GSK-3) reduced the mobility of human tau on SDS-PAGE, prevented binding of the monoclonal antibody (mAb), Tau.1, and induced binding of the mAb 8D8. Recombinant tau phosphorylated by GSK-3 aligned on SDS-PAGE with the abnormally phosphorylated tau (PHF-tau) associated with the paired helical filaments in Alzheimers disease brain. Phosphorylated serine396 (numbering of the largest human brain tau isoform) was identified as a binding site on tau for mAb 8D8. The localisation of GSK-3 within granular structures in pyramidal cells indicates that GSK-3 alpha and GSK-3 beta may have a role in the production of PHF-tau in Alzheimers disease.

Tau phosphorylation: the therapeutic challenge for neurodegenerative disease

The microtubule-associated protein tau is integral to the pathogenesis of Alzheimers disease (AD), as well as several related disorders, termed tauopathies, in which tau is deposited in affected brain regions. In the tauopathies, pathological tau is in an elevated state of phosphorylation and is aberrantly cleaved. It also exhibits abnormal conformations and becomes aggregated, resulting in neurofibrillary tau pathology. Recent evidence suggests that relatively early disease-associated changes in soluble tau proteins, including phosphorylation, are involved in the induction of neuronal death. Here, we summarize recent developments that suggest new therapeutic strategies to prevent or reduce the progression of pathology in the tauopathies. A list of tau phosphorylation sites identified in the tauopathies and in controls accompanies this review.

Alzheimer's disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells

BACKGROUND Paired helical filaments (PHFs) are a characteristic pathological feature of Alzheimers disease; their principal component is the microtubule-associated protein tau. The tau in PHFs (PHF-tau) is hyperphosphorylated, but the cellular mechanisms responsible for this hyperphosphorylation have yet to be elucidated. A number of kinases, including mitogen-activated protein (MAP) kinase, glycogen synthase kinase (GSK)-3 alpha, GSK-3 beta and cyclin-dependent kinase-5, phosphorylate recombinant tau in vitro so that it resembles PHF-tau as judged by its reactivity with a panel of antibodies capable of discriminating between normal tau and PHF-tau, and by a reduced electrophoretic mobility that is characteristic of PHF-tau. To determine whether MAP kinase, GSK-3 alpha and GSK-3 beta can also induce Alzheimers disease-like phosphorylation of tau in mammalian cells, we studied the phosphorylation status of tau in primary neuronal cultures and transfected COS cells following changes in the activities of MAP kinase and GSK-3. RESULTS Activating MAP kinase in cultures of primary neurons or transfected COS cells expressing tau isoforms did not increase the level of phosphorylation for any PHF-tau epitope investigated. But elevating GSK-3 activity in the COS cells by co-transfection with GSK-3 alpha or GSK-3 beta decreased the electrophoretic mobility of tau so that it resembled that of PHF-tau, and induced reactivity with eight PHF-tau-selective monoclonal antibodies. CONCLUSIONS Our data indicate that GSK-3 alpha and/or GSK-3 beta, but not MAP kinase, are good candidates for generating PHF-type phosphorylation of tau in Alzheimers disease. The involvement of other kinases in the generation of PHFs cannot, however, be eliminated. Our results suggest that aberrant regulation of GSK-3 may be a pathogenic mechanism in Alzheimers disease.

New Phosphorylation Sites Identified in Hyperphosphorylated Tau (Paired Helical Filament‐Tau) from Alzheimer's Disease Brain Using Nanoelectrospray Mass Spectrometry

Abstract: Paired helical filaments (PHFs) are the structural constituents of neurofibrillary tangles in Alzheimers 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 Alzheimers 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.

Novel Phosphorylation Sites in Tau from Alzheimer Brain Support a Role for Casein Kinase 1 in Disease Pathogenesis

Tau in Alzheimer disease brain is highly phosphorylated and aggregated into paired helical filaments comprising characteristic neurofibrillary tangles. Here we have analyzed insoluble Tau (PHF-tau) extracted from Alzheimer brain by mass spectrometry and identified 11 novel phosphorylation sites, 10 of which were assigned unambiguously to specific amino acid residues. This brings the number of directly identified sites in PHF-tau to 39, with an additional six sites indicated by reactivity with phosphospecific antibodies to Tau. We also identified five new phosphorylation sites in soluble Tau from control adult human brain, bringing the total number of reported sites to nine. To assess which kinases might be responsible for Tau phosphorylation, we used mass spectrometry to determine which sites were phosphorylated in vitro by several kinases. Casein kinase 1δ and glycogen synthase kinase-3β were each found to phosphorylate numerous sites, and each kinase phosphorylated at least 15 sites that are also phosphorylated in PHF-tau from Alzheimer brain. A combination of casein kinase 1δ and glycogen synthase kinase-3β activities could account for over three-quarters of the serine/threonine phosphorylation sites identified in PHF-tau, indicating that casein kinase 1δ may have a role, together with glycogen synthase kinase-3β, in the pathogenesis of Alzheimer disease.

