C. Hugh Reynolds

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.

Stress‐Activated Protein Kinase/c‐Jun N‐Terminal Kinase Phosphorylates τ Protein

Abstract: A proportion of the neuronal microtubule‐associated protein (MAP) τ is highly phosphorylated in foetal and adult brain, whereas the majority of τ in the neurofibrillary tangles of Alzheimers patients is hyperphosphorylated; many of the phosphorylation sites are serines or threonines followed by prolines. Several kinases phosphorylate τ at such sites in vitro. We have now shown that purified recombinant stress‐activated protein kinase/c‐Jun N‐terminal kinase, a proline‐directed kinase of the MAP kinase extended family, phosphorylates recombinant τ in vitro on threonine and serine residues. Western blots using antibodies to phosphorylation‐dependent τ epitopes demonstrated that phosphorylation occurs in both of the main phosphorylated regions of τ protein. Unlike glycogen synthase kinase‐3, the c‐Jun N‐terminal kinase readily phosphorylates Thr205 and Ser422, which are more highly phosphorylated in Alzheimer τ than in foetal or adult τ. Glycogen synthase kinase‐3 may preferentially phosphorylate the sites found physiologically, in foetal and to a smaller extent in adult τ, whereas stress‐activated/c‐Jun N‐terminal kinase and/or other members of the extended MAP kinase family may be responsible for pathological proline‐directed phosphorylations. Inflammatory processes in Alzheimer brain might therefore contribute directly to the pathological formation of the hyperphosphorylated τ found in neurofibrillary tangles.

Reactivating Kinase/p38 Phosphorylates τ Protein In Vitro

Abstract: Neurofibrillary tangles, one of the major pathological hallmarks of Alzheimer‐diseased brains, consist primarily of aggregated paired helical filaments (PHFs) of hyperphosphorylated τ protein. τ from normal brain and especially from foetal brain is also phosphorylated on some of the sites phosphorylated in PHFs, mainly at serines or threonines followed by prolines. A number of protein kinases can phosphorylate τ in vitro; those that require or accept prolines include GSK3 and members of the mitogen‐activated protein (MAP) kinase family, ERK1, ERK2, and SAP kinase‐β/JNK. In this report, we show that another member of the MAP kinase family, the stress‐activated kinase p38/RK, can phosphorylate τ in vitro. Western blots with phosphorylation‐sensitive antibodies showed that p38, like ERK2 and SAP kinase‐β/JNK, phosphorylated τ at sites found phosphorylated physiologically (Thr181, Ser202, Thr205, and Ser396) and also at Ser422, which is phosphorylated in neurofibrillary tangles but not in normal adult or foetal brain. These findings support the possibility that cellular stress might contribute to τ hyperphosphorylation during the formation of PHFs, and hence, to the development of τ pathology.

Tyrosine 394 Is Phosphorylated in Alzheimer's Paired Helical Filament Tau and in Fetal Tau with c-Abl as the Candidate Tyrosine Kinase

Tau is a major microtubule-associated protein of axons and is also the principal component of the paired helical filaments (PHFs) that comprise the neurofibrillary tangles found in Alzheimers disease and other tauopathies. Besides phosphorylation of tau on serine and threonine residues in both normal tau and tau from neurofibrillary tangles, Tyr-18 was reported to be a site of phosphorylation by the Src-family kinase Fyn. We examined whether tyrosine residues other than Tyr-18 are phosphorylated in tau and whether other tyrosine kinases might phosphorylate tau. Using mass spectrometry, we positively identified phosphorylated Tyr-394 in PHF-tau from an Alzheimer brain and in human fetal brain tau. When wild-type human tau was transfected into fibroblasts or neuroblastoma cells, treatment with pervanadate caused tau to become phosphorylated on tyrosine by endogenous kinases. By replacing each of the five tyrosines in tau with phenylalanine, we identified Tyr-394 as the major site of tyrosine phosphorylation in tau. Tyrosine phosphorylation of tau was inhibited by PP2 (4-amino-5-(4-chlorophenyl-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), which is known to inhibit Src-family kinases and c-Abl. Cotransfection of tau and kinases showed that Tyr-18 was the major site for Fyn phosphorylation, but Tyr-394 was the main residue for Abl. In vitro, Abl phosphorylated tau directly. Abl could be coprecipitated with tau and was present in pretangle neurons in brain sections from Alzheimer cases. These results show that phosphorylation of tau on Tyr-394 is a physiological event that is potentially part of a signal relay and suggest that Abl could have a pathogenic role in Alzheimers disease.

The Microtubule-Associated Protein Tau is Also Phosphorylated on Tyrosine

Tau protein is the principal component of the neurofibrillary tangles found in Alzheimers disease (AD), where it is hyperphosphorylated on serine and threonine residues. It is hypothesized that this hyperphosphorylation contributes to neurodegeneration through the destabilization of microtubules. There is now evidence that phosphorylation of tau can also occur on tyrosine residues. Human tau has five tyrosines numbered 18, 29, 197, 310, and 394, according to the sequence of the longest CNS isoform. Tyrosines 18, 197, and 394 have been shown to be phosphorylated in the brain of patients with AD whereas tyrosine 394 is the only residue that has been described to date that is phosphorylated in physiological conditions. Src family kinases and spleen tyrosine kinase (Syk) have been shown to phosphorylate tyrosine 18 while c-Abl is capable of phosphorylating tyrosine 394. Recently, a dual specificity kinase termed TTBK1 has been characterized in human brain and shown to be able to phosphorylate residue 197 of tau. Data about the role of tau tyrosine phosphorylation in neuronal physiology are still scarce and preliminary. In contrast, there is mounting evidence suggesting that tau tyrosine phosphorylation is an early event in the pathophysiology of AD and that Fyn and c-Abl are critical in the neurodegenerative process which occurs in tauopathies.

