Inability of tau to properly regulate neuronal microtubule dynamics: a loss-of-function mechanism by which tau might mediate neuronal cell death

Interest in the microtubule-associated protein tau stems from its critical roles in neural development and maintenance, as well as its role in Alzheimer's, FTDP-17 and related neurodegenerative diseases. Under normal circumstances, tau performs its functions by binding to microtubules and powerfully regulating their stability and growing and shortening dynamics. On the other hand, genetic analyses have established a clear cause-and-effect relationship between tau dysfunction/mis-regulation and neuronal cell death and dementia in FTDP-17, but the molecular basis of tau's destructive action(s) remains poorly understood. One attractive model suggests that the intracellular accumulation of abnormal tau aggregates causes cell death, i.e., a gain-of-toxic function model. Here, we describe the evidence and arguments for an alternative loss-of-function model in which tau-mediated neuronal cell death is caused by the inability of affected cells to properly regulate their microtubule dynamic due to mis-regulation by tau. In support of this model, our recent data demonstrate that missense FTDP-17 mutations that alter amino acid residues near tau's microtubule binding region strikingly modify the ability of tau to modulate microtubule dynamics. Additional recent data from our labs support the notion that the same dysfunction occurs in the FTDP-17 regulatory mutations that alter tau RNA splicing patterns. Our model posits that the dynamics of microtubules in neuronal cells must be tightly regulated to enable them to carry out their diverse functions, and that microtubules that are either over-stabilized or under-stabilized, that is, outside an acceptable window of dynamic activity, lead to neurodegeneration. An especially attractive aspect of this model is that it readily accommodates both the structural and regulatory classes of FTDP-17 mutations.     

Distinct FTDP-17 missense mutations in tau produce tau aggregates and other pathological phenotypes in transfected CHO cells

Multiple tau gene mutations are pathogenic for hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), with filamentous tau aggregates as the major lesions in the CNS of these patients. Recent studies have shown that bacterially expressed recombinant tau proteins with FTDP-17 missense mutations cause functional impairments, i.e., a reduced ability of mutant tau to bind to or promote the assembly of microtubules. To investigate the biological consequences of FTDP-17 tau mutants and assess their ability to form filamentous aggregates, we engineered Chinese hamster ovary cell lines to stably express tau harboring one or several different FTDP-17 mutations and showed that different tau mutants produced distinct pathological phenotypes. For example, delta K, but not several other single tau mutants (e.g., V337 M, P301L, R406W), developed insoluble amorphous and fibrillar aggregates, whereas a triple tau mutant (VPR) containing V337M, P301L, and R406W substitutions also formed similar aggregates. Furthermore, the aggregates increased in size over time in culture. Significantly, the formation of aggregated delta K and VPR tau protein correlated with reduced affinity of these mutants to bind microtubules. Reduced phosphorylation and altered proteolysis was also observed in R406W and delta K tau mutants. Thus, distinct pathological phenotypes, including the formation of insoluble filamentous tau aggregates, result from the expression of different FTDP-17 tau mutants in transfected Chinese hamster ovary cells and implies that these missense mutations cause diverse neurodegenerative FTDP-17 syndromes by multiple mechanisms.     

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.     

Regulation of tau isoform expression and dementia

In the central nervous system (CNS), aberrant changes in tau mRNA splicing and consequently in protein isoform ratios cause abnormal aggregation of tau and neurodegeneration. Pathological tau causes neuronal loss in Alzheimer's disease (AD) and a diverse group of disorders called the frontotemporal dementias (FTD), which are two of the most common forms of dementia and afflict more than 10% of the elderly population. Autosomal dominant mutations in the tau gene cause frontotemporal dementia with parkinsonism-chromosome 17 type (FTDP-17). Just over half the mutations affect tau protein function and decrease its affinity for microtubules (MTs) or increase self-aggregation. The remaining mutations occur within exon 10 (E10) and intron 10 sequences and alter complex regulation of E10 splicing by multiple mechanisms. FTDP-17 splicing mutations disturb the normally balanced levels of distinct protein isoforms that result in altered biochemical and structural properties of tau. In addition to FTDP-17, altered tau isoform levels are also pathogenically associated with other FTD disorders such as progressive supranuclear palsy (PSP), corticobasal degeneration and Pick's disease; however, the mechanisms remain undefined and mutations in tau have not been detected. FTDP-17 highlights the association between splicing mutations and the pronounced variability in pathology as well as phenotype that is characteristic of inherited disorders.     

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.     

