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Intraneuronal deposition of microtubule-associated protein tau in filamentous aggregates constitutes a pathological hallmark of neurofibrillary degeneration that is characteristic of Alzheimer's disease (AD) and related disorders known collectively as tauopathies. Formation of such fibril inclusions, consisting of hyperphosphorylated tau in multiple isoforms, correlates with the severity of cognitive decline in AD. How neurofibrillary pathology evolves in tauopathy remains unclear at present, but availability of a cellular model with robust tau aggregation will permit experimental scrutiny of the mechanistic process leading to such neurodegeneration. Through the use of a serial transfection strategy in conjunction with a tau minigene construct, we succeeded in generating conditional transfectants of human neuronal lineage that overproduce wild-type human brain tau in isoforms 4R0N, 3R1N and 4R1N via TetOff and ecdysone inducible expression mechanisms. Such transgenic overexpression of tau in multiple isoforms facilitated the assembly of filamentous tau aggregates that exhibit immunoreactivities, physicochemical properties, and ultrastructural attributes reminiscent of those found in human tauopathies. The conditional tau transfectants thus provide us with a useful tool to elucidate the molecular and cellular events leading to neurofibrillary degeneration and a convenient means to test hypothetical mechanisms implicated in the etiopathogenesis of AD and related tauopathies.

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

Frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) is a group of related disorders frequently characterized by the formation of tau inclusions in neurons and glial cells. To determine whether the formation of tau inclusions in FTDP-17 results from an alteration in the ability of mutant tau to maintain the microtubule (MT) system, we compared wild type four-repeat tau with three FTDP-17 mutants (P301L, V337M and R406W) for their ability to bind MT, promote MT assembly and bundling. According to in vitro binding and assembly assays, P301L is the only mutant that demonstrates a small, yet significant reduction, in its affinity for MT while both P301L and R406W have a small reduction in their ability to promote tubulin assembly. Based on studies of neuroblastoma and CHO cells transfected with GFP-tagged tau DNA constructs, both mutant and wild type tau transfectants were indistinguishable in the distribution pattern of tau in terms of co-localization with MT and generation of MT bundles. These results suggest that missense mutation of tau gene do not have an immediate impact on the integrity of MT system, and that exposure of affected neurons to additional insults or factors (e.g., aging) may be needed to initiate the formation of tau inclusions in FTDP-17.

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.

Tau is the major component of the neurofibrillar tangles that are a pathological hallmark of Alzheimers' disease. The identification of missense and splicing mutations in tau associated with the inherited frontotemporal dementia and Parkinsonism linked to chromosome 17 demonstrated that tau dysfunction can cause neurodegeneration. However, the mechanism by which tau dysfunction leads to neurodegeneration remains uncertain. Here, we present evidence that frontotemporal dementia and Parkinsonism linked to chromosome 17 missense mutations, P301L, V337M and R406W, cause an accelerated aggregation of tau into filaments. These results suggest one mechanism by which these mutations can cause neurodegeneration and frontotemporal dementia and Parkinsonism linked to chromosome 17.