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PubMed1997

Abnormal phosphorylation of tau and the mechanism of Alzheimer neurofibrillary degeneration: sequestration of microtubule-associated proteins 1 and 2 and the disassembly of microtubules by the abnormal tau.

The microtubule-associated protein (MAP) tau is abnormally hyperphosphorylated in Alzheimer disease and accumulates in neurons undergoing neurofibrillary degeneration. In the present study, the associations of the Alzheimer-hyperphosphorylated tau (AD P-tau) with the high molecular weight MAPs (HMW-MAPs) MAP1 and MAP2 were investigated. The AD P-tau was found to aggregate with MAP1 and MAP2 in solution. The association of AD P-tau to the MAPs resulted in inhibition of MAP-promoted microtubule assembly. However, unlike the coaggregation of AD P-tau and normal tau, the association between AD P-tau and the HMW-MAPs did not result in the formation of filaments/tangles. The affinity of the tau-AD P-tau association was higher than that of HMW-MAPs-AD P-tau because normal tau inhibited the latter binding. The association between AD P-tau and the HMW-MAPs also appeared to occur in situ because these proteins cosedimented from the Alzheimer brain extracts, and, in the sediment, the levels of the HMW-MAPs correlated with the levels of AD P-tau. These studies suggested that the abnormally phosphorylated tau can sequester both normal tau and HMW-MAPs and disassemble microtubules but, under physiological conditions, can form tangles of filaments only from tau.

Alzheimer Disease↗
PubMed2003

Microtubule-dependent oligomerization of tau. Implications for physiological tau function and tauopathies.

The accumulation of abnormal tau filaments is a pathological hallmark of many neurodegenerative diseases. In 1998, genetic analyses revealed a direct linkage between structural and regulatory mutations in the tau gene and the neurodegenerative disease, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). Importantly, the FTDP-17 phenotype is transmitted in a dominant rather than a recessive manner. However, the underlying molecular mechanisms causing disease remain uncertain. The most common molecular mechanism generating dominant phenotypes is the loss of function of a multimeric complex containing both mutant and wild-type subunits. Therefore, we sought to determine whether tau might normally function as a multimer. We co-incubated 35S-radiolabeled tau and biotinylated tau with taxol stabilized microtubules, at very low molar ratios of tau to tubulin. Subsequent covalent cross-linking followed by affinity-precipitation of the biotinylated tau revealed the formation of microtubule-dependent tau oligomers. We next used atomic force microscopy to independently assess this conclusion. Our results are consistent with the hypothesis that tau forms oligomers upon binding to microtubules. In addition to providing insights into normal tau action, our findings lead us to propose that one mechanism by which mutations in tau may cause cell death is through the formation of tau complexes containing mutant tau molecules in association with wild-type tau. These wild-type-mutant tau complexes may possess altered biological and/or biophysical properties that promote onset of the FTDP-17 phenotype, including neuronal cell death by either altering normal tau-mediated regulation of microtubule-dependent cellular functions and/or promoting the formation of pathological tau aggregates.

Animals↗
PubMed1995

Calpain-induced proteolysis of normal human tau and tau associated with paired helical filaments.