The natural osmolyte trimethylamine N-oxide (TMAO) restores the ability of mutant tau to promote microtubule assembly

Coding region and intronic mutations in the gene for microtubule-associated protein tau cause frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Most coding region mutations effect a reduced ability of tau protein to interact with microtubules and lead to the formation of a filamentous pathology made of hyperphosphorylated tau. Here we show that trimethylamine N-oxide (TMAO) restores the ability of tau with FTDP-17 mutations to promote microtubule assembly. To mimic phosphorylation, serine and threonine residues in tau were singly or multiply mutated to glutamic acid, resulting in a reduced ability of tau to promote microtubule assembly. With the exception of the most heavily substituted protein (27 glutamic acid residues), TMAO increased the ability of mutant tau to promote microtubule assembly. However, it had no significant effect on heparin-induced assembly of tau into filaments.     

Interaction of tau protein with the dynactin complex

Tau is an axonal microtubule-associated protein involved in microtubule assembly and stabilization. Mutations in Tau cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and tau aggregates are present in Alzheimer's disease and other tauopathies. The mechanisms leading from tau dysfunction to neurodegeneration are still debated. The dynein-activator complex dynactin has an essential role in axonal transport and mutations in its gene are associated with lower motor neuron disease. We show here for the first time that the N-terminal projection domain of tau binds to the C-terminus of the p150 subunit of the dynactin complex. Tau and dynactin show extensive colocalization, and the attachment of the dynactin complex to microtubules is enhanced by tau. Mutations of a conserved arginine residue in the N-terminus of tau, found in patients with FTDP-17, affect its binding to dynactin, which is abnormally distributed in the retinal ganglion cell axons of transgenic mice expressing human tau with a mutation in the microtubule-binding domain. These findings, which suggest a direct involvement of tau in axonal transport, have implications for understanding the pathogenesis of tauopathies.     

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.     

Effects of frontotemporal dementia FTDP-17 mutations on heparin-induced assembly of tau filaments

Missense mutations and intronic mutations in the gene for microtubule-associated protein tau cause frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Most missense mutations have as likely primary effect a reduced ability of tau to interact with microtubules. We report here an additional effect of several missense mutations, namely the stimulation of heparin-induced filament assembly of recombinant tau, despite the absence of any change in structure indicated by circular dichroism. These findings indicate that missense mutations in tau lead to frontotemporal dementia through potentially multiple mechanisms.     

Structure, microtubule interactions, and paired helical filament aggregation by tau mutants of frontotemporal dementias

We have studied biochemical and structural parameters of several missense and deletion mutants of tau protein (G272V, N279K, DeltaK280, P301L, V337M, R406W) found in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). The mutant proteins were expressed on the basis of both full-length tau (htau40) and constructs derived from the repeat domain. They were analyzed with respect to the capacity to enhance microtubule assembly, binding of tau to microtubules, secondary structure content, and aggregation into Alzheimer-like paired helical or straight filaments. We find that the mutations cause a moderate decrease in microtubule interactions and stabilization, and they show no gross structural changes compared with the natively unfolded conformation of the wild-type protein, but the aggregation into PHFs is strongly enhanced, particularly for the mutants DeltaK280 and P301L. This gain of pathological aggregation would be consistent with the autosomal dominant nature of the disease.     

Tau aggregation is driven by a transition from random coil to beta sheet structure

The abnormal aggregation of the microtubule associated protein tau into paired helical filaments (PHFs) is one the hallmarks of Alzheimer's disease. The soluble protein is one of the longest natively unfolded proteins, lacking significant amounts of secondary structure over a sequence of 441 amino acids in the longest isoform. Furthermore, the unfolded character is consistent with some notable features of the protein like stability towards heat and acid treatment. It is still unclear how these characteristics support the physiological function of binding to and stabilization of microtubules. We review here some recent studies on how an unfolded protein such as tau can adopt beta-structure, which then leads to the highly ordered morphology of the PHFs. The core sequence for both microtubule binding and PHF formation is the microtubule binding domain containing three or four repeats. This region alone is sufficient for PHF formation and mostly unfolded in the soluble state. A search for sequence motifs within this region crucial for PHF building revealed two hexapeptides in the second and the third repeat. Some of the genetically linked cases of FTDP-17 show missense mutations in or adjacent to these hexapeptide motifs. Proteins containing the P301L and the DeltaK280 mutations exhibit accelerated aggregation. The importance of the two hexapeptides stems from their capacity to undergo a conformational change from a random coil to a beta sheet structure. The increase of beta sheet structure is a typical feature of an amyloidogenic protein and is the basis of other characteristics like a decreased sensitivity towards proteolytic degradation and Congo red binding. PHFs aggregated in vitro and in vivo contain beta-sheet structure, as judged by circular dichroism (CD) spectroscopy, Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction.     