Physiological release of endogenous tau is stimulated by neuronal activity

Propagation of tau pathology is linked with progressive neurodegeneration, but the mechanism underlying trans‐synaptic spread of tau is unknown. We show that stimulation of neuronal activity, or AMPA receptor activation, induces tau release from healthy, mature cortical neurons. Notably, phosphorylation of extracellular tau appears reduced in comparison with intracellular tau. We also find that AMPA‐induced release of tau is calcium‐dependent. Blocking pre‐synaptic vesicle release by tetanus toxin and inhibiting neuronal activity with tetrodotoxin both significantly impair AMPA‐mediated tau release. Tau secretion is therefore a regulatable process, dysregulation of which could lead to the spread of tau pathology in disease.

Pathological inclusion bodies in tauopathies contain distinct complements of tau with three or four microtubule-binding repeat domains as demonstrated by new specific monoclonal antibodies.

Pathological inclusions containing fibrillar aggregates of hyperphosphorylated tau protein are a characteristic feature in the tauopathies, which include Alzheimers disease, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP‐17), progressive supranuclear palsy, corticobasal degeneration and Picks disease. Tau isoform composition and cellular and regional distribution as well as morphology of these inclusions vary in each disorder. Recently, several pathological missense and exon 10 splice‐donor site mutations of the tau gene were iden‐tified in FTDP‐17. Exon 10 codes for the second of four microtubule‐binding repeat domains. The splice‐site mutations result in increased inclusion of exon 10 which causes a relative increase in tau isoforms containing four microtubule‐binding repeat domains over those containing three repeat domains. This could be a central aetiological mechanism in FTDP‐17 and, perhaps, other related tauopathies. We have investigated changes in the ratio and distribution of three‐repeat and four‐repeat tau in the different tauopathies as a basis of the phenotypic range of these disorders and the selective vulnerability of different subsets of neurones. In this study, we have developed two monoclonal antibodies, RD3 and RD4 that effectively distinguish these closely related tau isoforms. These new isoform‐specific antibodies are useful tools for analysing tau isoform expression and distribution as well as pathological changes in the human brain.

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

Alzheimers disease (AD) is pathologically characterised by the age-dependent deposition of β-amyloid (Aβ) 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 Aβ-induced neuronal loss. We report that the presence of small numbers of astrocytes exacerbate Aβ-induced neuronal death, caspase-3 activation and the production of caspase-3-cleaved tau. Furthermore, we show that astrocytes are essential for the Aβ-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. Aβ-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 Aβ in models of AD, and suggest that mechanisms underlying pro-inflammatory cytokine release might be an important target for therapy.

Parkinson's disease alpha-synuclein mutations exhibit defective axonal transport in cultured neurons

α-Synuclein is a major protein constituent of Lewy bodies and mutations in α-synuclein cause familial autosomal dominant Parkinsons disease. One explanation for the formation of perikaryal and neuritic aggregates of α-synuclein, which is a presynaptic protein, is that the mutations disrupt α-synuclein transport and lead to its proximal accumulation. We found that mutant forms of α-synuclein, either associated with Parkinsons disease (A30P or A53T) or mimicking defined serine, but not tyrosine, phosphorylation states exhibit reduced axonal transport following transfection into cultured neurons. Furthermore, transfection of A30P, but not wild-type, α-synuclein results in accumulation of the protein proximal to the cell body. We propose that the reduced axonal transport exhibited by the Parkinsons disease-associated α-synuclein mutants examined in this study might contribute to perikaryal accumulation of α-synuclein and hence Lewy body formation and neuritic abnormalities in diseased brain.

Phosphorylation regulates tau interactions with Src homology 3 domains of phosphatidylinositol 3-kinase, phospholipase C gamma 1, Grb2, and Src family kinases

The microtubule-associated protein tau can associate with various other proteins in addition to tubulin, including the SH3 domains of Src family tyrosine kinases. Tau is well known to aggregate to form hyperphosphorylated filamentous deposits in several neurodegenerative diseases (tauopathies) including Alzheimer disease. We now report that tau can bind to SH3 domains derived from the p85α subunit of phosphatidylinositol 3-kinase, phospholipase Cγ1, and the N-terminal (but not the C-terminal) SH3 of Grb2 as well as to the kinases Fyn, cSrc, and Fgr. However, the short inserts found in neuron-specific isoforms of Src prevented the binding of tau. The experimentally determined binding of tau peptides is well accounted for when modeled into the peptide binding cleft in the SH3 domain of Fyn. After phosphorylation in vitro or in transfected cells, tau showed reduced binding to SH3 domains; no binding was detected with hyperphosphorylated tau isolated from Alzheimer brain, but SH3 binding was restored by phosphatase treatment. Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies including Alzheimer disease.