The major components of neurofibrillary tangles (NFT) in Alzheimer's disease are bundles of paired helical filaments (PHF) which are primarily composed of highly phosphorylated tau proteins (PHF-tau). To further understand the mechanism of PHF accumulation in NFT, we examined the calpain-induced proteolysis of highly purified and primarily non-aggregated PHF and normal tau proteins with various contents of phosphate isolated from either fetal (F-tau) or adult human brain (N-tau). The extent of proteolysis was determined by decreases in tau immunoreactivity using Western-blot analysis and a panel of site-specific tau antibodies (Alz 50, Tau-2, Tau 14, Tau-1, AT8, E-11, AH-1 and PHF-1). We found that full-size polypeptides of N-tau and F-tau were similarly and rapidly proteolyzed in vitro by calpain (calpain II, 3.3 units/mg protein) during a 10-min incubation at 30 degrees C, and that their half lives (t1/2) were 1.5 min and 1.8 min, respectively. Analysis of immunoblots suggests that full-length polypeptides of tau are first degraded into large fragments similar in size to that generated endogenously, then into smaller fragments. Since both endogenous and in-vitro-generated tau fragments retained N-terminal epitopes, the results suggest that most of the calpain-sensitive sites may be located in the C-terminal half of the tau molecule. In contrast, PHF were extremely resistant to degradation and only a fivefold higher concentration of calpain (16.7 units/mg protein) induced partial proteolysis of PHF. A major calpain-generated fragment was a 45-kDa polypeptide derived from the C-terminal region of PHF-tau, which forms a core of filaments. The results suggest that the inaccessibility of potential calpain-digestion sites in the filament core could contribute to the resistance of PHF to calpain and subsequently lead to the accumulation of PHF in Alzheimer's disease. The results also suggest that hyperphosphorylation of tau may be marginally involved in the resistance of PHF to degradation by calpain. Ultrastructural examination revealed that, in contrast to previous studies with trypsin, calpain did not alter the morphologic appearance of filaments; after incubation with calpain, the majority of PHF remained short and disperse and the number of PHF aggregated into NFT-like clusters was not significantly increased. The results suggest that the role of calpain in promoting the aggregation and clustering of filaments is limited.

Adult↗
PubMed2005

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 Alzheimer's 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 Alzheimer's disease.

Aged, 80 and over↗
PubMed2001

Mutated tau binds less avidly to microtubules than wildtype tau in living cells.

Some forms of genetically inherited dementia, including frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), are caused by mutations in tau. We have examined several mutations in the microtubule-binding portion of tau for their effect on microtubule binding, cellular distribution and cytoskeletal structure in mammalian cells. Using constructs coding for mutant (P301L and V337M) and wildtype human tau fused to a green fluorescent protein analog (EGFP) we followed the disposition of tau in live cells after transient transfection using confocal microscopy. Most of the tau protein localized to structures that resembled microtubules or microtubule bundles and co-localized with tubulin. At 3 days post-transfection mutant tau proteins showed a higher abundance of free tau in the cytoplasm than did wildtype tau. Cells expressing the P301L mutation showed proportionally more cytoplasmic localization of tau. Confirming these results, fractionated cells with mutant tau had a higher percentage of tau in the cytoplasmic compartment as compared to the cytoskeletal compartment. Cells with wildtype tau had most tau in the cytoskeletal fraction. Because the mutations (V337M, P301L) are associated with genetic tauopathies, these results suggest that a factor in disease etiology of genetic tauopathies and other dementias with altered tau is a greater abundance of tau in the cytoplasm due to decreased binding to microtubules. This increased cytoplasmic presence may be a significant factor in promoting tau aggregation.

Binding Sites↗
PubMed1993

Phosphorylation of tau protein in tau-transfected 3T3 cells.

The tau protein of Alzheimer paired helical filaments (PHFs) is aberrantly phosphorylated, as evidenced by its reactivity with several phosphate-dependent antibodies. We sought to identify whether this unusual phosphorylation state exists in tau expressed by transfected NIH 3T3 fibroblasts. Immunoblot analysis of cell clones transfected with constructs for either the 3-repeat or 4-repeat isoforms of tau revealed two tau bands, with the lower band migrating with unmodified tau in each case. Antibodies T3P and tau-1 were used to probe these bands, as they also react with PHF-tau in a phosphate-dependent manner. The epitopes for both antibodies were phosphorylated in both tau isoforms. Only the upper band was phosphorylated at the T3P site whereas phosphorylation at the tau-1 site was not always associated with a shift of tau mobility on gels. Tau in both bands was soluble, in contrast to PHF-tau, and was competent to bind to exogenously added bovine microtubules. Colchicine treatment of the cells resulted in an inhibition of phosphorylation at both sites, through an unknown mechanism. In conclusion human tau expressed in 3T3 cells was phosphorylated at the T3P and tau-1 sites as is PHF-tau, although no PHFs formed and the phosphorylated tau was competent to bind to microtubules.