Mutations causing neurodegenerative tauopathies

Tau is the major component of the intracellular filamentous deposits that define a number of neurodegenerative diseases. They include the largely sporadic Alzheimer's disease (AD), progressive supranuclear palsy, corticobasal degeneration, Pick's disease and argyrophilic grain disease, as well as the inherited frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). For a long time, it was unclear whether the dysfunction of tau protein follows disease or whether disease follows tau dysfunction. This was resolved when mutations in Tau were found to cause FTDP-17. Currently, 32 different mutations have been identified in over 100 families. About half of the known mutations have their primary effect at the protein level. They reduce the ability of tau protein to interact with microtubules and increase its propensity to assemble into abnormal filaments. The other mutations have their primary effect at the RNA level and perturb the normal ratio of three-repeat to four-repeat tau isoforms. Where studied, this resulted in a relative overproduction of tau protein with four microtubule-binding domains in the brain. Individual Tau mutations give rise to diseases that resemble progressive supranuclear palsy, corticobasal degeneration or Pick's disease. Moreover, the H1 haplotype of Tau has been identified as a significant risk factor for progressive supranuclear palsy and corticobasal degeneration. At a practical level, the new work is leading to the production of experimental animal models that reproduce the essential molecular and cellular features of the human tauopathies, including the formation of abundant filaments made of hyperphosphorylated tau protein and nerve cell degeneration.     

Functional effects of tau gene mutations deltaN296 and N296H

Mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome-17 (FTDP-17). Functionally, about half of the known mutations increase the alternative mRNA splicing of exon 10 of the tau gene, resulting in the overproduction of tau isoforms with four microtubule-binding repeats. The other mutations reduce the ability of tau to interact with microtubules, with some mutations also increasing the propensity of tau to assemble into filaments. Here we have examined the functional effects of the recently described tau gene mutations deltaN296 and N296H. Both mutations reduced the ability of tau to promote microtubule assembly, without having a significant effect on tau filament formation. By exon trapping, they increased the splicing of exon 10. DeltaN296 and N296H thus define a class of tau mutations with effects at both the RNA and the protein level.     

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.     

Age-dependent emergence and progression of a tauopathy in transgenic mice overexpressing the shortest human tau isoform

Filamentous tau aggregates are hallmarks of tauopathies, e.g., frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) and amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC). Since FTDP-17 tau gene mutations alter levels/functions of tau, we overexpressed the smallest human tau isoform in the CNS of transgenic (Tg) mice to model tauopathies. These mice acquired age-dependent CNS pathology similarto FTDP-17 and ALS/PDC, including insoluble, hyperphosphorylated tau and argyrophilic intraneuronal inclusions formed by tau-immunoreactive filaments. Inclusions were present in cortical and brainstem neurons but were most abundant in spinal cord neurons, where they were associated with axon degeneration, diminished microtubules (MTs), and reduced axonal transport in ventral roots, as well as spinal cord gliosis and motor weakness. These Tg mice recapitulate key features of tauopathies and provide models for elucidating mechanisms underlying diverse tauopathies, including Alzheimer's disease (AD).     

FTDP-17 mutations compromise the ability of tau to regulate microtubule dynamics in cells

The neural microtubule-associated protein Tau binds directly to microtubules and regulates their dynamic behavior. In addition to being required for normal development, maintenance, and function of the nervous system, Tau is associated with several neurodegenerative diseases, including Alzheimer disease. One group of neurodegenerative dementias known as FTDP-17 (fronto-temporal dementia with Parkinsonism linked to chromosome 17) is directly linked genetically to mutations in the tau gene, demonstrating that Tau misfunction can cause neuronal cell death and dementia. These mutations result either in amino acid substitutions in Tau or in altered Tau mRNA splicing that skews the expression ratio of wild-type 3-repeat and 4-repeat Tau isoforms. Because wild-type Tau regulates microtubule dynamics, one possible mechanism underlying Tau-mediated neurodegeneration is aberrant regulation of microtubule behavior. In this study, we microinjected normal and mutated Tau protein into cultured cells expressing fluorescent tubulin and measured the effects on the dynamic instability of individual microtubules. We found that the FTDP-17 amino acid substitutions G272V (in both 3-repeat and 4-repeat Tau contexts), DeltaK280, and P301L all exhibited markedly reduced abilities to regulate dynamic instability relative to wild-type Tau. In contrast, the FTDP-17 R406W mutation (which maps in a regulatory region outside the microtubule binding domain of Tau) did not significantly alter the ability of 3-repeat or 4-repeat Tau to regulate microtubule dynamics. Overall, these data are consistent with a loss-of-function model in which both amino acid substitutions and altered mRNA splicing in Tau lead to neurodegeneration by diminishing the ability of Tau to properly regulate microtubule dynamics.     

