Functional characterization of FTDP-17 tau gene mutations through their effects on Xenopus oocyte maturation.

tau gene mutations cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Here we have used Xenopus oocyte maturation as an indicator of microtubule function. We show that wild-type four-repeat Tau protein inhibits maturation in a concentration-dependent manner, whereas three-repeat Tau has no effect. Of the seven four-repeat Tau proteins with FTDP-17 mutations tested, five (G272V, DeltaK280, P301L, P301S, and V337M) failed to interfere significantly with oocyte maturation, demonstrating a greatly reduced ability to interact with microtubules. One mutant protein (R406W) almost behaved like wild-type Tau, and one (S305N) inhibited maturation more strongly than wild-type Tau. With the exception of R406W, wild-type Tau and all the mutants studied were similarly phosphorylated during the Xenopus oocyte maturation, and this was independent of their effects on this process. Data obtained with R406W and S305N may be related to charge changes (phosphorylation and basic amino acids). Our results demonstrate variable effects of FTDP-17 mutations on microtubules in an intact cell situation. Those findings establish Xenopus oocyte maturation as a system allowing the study of the functional effects of tau gene mutations in a quantitative manner.     

Protein kinase C and calcium/calmodulin-dependent protein kinase II phosphorylate three-repeat and four-repeat tau isoforms at different rates

All six isoforms of the microtubule-associated protein tau are present in hyperphosphorylated states in the brains of patients with Alzheimer's disease (AD). It is presently unclear how such hyperphosphorylation of tau is controlled. In a previous study (Singh et al. Arch Biochem Biophys 328: 43-50, 1996) we have shown that three-repeat taus containing two N-terminal inserts were phosphorylated to higher levels and at different sites compared to those either lacking or containing only one such insert. We have extended these observations in this study by comparing the phosphorylation of tau isoforms containing three-repeats (t3, t3L) and four-repeats (t4, t4L). In the absence of N-terminal inserts in tau structure (t3, t4) both CaM kinase II and C-kinase phosphorylated four-repeat tau (t4) to a higher extent than three-repeat tau (t3). When two N-terminal inserts are present in tau structure (t3L, t4L), then three-repeat tau (t3L) is phosphorylated to a higher extent than four-repeat tau (t4L) by these kinases. CK-1 and GSK-3 phosphorylated each of the above pairs of three-repeat and four-repeat taus to the same extents. However, after an initial prephosphorylation of the taus by CaM kinase II, GSK-3 differentially phosphorylated three-repeat and four-repeat taus. Under these conditions thr 231, ser 235, ser 396, and ser 404 were phosphorylated to greater extents in four-repeat tau (t4) compared to three-repeat tau (t3) in the absence of N-terminal inserts. In the presence of such inserts these sites were phosphorylated to greater extents in three-repeat (t3L) compared to four-repeat (t4L) tau. Our results indicate that the extents to which tau isoforms are phosphorylated in normal and AD brain depends on (a) the number of repeats (3 or 4), (b) the number of N-terminal inserts (0, 1, or 2), and (c) the initial phosphorylation state of tau.     

Development and assessment of sensitive immuno‐PCR assays for the quantification of cerebrospinal fluid three‐ and four‐repeat tau isoforms in tauopathies

Characteristic tau isoform composition of the insoluble fibrillar tau inclusions define tauopathies, including Alzheimer's disease (AD), progressive supranuclear palsy (PSP) and frontotemporal dementia with parkinsonism linked to chromosome 17/frontotemporal lobar degeneration‐tau (FTDP‐17/FTLD‐tau). Exon 10 splicing mutations in the tau gene, MAPT, in familial FTDP‐17 cause elevation of tau isoforms with four microtubule‐binding repeat domains (4R‐tau) compared to those with three repeats (3R‐tau). On the basis of two well‐characterised monoclonal antibodies against 3R‐ and 4R‐tau, we developed novel, sensitive immuno‐PCR assays for measuring the trace amounts of these isoforms in CSF. This was with the aim of assessing if CSF tau isoform changes reflect the pathological changes in tau isoform homeostasis in the degenerative brain and if these would be relevant for differential clinical diagnosis. Initial analysis of clinical CSF samples of PSP (n = 46), corticobasal syndrome (CBS; n = 22), AD (n = 11), Parkinson's disease with dementia (PDD; n = 16) and 35 controls revealed selective decreases of immunoreactive 4R‐tau in CSF of PSP and AD patients compared with controls, and lower 4R‐tau levels in AD compared with PDD. These decreases could be related to the disease‐specific conformational masking of the RD4‐binding epitope because of abnormal folding and/or aggregation of the 4R‐tau isoforms in tauopathies or increased sequestration of the 4R‐tau isoforms in brain tau pathology.     

