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.     

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.     

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.     

Phosphorylation of FTDP-17 mutant tau by cyclin-dependent kinase 5 complexed with p35, p25, or p39

One of the major pathological hallmarks of Alzheimer disease is neurofibrillary tangles. Neurofibrillary tangles are bundles of paired helical filaments composed of hyperphosphorylated tau. Cyclin-dependent kinase 5 (Cdk5) is one of the tau protein kinases that increase paired helical filament epitopes in tau by phosphorylation. Recently, various mutations of tau have been identified in frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Here, we investigated the phosphorylation of FTDP-17 mutant tau proteins, K257T, P301L, P301S, and R406W, by Cdk5 complexed with p35, p25, or p39 in vitro and in cultured cells. The extent of phosphorylation by all Cdk5 species was slightly lower in mutant tau than in wild-type tau. Major phosphorylation sites, including Ser202, Ser235, and Ser404, were the same among the wild-type, K257T, P301L, and P301S tau proteins phosphorylated by any Cdk5. On the other hand, R406W tau was less phosphorylated at Ser404 than were the other variants. This was not due to the simple replacement of amino acid Arg406 with Trp close to the phosphorylation site, because Ser404 in a R406W peptide was equally phosphorylated in a wild-type peptide. The decreased phosphorylation of mutant tau by Cdk5s was canceled when tau protein bound to microtubules was phosphorylated. These results indicate that FTDP-17 mutations do not affect the phosphorylatability of tau by Cdk5 complexed with p35, p25, or p39 and may explain part of the discrepancy reported previously between in vivo and in vitro phosphorylation of FTDP-17 tau mutants.     

Fibrillogenic nuclei composed of P301L mutant tau induce elongation of P301L tau but not wild-type tau

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), an autosomal, dominantly inherited neurodegenerative disorder caused by tau gene mutations, is neuropathologically characterized by intraneuronal filamentous inclusions of hyperphosphorylated tau protein. Biochemical and immunocytochemical analyses have shown that only mutant tau is deposited in patients harboring P301L missense mutation, whereas both wild-type and mutant tau are deposited in patients harboring R406W mutation (Miyasaka, T., Morishima-Kawashima, M., Ravid, R., Kamphorst, W., Nagashima, K., and Ihara, Y. (2001) J. Neuropathol. Exp. Neurol. 60, 872- 884 and Miyasaka, T., Morishima-Kawashima, M., Ravid, R., Heutink, P., van Swieten, J. C., Nagashima, K., and Ihara, Y. (2001) Am. J. Pathol. 158, 373-379). Here we have tested the nucleation ability of monomeric tau and the seeding ability of fibrillogenic nuclei obtained from bacterially expressed human tau. P301L mutant tau showed a higher nucleation ability than wild-type tau, whereas R406W mutant tau shows similar ability to wild-type tau. Surprisingly, fibrillogenic nuclei composed of P301L mutant tau enhanced the assembly of P301L mutant tau into filaments but did not promote filament formation from wild-type tau. In contrast, nuclei composed of R406W mutant tau supported filament formation from both wild-type tau and R406W mutant tau, as did nuclei composed of wild-type tau. Proteolytic analyses indicated that the substructure of nuclei composed of P301L mutant tau was different from that of nuclei composed of wild-type or R406W mutant tau. Thus, the interaction between fibrillogenic nuclei and monomeric protein appears to play an important role in the mechanism of tau filament assembly.     

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.     

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.     

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.     

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.     

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.     

Structural and microtubule binding properties of tau mutants of frontotemporal dementias

Several mutations in the gene encoding the microtubule-associated protein tau are responsible for the formation of neurofibrillary inclusions in frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Here we present the high-resolution characterization of the conformational properties of two FTDP-17 mutants of the four-repeat domain of tau, P301L and DeltaK280, and their properties for binding to polyanions and microtubules. Multidimensional NMR spectroscopy shows that the mutations do no lead to a significant increase in the level of beta-structure in their monomeric state, even though the mutations strongly promote beta-structure during aggregation. However, local structural changes are induced in the second repeat. These changes only weakly affect the binding to the polyanion heparin, which promotes paired helical filament formation. The extent of binding to microtubules, however, is strongly decreased. Our results demonstrate that the reversible binding of tau to microtubules involves specific interactions, which are not essential for binding to polyanions.     

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.     

In vitro polymerization of tau protein monitored by laser light scattering: method and application to the study of FTDP-17 mutants

Tau polymerization into the filaments that compose neurofibrillary tangles is seminal to the development of many neurodegenerative diseases. It is therefore important to understand the mechanisms involved in this process. However, a consensus method for monitoring tau polymerization in vitro has been lacking. Here we demonstrate that illuminating tau polymerization reactions with laser light and measuring the increased scattering at 90 degrees to the incident beam with a digital camera results in data that closely approximate the mass of tau polymer formation in vitro. The validity of the technique was demonstrated over a range of tau concentrations and through multiple angle scattering measurements. In addition, laser light scattering data closely correlated with quantitative electron microscopy measurements of the mass of tau filaments. Laser light scattering was then used to measure the efficiency with which the mutant tau proteins found in frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) form filamentous structures. Several of these mutant proteins display enhanced polymerization in the presence of arachidonic acid, suggesting a direct role for these mutations in tau the filament formation that characterizes FTDP-17.     