3T3 Cells↗
PubMed1994

Tau phosphorylation in human, primate, and rat brain: evidence that a pool of tau is highly phosphorylated in vivo and is rapidly dephosphorylated in vitro.

The extent of tau phosphorylation is thought to regulate the binding of tau to microtubules: Highly phosphorylated tau does not bind to tubules, whereas dephosphorylated tau can bind to microtubules. It is interesting that the extent of tau phosphorylation in vivo has not been accurately determined. Tau was rapidly isolated from human temporal neocortex and hippocampus, rhesus monkey temporal neocortex, and rat temporal neocortex and hippocampus under conditions that minimized dephosphorylation. In brain slices, we observed that tau isolated under such conditions largely existed in several phosphorylated states, including a pool that was highly phosphorylated; this was determined using epitope-specific monoclonal and polyclonal antibodies. This highly phosphorylated tau was dephosphorylated during a 120-min time course in vitro, presumably as a result of neuronal phosphatase activity. The slow-mobility forms of tau were shifted to faster-mobility forms following in vitro incubation with alkaline phosphatase. Laser densitometry was used to estimate the percent of tau in slow-mobility, highly phosphorylated forms. Approximately 25% of immunoreactive tau was present as slow-mobility (66- and 68-kDa) forms of tau. The percentage of immunoreactive tau in faster-mobility pools (42-54 kDa) increased in proportion to the decrease in content of 66-68-kDa tau as a function of neuronal phosphatases or alkaline phosphatase treatment. These data suggest that the turnover of phosphorylated sites on tau is rapid and depends on neuronal phosphatases. Furthermore, tau is highly phosphorylated in normal-appearing human, primate, and rodent brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed2005

Disease-related modifications in tau affect the interaction between Fyn and Tau.

Microtubule-associated protein tau is the major component of the neurofibrillary tangles of Alzheimer disease (AD) and is genetically linked to frontotemporal dementias (FTDP-17). We have recently shown that tau interacts with the SH3 domain of Fyn, an Src family non-receptor tyrosine kinase, and is tyrosine-phosphorylated by Fyn on Tyr-18. Also, tyrosine-phosphorylated tau is present in the neuropathology of AD. To determine whether alterations in the tau-Fyn interaction might correlate with disease-related factors in AD and FTDP-17, we have performed real-time surface plasmon resonance studies on a panel of 21 tau constructs with Fyn SH3. We report that the interaction between Fyn SH3 and 3R-tau was 20-fold higher than that with 4R-tau. In addition, the affinity between 4R-tau and Fyn SH3 was increased 25-45-fold by phosphorylation-mimicking mutations or by FTDP-17 mutations. In vitro kinase reactions show that tau, with lower affinity SH3 interactions, exhibited a lower level of Tyr-18 phosphorylation under our reaction conditions. Lastly, we have demonstrated that tau is phosphorylated on Tyr-18 in the tau P301L mouse model for tauopathy (JNPL3). In summary, our results suggest that disease-related phosphorylation and missense mutations of tau increase association of tau with Fyn. Because these effects are mediated through the 4R component of the tau population, these results also have implications for the FTDP-17 diseases caused by increased expression of 4R-tau. Our data support a role for the Fyn-tau interaction in neurodegeneration.

Alternative Splicing↗
PubMed2006

Microtubule binding and clustering of human Tau-4R and Tau-P301L proteins isolated from yeast deficient in orthologues of glycogen synthase kinase-3beta or cdk5.