Tangle evolution linked to differential 3- and 4-repeat tau isoform deposition: a double immunofluorolabeling study using two monoclonal antibodies

Double immunofluorolabeling for 3-repeat (3R) and 4-repeat (4R) tau was performed with two monoclonal antibodies, RD3 and RD4, after an additional pretreatment with potassium permanganate and oxalic acid to eliminate nonspecific 3R tau cytoplasmic staining. This method involves hyperdilution of one of the primary monoclonal antibodies (≥100-fold), making it undetectable by usual secondary antibodies. The hyperdiluted primary antibody can then only be detected after tyramide amplification. Subsequent application of the other monoclonal antibody at its usual concentration allows double immunofluorolabeling without cross-reaction. This novel method revealed that tau immunoreactivity (IR) in the hippocampal pyramidal neurons of Alzheimer’s disease (AD) brains is heterogeneous in that pretangle neurons exhibit 4R-selective (3R−/4R+) IR, ghost tangles exhibit 3R-selective (3R+/4R−) IR, and neurofibrillary tangles exhibit both 3R and 4R (3R+/4R+) IR. Some nigral neurons exhibited RD3 IR in both AD and corticobasal degeneration/progressive supranuclear palsy (CBD/PSP) brains. However, in CBD/PSP cases, 3R IR was always superimposed on 4R IR, while 3R-selective neurons were present in AD cases. These differential isoform profiles may provide a pivotal molecular reference, closely related to the morphological evolution of tau-positive neurons, which may be variable according to disease (CBD/PSP vs. AD), lesion site (cerebral cortex and substantia nigra), or the stage of evolution (from pretangles to ghost tangles). These findings should provide a more comprehensive understanding of the histological differentiation of various tau deposits in human neurodegenerative disease.     

Yeast surface display of full-length human microtubule-associated protein tau

Microtubule-associated protein tau is an intrinsically disordered, highly soluble protein found primarily in neurons. Under normal conditions, tau regulates the stability of axonal microtubules and intracellular vesicle transport. However, in patients of neurodegeneration such as Alzheimer's disease (AD), tau forms neurofibrillary deposits, which correlates well with the disease progression. Identifying molecular signatures in tau, such as posttranslational modification, truncation, and conformational change has great potential to detect earliest signs of neurodegeneration and develop therapeutic strategies. Here, we show that full-length human tau, including the longest isoform found in the adult brain, can be robustly displayed on the surface of yeast Saccharomyces cerevisiae. Yeast-displayed tau binds to anti-tau antibodies that cover epitopes ranging from the N-terminus to the 4R repeat region. Unlike tau expressed in the yeast cytosol, surface-displayed tau was not phosphorylated at sites found in AD patients (probed by antibodies AT8, AT270, AT180, and PHF-1). However, yeast-displayed tau showed clear binding to paired helical filament (PHF) tau conformation-specific antibodies Alz-50, MC-1, and Tau-2. Although the tau possessed a conformation found in PHFs, oligomerization or aggregation into larger filaments was undetected. Taken together, yeast-displayed tau enables robust measurement of protein interactions and is of particular interest for characterizing conformational change.     