Sites of tau important for aggregation populate {beta}-structure and bind to microtubules and polyanions

The aggregation of the microtubule-associated tau protein and formation of "neurofibrillary tangles" is one of the hallmarks of Alzheimer disease. The mechanisms underlying the structural transition of innocuous, natively unfolded tau to neurotoxic forms and the detailed mechanisms of binding to microtubules are largely unknown. Here we report the high-resolution characterization of the repeat domain of soluble tau using multidimensional NMR spectroscopy. NMR secondary chemical shifts detect residual beta-structure for 8-10 residues at the beginning of repeats R2-R4. These regions correspond to sequence motifs known to form the core of the cross-beta-structure of tau-paired helical filaments. Chemical shift perturbation studies show that polyanions, which promote paired helical filament aggregation, as well as microtubules interact with tau through positive charges near the ends of the repeats and through the beta-forming motifs at the beginning of repeats 2 and 3. The high degree of similarity between the binding of polyanions and microtubules supports the hypothesis that stable microtubules prevent paired helical filament formation by blocking the tau-polyanion interaction sites, which are crucial for paired helical filament formation.     

Tau mutations in frontotemporal dementia FTDP-17 and their relevance for Alzheimer's disease

Alzheimer's disease is characterised by the degeneration of selected populations of nerve cells that develop filamentous inclusions prior to degeneration. The neuronal inclusions of Alzheimer's disease are made of the microtubule-associated protein tau, in a hyperphosphorylated state. Abundant filamentous tau inclusions are not limited to Alzheimer's disease. They are the defining neuropathological characteristic of frontotemporal dementias, such as Pick's disease, and of progressive supranuclear palsy and corticobasal degeneration. The discovery of mutations in the tau gene in familial frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has provided a direct link between tau dysfunction and dementing disease. Known mutations produce either a reduced ability of tau to interact with microtubules, or an overproduction of tau isoforms with four microtubule-binding repeats. This leads in turn to the assembly of tau into filaments similar or identical to those found in Alzheimer's disease brain. Several missense mutations also have a stimulatory effect on heparin-induced tau filament formation. Assembly of tau into filaments may be the gain of toxic function that is believed to underlie the demise of affected brain cells.     

Alteration in calcium channel properties is responsible for the neurotoxic action of a familial frontotemporal dementia tau mutation

Tau, a microtubule binding protein, is not only a major component of neurofibrillary tangles in Alzheimer's disease, but also a causative gene for hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We show here that an FTDP-17 tau mutation (V337M) in SH-SY5Y cells reduces microtubule polymerization, increases voltage-dependent calcium current (ICa) density, and decreases ICa rundown. The reduced rundown of ICa by V337M was significantly inhibited by nifedipine (L-type Ca channel blocker), whereas omega-conotoxin GVIA (N-type Ca channel blocker) showed smaller effects, indicating that tau mutations affect L-type calcium channel activity. The depolarization-induced increase in intracellular calcium was also significantly augmented by the V337M tau mutation. Treatment with a microtubule polymerizing agent (taxol), an adenylyl cyclase inhibitor, or a protein kinase A (PKA) inhibitor, counteracted the effects of mutant tau on ICa. Taxol also attenuated the Ca2+ response to depolarization in cells expressing mutant tau. Apoptosis in SH-SY5Y cells induced by serum deprivation was exacerbated by the V337M mutation, and nifedipine, taxol, and a PKA inhibitor significantly protected cells against apoptosis. Our results indicate that a tau mutation which decreases its microtubule-binding ability augments calcium influx by depolymerizing microtubules and activating adenylyl cyclase and PKA.     

FTDP-17 tau mutations decrease the susceptibility of tau to calpain I digestion

Frontal temporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) is caused by splice site and missense mutations in the tau gene, and characterized by the accumulation of filamentous tau in cerebral neurons and glia. The missense mutations reduce the ability of tau to promote microtubule assembly and increase the ability of tau to form filaments. In this report we demonstrate that mutants V337M and R406W are less susceptible than mutant P301L or corresponding wild type tau to degradation by calpain I. The differences were at least in part due to changes in accessibility of a cleavage site located about 100 amino acids off the carboxy-terminus. The results suggest that the pathogenesis of some forms of FTDP-17 may involve tau accumulation due to decreased proteolytic degradation.