Phosphorylation of Tau protein and binding to microtubules is complex in neurons and was therefore studied in the less complicated model of humanized yeast. Human Tau was readily phosphorylated at pathological epitopes, but in opposite directions regulated by kinases Mds1 and Pho85, orthologues of glycogen synthase kinase-3beta and cdk5, respectively (1). We isolated recombinant Tau-4R and mutant Tau-P301L from wild type, Delta mds1 and Delta pho85 yeast strains and measured binding to Taxol-stabilized mammalian microtubules in relation to their phosphorylation patterns. Tau-4R isolated from yeast lacking mds1 was less phosphorylated and bound more to microtubules than Tau-4R isolated from wild type yeast. Paradoxically, phosphorylation of Tau-4R isolated from kinase Pho85-deficient yeast was dramatically increased resulting in very poor binding to microtubules. Dephosphorylation promoted binding to microtubules to uniform high levels, excluding other modifications. Isolated hyperphosphorylated, conformationally altered Tau-4R completely failed to bind microtubules. In parallel to Tau-4R, we expressed, isolated, and analyzed mutant Tau-P301L. Total dephosphorylated Tau-4R and Tau-P301L bound to microtubules very similarly. Surprisingly, Tau-P301L isolated from all yeast strains bound to microtubules more extensively than Tau-4R. Atomic force microscopy demonstrated, however, that the high apparent binding of Tau-P301L was due to aggregation on the microtubules, causing their deformation and bundling. Our data explain the pathological presence of granular Tau aggregates in neuronal processes in tauopathies.

Cyclin-Dependent Kinase 5↗
PubMed2005

Tau protein is cross-linked by transglutaminase in P301L tau transgenic mice.

The microtubule-associated protein tau is highly soluble under physiological conditions. However, in tauopathies, tau protein aggregates into insoluble filaments and neurofibrillary tangles (NFTs). The mechanisms underlying the formation of tau filaments and NFTs in tauopathies remain unclear. Several lines of evidence suggest that transglutaminase may cross-link tau into stable, insoluble aggregates, leading to the formation of NFTs in Alzheimer's disease and progressive supranuclear palsy. To further determine the contribution of transglutaminase in the formation of NFTs, we compared the levels of cross-linked tau protein from P301L tau transgenic mice that develop NFTs to four-repeat wild-type (4RWT) tau transgenic and nontransgenic mice that do not develop NFT pathology. Immunoprecipitation and immunoblotting experiments show that transglutaminase cross-links phosphorylated tau in the hindbrain of P301L tau transgenic mice but not in mice overexpressing 4RWT tau and nontransgenic mice. Cross-linked, phosphorylated tau from P301L tau transgenic mice runs as high-molecular mass aggregates on Western blots, similar to cross-linked tau from paired helical filaments of Alzheimer's disease. We also used double-label immunofluorescence to demonstrate colocalization of PHF-1-immunoreactive tau and the transglutaminase-catalyzed cross-link in the hindbrain, spinal cord, and cortex of P301L tau transgenic mice. In the spinal cord, 87% of PHF-1-labeled cells colocalize with the transglutaminase-catalyzed cross-link. Additionally, transglutaminase enzymatic activity is significantly elevated in the spinal cord of P301L tau transgenic mice. These studies further implicate transglutaminase in the formation and/or stabilization of NFT and paired helical filaments and provide a model system to investigate the therapeutic potential of transglutaminase inhibitors in tauopathies.

Amino Acid Substitution↗
PubMed2006

Tau aggregation and progressive neuronal degeneration in the absence of changes in spine density and morphology after targeted expression of Alzheimer's disease-relevant tau constructs in organotypic hippocampal slices.

Alzheimer's disease (AD) is characterized by progressive loss of neurons in selected brain regions, extracellular accumulations of amyloid beta, and intracellular fibrils containing hyperphosphorylated tau. Tau mutations in familial tauopathies confirmed a central role of tau pathology; however, the role of tau alteration and the sequence of tau-dependent neurodegeneration in AD remain elusive. Using Sindbis virus-mediated expression of AD-relevant tau constructs in hippocampal slices, we show that disease-like tau modifications affect tau phosphorylation at selected sites, induce Alz50/MC1-reactive pathological tau conformation, cause accumulation of insoluble tau, and induce region-specific neurodegeneration. Live imaging demonstrates that tau-dependent degeneration is associated with the development of a "ballooned" phenotype, a distinct feature of cell death. Spine density and morphology is not altered as judged from algorithm-based evaluation of dendritic spines, suggesting that synaptic integrity is remarkably stable against tau-dependent degeneration. The data provide evidence that tau-induced cell death involves apoptotic as well as nonapoptotic mechanisms. Furthermore, they demonstrate that targeted expression of tau in hippocampal slices provides a novel model to analyze tau modification and spatiotemporal dynamics of tau-dependent neurodegeneration in an authentic CNS environment.