A thiol-based intramolecular redox switch in four-repeat tau controls fibril assembly and disassembly

Oxidative stress has been implicated in the pathogenesis and progression of several tauopathies, including Alzheimer''s disease. The deposition of fibrillar inclusions made of tau protein is one of the pathological hallmarks of these disorders. Although it is becoming increasingly evident that the specific fibril structure may vary from one tauopathy to another and it is recognized that different types of isoforms (three-repeat and four-repeat tau) can be selectively deposited, little is known about the role oxidation may play in aggregation. Four-repeat tau contains two cysteines that can form an intramolecular disulfide bond, resulting in a structurally restrained compact monomer. There is discrepancy as to whether this monomer can aggregate or not. Using isolated four-repeat tau monomers (htau40) with intramolecular disulfide bonds, we demonstrate that these proteins form fibrils. The fibrils are less stable than fibrils formed under reducing conditions but are highly effective in seeding oxidized tau monomers. Conversely, a strong seeding barrier prevents incorporation of reduced tau monomers, tau mimics in which the cysteines have been replaced by alanines or serines, and three-repeat tau (htau23), a single-cysteine isoform. The barrier also holds true when seed and monomer types are reversed, indicating that oxidized and reduced tau are incompatible with each other. Surprisingly, fibrils composed of compact tau disaggregate upon reduction, highlighting the importance of the intramolecular disulfide bond for fibril stability. The findings uncover a novel binary redox switch that controls the aggregation and disaggregation of these fibrils and extend the conformational spectrum of tau aggregates.     

Competition for microtubule-binding with dual expression of tau missense and splice isoforms

How tau mutations lead to neurodegeneration is unknown but may be related to altered microtubule binding properties of mutant tau protein. The tendency for the mutations to cluster around the microtubule-binding domain of tau or to alter the ratios of those splice isoforms that affect binding supports the view that the tau/microtubule interaction is critical and finely regulated. In cells transfected with both mutant and wild-type tau isoforms fused to either yellow fluorescent protein or cyan fluorescent protein we can observe tau fusion proteins that differ by a single amino acid or by the inclusion or exclusion of exon 10. With coexpression of mutant and wild-type tau, the mutant isoform appears diffuse throughout the cytoplasm; however, when mutant tau is expressed alone, it appears mostly bound to the microtubules. Dual imaging of the three- and four-repeat tau isoforms indicated that the expression of four-repeat tau displaced three-repeat tau from the microtubules. These results suggest that altered kinetic competition among the isoforms for microtubule binding could be a disease precipitant.     

Three- and four-repeat tau regulate the dynamic instability of two distinct microtubule subpopulations in qualitatively different manners. Implications for neurodegeneration

The microtubule-associated protein tau is implicated in the pathogenesis of many neurodegenerative diseases, including fronto-temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), in which both RNA splicing and amino acid substitution mutations in tau cause dominantly inherited early onset dementia. RNA-splicing FTDP-17 mutations alter the wild-type approximately 50:50 3-repeat (3R) to 4-repeat (4R) tau isoform ratio, usually resulting in an excess of 4R tau. To examine further how splicing mutations might cause dysfunction by misregulation of microtubule dynamics, we used video microscopy to determine the in vitro behavior of individual microtubules stabilized by varying amounts of human 4R and 3R tau. At low tau:tubulin ratios (1:55 and 1:45), all 3R isoforms reduced microtubule growth rates relative to the no-tau control, whereas all 4R isoforms increased them; however, at a high tau:tubulin ratio (1:20), both 4R and 3R tau increased the growth rates. Further analysis revealed two distinct subpopulations of growing microtubules in the absence of tau. Increasing concentrations of both 4R and 3R tau resulted in an increase in the size of the faster growing subpopulation of microtubules; however, 4R tau caused a redistribution to the faster growing subpopulation at lower tau:tubulin ratios than 3R tau. This modulation of discrete growth rate subpopulations by tau suggests that tau causes a conformational shift in the microtubule resulting in altered dynamics. Quantitative and qualitative differences observed between 4R and 3R tau are consistent with a "microtubule misregulation" model in which abnormal tau isoform expression results in the inability to properly regulate microtubule dynamics, leading to neuronal death and dementia.     