Transgenic mice expressing mutant (N279K) human tau show mutation dependent cognitive deficits without neurofibrillary tangle formation

Mutations in the tau gene, which is located on chromosome 17, were found causative for autosomal dominantly inherited frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). To determine if cognitive deficits could be caused by tau mutations, two transgenic mouse lines were generated expressing a four-repeat isoform of human tau or its mutant, containing one of the FTDP-17 mutations (WILD mice and N279K mice). In open field test, N279K mice showed hyperactivity in locomotion and rearing. In prepulse inhibition test, N279K mice but not Wild mice showed significant deficits. Both transgenic mice, especially N279K mice, showed impairment in acquisition of spatial learning in Morris water maze. Although both N279K mice and Wild mice acquired passive avoidance as well as non-transgenic mice, N279K mice but not Wild mice showed severe deficits in acquisition of active avoidance. Histological analysis of the present mutant mice did not show any signs of neurofibrillary tangle formations in the brain, and cognitive dysfunction seemed to precede such neuropathological changes or occur independently from them. The behavioral phenotype of N279K mice mimics features of human FTDP-17 and provides a basic model for elucidating mechanisms underlying cognitive deficits in not only FTDP-17, but also diverse tauopathies.     

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.     

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.     

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.     

Mutant (R406W) human tau is hyperphosphorylated and does not efficiently bind microtubules in a neuronal cortical cell model

Frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) is an autosomal dominant neurodegenerative disorder caused by mutations in the gene that encodes for tau, a microtubule-binding protein. Neuropathologically the disease is characterized by extensive neuronal loss in the frontal and temporal lobes and the filamentous accumulation of hyperphosphorylated tau. The R406W missense mutation was originally described in an American and a Dutch family. Although R406W tau is hyperphosphorylated in FTDP-17 cases, R406W tau expressed in cell model systems has not shown increased phosphorylation. The purpose of this study was to establish a neuronal model system in which the phosphorylation of R406W tau is increased and thus more representative of the in vivo situation. To accomplish this goal immortalized mouse cortical cells that express low levels of endogenous tau were stably transfected with human wild type or R406W tau. In this neuronal model R406W tau was more highly phosphorylated at numerous epitopes and showed decreased microtubule binding compared with wild type tau, an effect that could be reversed by dephosphorylation. In addition the expression of R406W tau in the cortical cells resulted in increased cell death as compared with wild type tau-expressing cells when the cells were exposed to an apoptotic stressor. These results indicate that in an appropriate cellular context R406W tau is hyperphosphorylated, which leads to decreased microtubule binding. Furthermore, expression of R406W tau sensitized cells to apoptotic stress, which may contribute to the neuronal cell loss that occurs in this FTDP-17 tauopathy.     

Interactions between Aβ and Mutated Tau Lead to Polymorphism and Induce Aggregation of Aβ-Mutated Tau Oligomeric Complexes

One of the main hallmarks of the fronto-temporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) is the accumulation of neurofibrillary tangles in the brain as an outcome of the aggregation of mutated tau protein. This process occurs due to a number of genetic mutations in the MAPT gene. One of these mutations is the ∆K280 mutation in the tau R2 repeat domain, which promotes the aggregation vis-à-vis that for the wild-type tau. Experimental studies have shown that in Alzheimer’s disease Aβ peptide forms aggregates both with itself and with wild-type tau. By analogy, in FTDP-17, it is likely that there are interactions between Aβ and mutated tau, but the molecular mechanisms underlying such interactions remain to be elucidated. Thus, to investigate the interactions between Aβ and mutated tau, we constructed fourteen ∆K280 mutated tau-Aβ_17-42 oligomeric complexes. In seven of the mutated tau-Aβ_17-42 oligoemric complexes the mutated tau oligomers exhibited hydrophobic interactions in their core domain, and in the other seven mutated tau-Aβ_17-42 oligoemric complexes the mutated tau oligomers exhibited salt-bridge interactions in their core domain. We considered two types of interactions between mutated tau oligomers and Aβ oligomers: interactions of one monomer of the Aβ oligomer with one monomer of the mutated tau oligomer to form a single-layer conformation, and interactions of the entire Aβ oligomer with the entire mutated tau oligomer to form a double-layer conformation. We also considered parallel arrangements of Aβ trimers alternating with mutated tau trimers in a single-layer conformation. Our results demonstrate that in the interactions of Aβ and mutated tau oligomers, polymorphic mutated tau-Aβ_17-42 oligomeric complexes were observed, with a slight preference for the double-layer conformation. Aβ trimers alternating with mutated tau trimers constituted a structurally stable confined β-structure, albeit one that was energetically less stable than all the other constructed models.     

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.