Tyrosine phosphorylation of tau regulates its interactions with Fyn SH2 domains, but not SH3 domains, altering the cellular localization of tau

Recent reports have demonstrated that interactions between the microtubule‐associated protein tau and the nonreceptor tyrosine kinase Fyn play a critical role in mediating synaptic toxicity and neuronal loss in response to β‐amyloid (Aβ) in models of Alzheimer’s disease. Disruption of interactions between Fyn and tau may thus have the potential to protect neurons from Aβ‐induced neurotoxicity. Here, we investigated tau and Fyn interactions and the potential implications for positioning of these proteins in membrane microdomains. Tau is known to bind to Fyn via its Src‐homology (SH)3 domain, an association regulated by phosphorylation of PXXP motifs in tau. Here, we show that Pro216 within the PXXP(213–216) motif in tau plays an important role in mediating the interaction of tau with Fyn‐SH3. We also show that tau interacts with the SH2 domain of Fyn, and that this association, unlike that of Fyn‐SH3, is influenced by Fyn‐mediated tyrosine phosphorylation of tau. In particular, phosphorylation of tau at Tyr18, a reported target of Fyn, is important for mediating Fyn‐SH2–tau interactions. Finally, we show that tyrosine phosphorylation influences the localization of tau to detergent‐resistant membrane microdomains in primary cortical neurons, and that this trafficking is Fyn‐dependent. These findings may have implications for the development of novel therapeutic strategies aimed at disrupting the tau/Fyn‐mediated synaptic dysfunction that occurs in response to elevated Aβ levels in neurodegenerative disease.

Oxidative Stress Induces Dephosphorylation of τ in Rat Brain Primary Neuronal Cultures

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

Stimulation of MAP kinase by v-raf transformation of fibroblasts fails to induce hyperphosphorylation of transfected tau.

A proportion of the microtubule‐associated protein, tau, is in an elevated state of phosphorylation in foetal and adult brain whereas all of the tau in paired helical filaments, which are characteristic of Alzheimers disease is hyperphosphorylated; it is important therefore to elucidate the mechanisms that regulate tau phosphorylation. Here we describe results that show that although MAP kinase can hyperphosphorylate tau in vitro, activation of MAP kinase in transformed fibroblasts does not result in hyperphosphorylation of transfected tau, whereas glycogen synthase kinase‐3β (GSK‐3β) when co‐transfected with tau does result in tau hyperphosphorylation. The findings imply that GSK‐3β may be a stronger candidate than MAP kinase for inducing tau hyperphosphorylation in vivo.

GSK3α exhibits β‐catenin and tau directed kinase activities that are modulated by Wnt

In the presence of a Wnt signal β‐catenin is spared from proteasomal degradation through a complex mechanism involving GSK3β, resulting in the transcription of Wnt target genes. In this study we have explored whether GSK3α, a related isoform, can also regulate nuclear β‐catenin levels and whether this and the tau‐directed kinase activity of GSK3α are modulated by Wnt. GSK3α or GSK3β and their substrates, β‐catenin and tau, were transiently expressed in mammalian cells. Immunoblotting revealed that GSK3α reduces nuclear levels of β‐catenin, whilst reporter gene assays demonstrated that GSK3α inhibits β‐catenin‐directed Tcf/Lef‐dependent transcription. Moreover, activation of the Wnt pathway was found to attenuate both the β‐catenin‐ and the tau‐directed kinase activities of GSK3α and GSK3β. By immunoprecipitation we also found that axin‐1, the β‐catenin destruction complex scaffold protein, binds GSK3α. In the light of these findings GSK3α warrants further investigation regarding its involvement in Wnt signalling and tauopathies such as Alzheimers disease.

Tyrosine Phosphorylation of Tau by the Src Family Kinases Lck and Fyn

BackgroundTau protein is the principal component of the neurofibrillary tangles found in Alzheimers disease, where it is hyperphosphorylated on serine and threonine residues, and recently phosphotyrosine has been demonstrated. The Src-family kinase Fyn has been linked circumstantially to the pathology of Alzheimers disease, and shown to phosphorylate Tyr18. Recently another Src-family kinase, Lck, has been identified as a genetic risk factor for this disease.ResultsIn this study we show that Lck is a tau kinase. In vitro, comparison of Lck and Fyn showed that while both kinases phosphorylated Tyr18 preferentially, Lck phosphorylated other tyrosines somewhat better than Fyn. In co-transfected COS-7 cells, mutating any one of the five tyrosines in tau to phenylalanine reduced the apparent level of tau tyrosine phosphorylation to 25-40% of that given by wild-type tau. Consistent with this, tau mutants with only one remaining tyrosine gave poor phosphorylation; however, Tyr18 was phosphorylated better than the others.ConclusionsFyn and Lck have subtle differences in their properties as tau kinases, and the phosphorylation of tau is one mechanism by which the genetic risk associated with Lck might be expressed pathogenically.