Promotion of hyperphosphorylation by frontotemporal dementia tau mutations

Mutations in the tau gene are known to cosegregate with the disease in frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). However, the molecular mechanism by which these mutations might lead to the disease is not understood. Here, we show that four of the FTDP-17 tau mutations, R406W, V337M, G272V, and P301L, result in tau proteins that are more favorable substrates for phosphorylation by brain protein kinases than the wild-type, largest four-repeat protein tau4L and tau4L more than tau3L. In general, at all the sites studied, mutant tau proteins were phosphorylated faster and to a higher extent than tau4L and tau4L > tau3L. The most dramatic difference found was in the rate and level of phosphorylation of tau4L(R406W) at positions Ser-396, Ser-400, Thr-403, and Ser-404. Phosphorylation of this mutant tau was 12 times faster and 400% greater at Ser-396 and less than 30% at Ser-400, Thr-403, and Ser-404 than phosphorylation of tau4L. The mutated tau proteins polymerized into filaments when 4-6 mol of phosphate per mol of tau were incorporated, whereas wild-type tau required approximately 10 mol of phosphate per mol of protein to self-assemble. Mutated and wild-type tau proteins were able to sequester normal tau upon incorporation of approximately 4 mol of phosphate per mol of protein, which was achieved at as early as 30 min of phosphorylation in the case of mutant tau proteins. These findings taken together suggest that the mutations in tau might cause neurodegeneration by making the protein a more favorable substrate for hyperphosphorylation.     

Variations in filament conformation dictate seeding barrier between three- and four-repeat tau

Tau filaments are the pathological hallmark of >20 neurodegenerative diseases including Alzheimer's disease. Six tau isoforms exist that can be grouped into 4-repeat (4R) tau and 3-repeat (3R) tau based on the presence or absence of the second of four microtubule binding repeats. Recent evidence suggests that tau filaments can transfer between cells and spread through the brain. Here we demonstrate in vitro that seeded filament growth, a prerequisite for tau spreading, is crucially dependent on the isoform composition of individual seeds. Seeds of 3R tau and 3R/4R tau recruit both types of isoforms. Seeds of 4R tau recruit 4R tau, but not 3R tau, establishing an asymmetric barrier. Conformational templating of 4R tau onto 3R tau seeds eliminates this barrier, giving rise to a new type of tau filament. These findings provide fundamental mechanistic insights into the seeding, propagation, and diversification of tau filaments.     

Aggregation analysis of the microtubule binding domain in tau protein by spectroscopic methods

The microtubule-associated protein tau is a highly soluble protein that shows hardly any tendency to assemble under physiological conditions. In the brains of Alzheimer's disease (AD) patients, however, tau dissociates from the axonal microtubule and abnormally aggregates to form paired helical filaments (PHFs). One of the priorities in Alzheimer research is to clarify the mechanism of PHF formation. In recent years, several factors regulating tau assembly have come to light, yet some important questions remain to be answered. In this work, the His-tagged gene constructs of the four-repeat microtubule binding domain (4RMBD) in tau protein and its three mutants, 4RMBD S305N, N279K, and P301L, were expressed in E. coli and purified. Gel filtration chromatography and dynamic light scattering measurement yielded a Stokes radius of 3.1 nm, indicating that the His-tagged 4RMBD normally exists in buffer solution in a dimer state, which is formed by non-covalent intermolecular interactions. This non-covalent dimer can further polymerize to form filaments in the presence of polyanions such as heparin. The kinetics of the in vitro aggregation was monitored by thioflavine S dye fluorescence and CD measurements. The aggregation of 4RMBD was suggested to be a nucleation-dependent process, where the non-covalent dimer acts as an effective structural unit. The aggregation rate was strongly affected by the point mutation. Among the 4RMBD mutants, the rate of S305N was exceptionally fast, whereas N279K was the slowest, even slower than the wild-type. The aggregations were optimal in a weakly reducing environment for all the mutants and the wild type. However, the aggregations were affected differently by buffer pH, depending on the 4RMBD mutation.     