Alzheimer Disease↗
PubMed1993

Locations and immunoreactivities of phosphorylation sites on bovine and porcine tau proteins and a PHF-tau fragment.

Tau protein is a phosphorylated neuronal microtubule-associated protein. Tau protein is also present in the major pathological lesions of Alzheimer's disease in an insoluble hyperphosphorylated state as paired helical filaments (PHFs). We have investigated the phosphorylation state of control taus and a fragment of PHF-tau. Tau samples were digested with protease, separated by reversed-phase high-performance liquid chromatography, and analyzed by mass spectrometry and Edman microsequencing. The serine homologous with S404 of human tau 441 was phosphorylated on bovine and porcine tau and up to two phosphates were present on a peptide of amino acids 182-240 of bovine tau (193-251 of human tau 441). The serine within the KSPV motif was not phosphorylated on bovine or porcine tau. PHF-tau fragments, isolated from pronase-treated PHFs encompassed a 93-amino acid region within the microtubule binding domain. Enzymatic digestion and mass spectrometric analysis showed no phosphate was present and a second carboxyl terminus was identified at E380. Antibodies T3P and SMI34, which recognize PHF-tau and peptides phosphorylated at the sequence KSPV, both reacted with bovine and porcine tau even though the KSPV sequence was not phosphorylated. These data indicate that the 93-amino acid sequence of F5.5 tau from PHFs is not phosphorylated, and the serine equivalent to S404 of human tau is phosphorylated in bovine and porcine tau. Antibodies T3P and SMI34 react with phosphorylated epitopes that are not unique to PHF-tau and that are not necessarily at the KSPV site.

Amino Acid Sequence↗
PubMed1997

Phospholipids alter tau conformation, phosphorylation, proteolysis, and association with microtubules: implication for tau function under normal and degenerative conditions.

Discerning the in situ functions of the microtubule-associated protein (MAP) tau is of interest both in terms of neuronal differentiation and homeostasis as well as in terms of neurodegenerative conditions such as Alzheimer's disease. In the present study, exposure to excess phosphatidyl serine (PS) for < 1 min induced antigenic alterations in multiple N-terminal, C-terminal and central epitopes of purified human brain tau. Notably, "AD-like" epitopes (PHF-1, ALZ-50, AT-8) were decreased by PS; other epitopes (e.g., 5E2, Tau-1) increased and others remained relatively unchanged. Inclusion of gamma-AT[32P] during incubations did not reveal any contaminating kinase activity. Direct addition of chloroform:methanol (CM; the initial PS solvent) demonstrated that these changes were not derived from CM-mediated tau denaturation. Phosphatidyl choline induced similar antigenic changes, while phosphatidyl inositol did not. PS inhibited MAP-kinase generation of phospho-dependent tau epitopes and incorporation of phosphates by tau. Inclusion of PS during coincubation of tau and tubulin reduced the extent of cosedimentation of tau with MTs. Finally, PS enhanced the ability of calpain-mediated tau proteolysis. These data suggest that tau antigenicity in situ may be derived from phospholipid-dependent alterations in tau conformation in addition to tau phosphorylation state. These data further suggest that disruption of the normal association of tau with phospholipids may foster accumulation of tau and, in doing so, render tau more susceptible to hyperphosphorylation.

Alzheimer Disease↗
PubMed2002

Transient expression of fluorescent tau proteins promotes process formation in PC12 cells: contributions of the tau C-terminus to this process.