Linkage-dependent contribution of repeat peptides to self-aggregation of three- or four-repeat microtubule-binding domains in tau protein

Although one of the priorities in Alzheimer's research is to clarify the filament formation mechanism for the tau protein, it is still unclear how it is transformed from a normal structure in a neuron. To examine the linkage-dependent contribution of each repeat peptide (R1-R4) to filament formation of the three- or four-repeat microtubule-binding domain (MBD) in the tau protein, four two-repeat peptides (R12, R13, R23 and R34) and two three-repeat peptides (R123 and R234) were prepared, and their in vitro self-aggregation was investigated by thioflavin S fluorescence and circular dichroism measurements, and by electron microscopy in neutral buffer (pH 7.6). Comparison of these aggregation behaviors with previous results for single-repeat peptides and wild-type 3RMBD (R134) and 4RMBD (R1234) indicated that (a) the two-repeat R23, not the R2 or R3 single repeat, forms the core structure in self-aggregation of 4RMBD, whereas that of 3RMBD comprises the R3 single repeat, (b) co-existence of R1 and R4 repeats is necessary for the aggregation behavior inherent in 3RMBD and 4RMBD, whereas the R1 or R4 repeat alone functions as a repressor or modifier of the filament formation, (c) 4RMBD aggregation is accompanied by R1-driven transition from random and alpha-helix structures to a beta-sheet structure, whereas 3RMBD aggregation involves three-repeat R134-specific transition from a random structure to an alpha-helix structure without the participation of a beta-sheet structure, and (d) the peptides that include the R1 repeat form a long filament irrespective of the absence or presence of the R4 repeat, whereas those that include the R4 repeat, but not the R1 repeat, form a relatively short filament. To the best of our knowledge, a systematic study of the linkage-dependent contribution of each repeat peptide to the paired helical filament formation of tau MBD has not been carried out previously, and thus the present information is useful for understanding the essence of the filament formation of tau MBD.     

Reduced binding of protein phosphatase 2A to tau protein with frontotemporal dementia and parkinsonism linked to chromosome 17 mutations

Coding region and intronic mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We have previously reported that ABalphaC, a major form of protein phosphatase 2A (PP2A) in brain, binds tightly to tau protein in vitro and is a major tau phosphatase in vivo. Using in vitro assays, we show here that the FTDP-17 mutations G272V, DeltaK280, P301L, P301S, S305N, V337M, G389R, and R406W inhibit by approximately 20-95% the binding of recombinant three-repeat and four-repeat tau isoforms to the ABalphaC holoenzyme and the AC core enzyme of PP2A. Reduction in binding was maximal for tau proteins with the G272V, DeltaK280, and V337M mutations. We also show that tau protein can be specifically coimmunoprecipitated with endogenous PP2A from both rat brain and transfected cell extracts. It is significant that, by using similar coimmunoprecipitation assays, we show that all FTDP-17 mutations tested, including the N279K mutation, alter the ability of tau to associate with cellular PP2A. Taken together, these results indicate that FTDP-17 mutations induce a significant decrease in the binding affinity of tau for PP2A in vivo. We propose that altered protein-protein interactions between PP2A and tau may contribute to FTDP-17 pathogenesis.     

Possible role of each repeat structure of the microtubule-binding domain of the tau protein in in vitro aggregation

Although one of the priorities in Alzheimer's research is to clarify the filament formation mechanism of the tau protein, it is currently unclear how it is transformed from a normal structure in a neuron. To examine which part and what structural change in the tau protein are involved in its transformation into a pathological entity, the initial in vitro self-aggregation features of each repeat peptide (R1-R4) constituting a three- or four-repeat microtubule-binding domain (3RMBD or 4RMBD) in the tau protein was investigated by measuring both the fluorescence and light scattering (LS) spectra on the same instrument, because these MBD domains constitute the core moiety of the tau paired helical filament (PHF) structure. The conformational features of the R1 and R4 peptides in trifluoroethanol were also investigated by (1)H-NMR and molecular modeling analyses and compared with those of the R2 and R3 peptides. The analyses of the LS spectra clarified (i) the self-aggregation rates of R1-R4, 3RMBD and 4RMBD at a fixed concentration (15 mM), (ii) their minimum concentrations for starting filament extension, and (iii) the concentration dependence of their self-aggregations. The fluorescence analyses showed that the R2 and R3 peptides have high self-aggregation abilities at the extension and nucleation steps, respectively, in their filament formation processes. It was shown that the R2 repeat exhibits a positive synergistic effect on the aggregation of 4RMBD. The R1 and R4 repeats, despite their weak self-aggregation abilities, are necessary for the intact PHF formation of tau MBD, whereas they exerted a negative effect on the R3-driven aggregation of 3RMBD. The conformational analyses showed the importance of the amphipathic conformational features of the R1 to R4 peptides, and the intermolecular disulfide bonding abilities of the R2 and R3 peptides for the PHF formation. On the basis of the present spectral and conformational results, the possible role of each repeat structure in the dimeric formation of MBD at the initial in vitro aggregation stage is discussed.     

Dual vulnerability of tau to calpains and caspase-3 proteolysis under neurotoxic and neurodegenerative conditions

Axonally specific microtubule-associated protein tau is an important component of neurofibrillary tangles found in AD (Alzheimer''s disease) and other tauopathy diseases such as CTE (chronic traumatic encephalopathy). Such tau aggregate is found to be hyperphosphorylated and often proteolytically fragmented. Similarly, tau is degraded following TBI (traumatic brain injury). In the present study, we examined the dual vulnerability of tau to calpain and caspase-3 under neurotoxic and neurodegenerative conditions. We first identified three novel calpain cleavage sites in rat tau (four-repeat isoform) as Ser¹³⁰↓Lys¹³¹, Gly¹⁵⁷↓Ala¹⁵⁸ and Arg³⁸⁰↓Glu³⁸¹. Fragment-specific antibodies to target the major calpain-mediated TauBDP-35K (35 kDa tau-breakdown product) and the caspase-mediated TauBDP-45K respectively were developed. In rat cerebrocortical cultures treated with excitotoxin [NMDA (N-methyl-d-aspartate)], tau is significantly degraded into multiple fragments, including a dominant signal of calpain-mediated TauBDP-35K with minimal caspase-mediated TauBDP-45K. Following apoptosis-inducing EDTA treatment, tau was truncated only to TauBDP-48K/45K-exclusively by caspase. Cultures treated with another apoptosis inducer STS (staurosporine), dual fragmentation by calpain (TauBDP-35K) and caspase-3 (TauBDP-45K) was observed. Tau was also fragmented in injured rat cortex following TBI in vivo to BDPs of 45–42 kDa (minor), 35 kDa and 15 kDa, followed by TauBDP-25K. Calpain-mediated TauBDP-35K-specific antibody confirmed robust signals in the injured cortex, while caspase-mediated TauBDP-45K-specific antibody only detected faint signals. Furthermore, intravenous administration of a calpain-specific inhibitor SNJ-1945 strongly suppressed the TauBDP-35K formation. Taken together, these results suggest that tau protein is dually vulnerable to calpain and caspase-3 proteolysis under different neurotoxic and injury conditions.     

Monte carlo simulations of tau proteins: effect of phosphorylation

We perform Monte Carlo simulations of tau proteins bound to a cylinder that mimics a microtubule (MT), and then study them in solution. Tau protein binds to a highly anionic MT surface to stabilize the cylindrical structure of MT. The negatively charged tail domain floats away from the anionic MT surface while positively charged tau segments localize near the MT surface. Monte Carlo simulations demonstrate that, in 3RS tau isoform (which has three imperfect repeats (R) short (S) isoform), amino acids are more condensed near a highly charged interface compared to 4RL isoform (which has four imperfect repeats (R) long (L) isoform). In 4RL isoform, amino acids in tail domain stay mostly apart from the MT surface. In the bulk solution, dephosphorylated taus are separated due to Coulomb repulsion between similarly charged isoforms. Moderate phosphorylation of 3RS isoform decreases average intermolecular distance between dephosphorylated and phosphorylated taus and lead to their overlap. Further phosphorylation does not change noticeably the intermolecular distances.     