The neuronal microtubule-associated protein tau promotes microtubule assembly and has been implicated in the development of axonal morphology. In this study, PC12 cells were transiently transfected with constructs coding fusion proteins of human tau with green fluorescent protein (GFP). Expression of tau constructs actively stabilized microtubules. Expression of the C-terminus of tau can mimic this effect in living cells, though to a lesser extent because of the absence of the tau N-terminus. However, tau colocalization with microtubules did not require the presence of the tau N-terminus. Transient expression of tau (including tau24, a four-repeat human tau isoform encoded in 383 residues, and tau23, human fetal tau isoform encoded in 352 residues) stimulated process formation in PC12 cells, and this occurred faster with tau24 than with tau23. The residues (residues 154-172 in tau23) that confer microtubule nucleation activity of tau in vitro are not required for tau-directed process formation. However, when tau induces the formation of cellular processes in response to cortical breakdown by cytochalasin B, residues 154-172 must be present. Thus, it appears that tau may serve to promote cellular process outgrowth in cultured neuronal cells and that C-terminus of tau is essential to this process.

Animals↗
PubMed1995

Developmental expression of tau proteins in the chicken and rat brain: rapid down-regulation of a paired helical filament epitope in the rat cerebral cortex coincides with the transition from immature to adult tau isoforms.

The monoclonal antibodies TAU-1 and AT8 are directed at human microtubule-associated protein tau epitopes that contain a dephosphorylated and phosphorylated Ser202, respectively, while AT180 and AT270 are anti-tau monoclonals with epitopes that require phosphorylated Thr181 and Thr231, respectively. We used these antibodies to study the developmental profiles of tau proteins in rat cerebral cortex and chicken optic lobes. In tau extracts from perinatal rat cerebral cortex. AT8 recognized one major protein band of approximately 50 kDa that peaks on postnatal day 6 and declines rapidly to lower levels at day 12. At later stages, the AT8 epitope was expressed by several adult tau isoforms that were, however, stained only very faintly in highly enriched samples. Two additional tau epitopes recognized by AT180 and AT270 were found to be expressed by one or two protein bands up to about postnatal day 19 and then declined. Unlike the AT8 epitope, in the mature brain these epitopes were stained strongly in enriched samples, where they were expressed by a greater number of adult isoforms. Between embryonic day 19 and postnatal day 12, TAU-1 was found to recognize one major protein band of approximately 50 kDa which migrated slightly faster than the AT8-binding band. At postnatal day 19 and all older stages (including adult cortex), at least three additional TAU-1-binding isoforms with higher apparent molecular weights were present. Hence, the transition from one immature to several adult TAU-1-binding tau isoforms between postnatal day 12 and 19 in rat cerebral cortex coincides with the phase of rapid down-regulation of the AT8 epitope. As in the rat cerebrum, in chicken optic lobes there is a developmental decrease of AT8-binding proteins which is paralleled by striking changes in the electrophoretic pattern of tau isoforms recognized by TAU-1. In both rat cerebral cortex and chicken optic lobes, the period of maximal expression of AT8-binding tau is morphologically characterized by intense axonal growth and beginning synaptogenesis, whereas its subsequent rapid down-regulation and the appearance of novel TAU-1-binding isoforms correlates with synaptic maturation, the onset of spontaneous electrical activity and the beginning of myelination.

Animals↗
PubMed2005

Increase in tau tyrosine phosphorylation correlates with the formation of tau aggregates.