Pseudohyperphosphorylation has differential effects on polymerization and function of tau isoforms

The microtubule-associated protein tau exists as six isoforms created through the splicing of the second, third, and tenth exons. The isoforms are classified by their number of N-terminal exons (0N, 1N, or 2N) and by their number of microtubule-binding repeat regions (3R or 4R). Hyperphosphorylated isoforms accumulate in insoluble aggregates in Alzheimer's disease and other tauopathies. These neurodegenerative diseases can be categorized based on the isoform content of the aggregates they contain. Hyperphosphorylated tau has the general characteristics of an upward electrophoretic shift, decreased microtubule binding, and an association with aggregation. Previously we have shown that a combination of seven pseudophosphorylation mutations at sites phosphorylated by GSK-3β, referred to as 7-Phos, induced several of these characteristics in full-length 2N4R tau and led to the formation of fewer but longer filaments. We sought to determine whether the same phosphorylation pattern could cause differential effects in the other tau isoforms, possibly through varied conformational effects. Using in vitro techniques, we examined the electrophoretic mobility, aggregation properties, and microtubule stabilization of all isoforms and their pseudophosphorylated counterparts. We found that pseudophosphorylation affected each isoform, but in several cases certain isoforms were affected more than others. These results suggest that hyperphosphorylation of tau isoforms could play a major role in determining the isoform composition of tau aggregates in disease.     

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.     

Three- and four-repeat Tau coassemble into heterogeneous filaments: an implication for Alzheimer disease

Tau filaments are the pathological hallmark of numerous neurodegenerative diseases including Alzheimer disease, Pick disease, and progressive supranuclear palsy. In the adult human brain, six isoforms are expressed that differ by the presence or absence of the second of four semiconserved repeats. As a consequence, half of the tau isoforms have three repeats (3R tau), whereas the other half of the isoforms have four repeats (4R tau). Tauopathies can be characterized based on the isoform composition of their filaments. Alzheimer disease filamentous inclusions contain all isoforms. Pick disease filaments contain 3R tau. Progressive supranuclear palsy filaments contain 4R tau. Here, we used site-directed spin labeling of recombinant tau in conjunction with electron paramagnetic resonance spectroscopy to obtain structural insights into these filaments. We find that filaments of 4R tau and 3R tau share a highly ordered core structure in the third repeat with parallel, in-register arrangement of β-strands. This structure is conserved regardless of whether full-length isoforms (htau40 and htau23) or truncated constructs (K18 and K19) are used. When mixed, 3R tau and 4R tau coassemble into heterogeneous filaments. These filaments share the highly ordered core in the third repeat; however, they differ in their overall composition. Our findings indicate that at least three distinct types of filaments exist: homogeneous 3R tau, homogeneous 4R tau, and heterogeneous 3R/4R tau. These results suggest that individual filaments found in Alzheimer disease are structurally distinct from those in the 3R and 4R tauopathies.     

Changed conformation of mutant Tau-P301L underlies the moribund tauopathy, absent in progressive, nonlethal axonopathy of Tau-4R/2N transgenic mice

Protein tau-3R/4R isoform ratio and phosphorylation regulates binding to microtubules and, when disturbed by aging or mutations, results in diverse tauopathies and in neurodegeneration. The underlying mechanisms were studied here in three transgenic mouse strains with identical genetic background, all expressing the tau-4R/2N isoform driven specifically in neurons by the thy1 gene promoter. Two strains, expressing human tau-4R/2N or mutant tau-4R/2N-P301L at similar, moderate levels, developed very different phenotypes. Tau-4R/2N mice became motor-impaired already around age 6-8 weeks, accompanied by axonopathy (dilatations, spheroids), but no tau aggregates, and surviving normally. In contrast, tau-P301L mice developed neurofibrillary tangles from age 6 months, without axonal dilatations and, despite only minor motor problems, all succumbing before the age of 13 months. The third strain, obtained by tau knock-out/knock-in (tau-KOKI), expressed normal levels of wild-type human tau-4R/2N replacing all mouse tau isoforms. Tau-KOKI mice survived normally with minor motor problems late in life and without any obvious pathology. Biochemically, a fraction of neuronal tau in aging tau-P301L mice was hyperphosphorylated concomitant with conformational changes and aggregation, but overall, tau-4R/2N was actually more phosphorylated than tau-P301L. Significantly, tau with changed conformation and with hyperphosphorylation colocalized in the same neurons in aging tau-P301L mice. Taken together, we conclude that excessive binding of tau-4R/2N as opposed to reduced binding of tau-P301L to microtubules is responsible for the development of axonopathy and tauopathy, respectively, in tau-4R/2N and tau-P301L mice and that the conformational change of tau-P301L is a major determinant in triggering the tauopathy.