Tauopathies are neurodegenerative disorders characterized by aberrant intracellular aggregation of hyperphosphorylated tau. It has been shown that aggregated tau is phosphorylated at serine, threonine, and tyrosine residues. However, the occurrence of tyrosine phosphorylation on tau proteins at different states of tau aggregation has not been shown. In this report, we utilized the tauopathy mouse model JNPL3 that expresses human 0N4R tau isoform bearing the missense P301L mutation to study the occurrence of tau tyrosine phosphorylation in the course of the development of tau aggregation. These mice develop behavioral and motor deficits and form sarkosyl-insoluble hyperphosphorylated tau in an age-dependent manner. Mass spectrometry analyses of immunopurified brain tau proteins from JNPL3 and Alzheimer's disease affected individual uncovered novel tau tyrosine-phosphorylated sites. Further studies demonstrated that the abundance of tyrosine-phosphorylated tau increases in an age-dependent manner in JNPL3 mice. Tyrosine-phosphorylated tau was detected in both soluble and sarkosyl-insoluble preparations derived from brain and spinal cord, and localized in neurons containing aggregated tau. The phosphorylation of tyrosine residues in tau appeared to occur along with that of serine and threonine residues and was not detectable in non-transgenic littermates and transgenic mice expressing 0N4R wild-type human tau. The results suggest that tyrosine phosphorylation is as important as phosphorylation of other residues in tauopathy.

Aging↗
PubMed2002

Tau assembly in inducible transfectants expressing wild-type or FTDP-17 tau.

Conditional expression systems for 4-repeat wild-type (WT) tau or the corresponding mutants V337M and R406W were established in human neuroglioma H4 cells to study the effect of tau mutations on the physicochemical properties of tau, and to develop a cellular model for the formation of filamentous tau characteristic of frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) and Alzheimer's disease. Upon induction tau expression increased, reaching maximal levels at 5 to 7 days. WT tau was phosphorylated at amino acids T181, S202/T205, T231, and S396/S404. The R406W mutation decreased tau phosphorylation at each of these sites as did the V337M mutation except for S396/S404 sites that increased. Most tau in postnuclear cell lysates was recovered in the supernatant fraction after centrifugation at 200,000 x g. The amount of tau in the pellet fraction increased more in mutant transfectants compared to WT when the induction was extended beyond 5 days. This particulate tau could be partially extracted with salt, Triton X-100, or sarkosyl. Of the transfectants, R406W had the highest proportion of sarkosyl-insoluble tau by day 7. This insoluble fraction was thioflavin S-positive and contained 15- to 5-nm-wide filaments with tau immunoreactivities. The R406W filaments were more abundant than those detected in similar preparations from WT or V337M transfectants. At the light microscopy level, most tau was found with microtubules, or diffusely distributed in the cytoplasm, but none of this appeared thioflavin S-positive. The results suggest that conditional tau transfectants are in a pretangle stage making them an attractive model system for studying intracellular tangle accumulation and for testing potential therapeutic agents as inhibitors for tau aggregation.

Chromosomes, Human, Pair 17↗
PubMed1997

Phosphorylation of tau by glycogen synthase kinase 3beta affects the ability of tau to promote microtubule self-assembly.

To study the effects of phosphorylation by glycogen synthase kinase-3beta (GSK-3beta) on the ability of the microtubule-associated protein tau to promote microtubule self-assembly, tau isoform 1 (foetal tau) and three mutant forms of this tau isoform were investigated. The three mutant forms of tau had the following serine residues, known to be phosphorylated by GSK-3, replaced with alanine residues so as to preclude their phosphorylation: (1) Ser-199 and Ser-202 (Ser-199/202-->Ala), (2) Ser-235 (Ser-235-->Ala) and (3) Ser-396 and Ser-404 (Ser-396/404-->Ala). Wild-type tau and the mutant forms of tau were phosphorylated with GSK-3beta, and their ability to promote microtubule self-assembly was compared with the corresponding non-phosphorylated tau species. In the non-phosphorylated form, wild-type tau and all of the mutants affected the mean microtubule length and number concentrations of assembled microtubules in a manner consistant with enhanced microtubule nucleation. Phosphorylation of these tau species with GSK-3beta consistently reduced the ability of a given tau species to promote microtubule self-assembly, although the affinity of the tau for the microtubules was not greatly affected by phosphorylation since the tau species remained largely associated with the microtubules. This suggests that the regulation of microtubule assembly can be controlled by phosphorylation of tau at sites accessible to GSK-3beta by a mechanism that does not necessarily involve the dissociation of tau from the microtubules.

Alzheimer Disease↗