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Alzheimer's disease (AD) and Alzheimer's disease-related dementias (ADRDs) affect 6 million Americans, and they are projected to have an estimated health care cost of $355 billion for 2021. A histopathological hallmark of AD and many ADRDs is the aberrant intracellular accumulation of the microtubule-associated protein tau. These neurodegenerative disorders that contain tau aggregates are collectively known as tauopathies, and recent structural studies have shown that different tauopathies are characterized by different "strains" of tau filaments. In addition, mutations in the gene that encodes for tau protein expression have been associated with a group of tauopathies known as frontotemporal dementias with parkinsonism linked to chromosome 17 (FTDP-17 or familial frontotemporal dementia). In vitro studies often use small molecules to induce tau aggregation as tau is extremely soluble and does not spontaneously aggregate under typical laboratory conditions, and the use of authentic filaments to conduct in vitro studies is not feasible. This study highlights how different inducer molecules can have fundamental disparities to how disease-related mutations affect the aggregation dynamics of tau. Using three different classes of tau aggregation inducer molecules, we characterized disease-relevant mutations in tau's PGGG motifs at positions P301S, P332S, and P364S. When comparing these mutations to wild-type tau, we found that depending on the type of inducer molecule used, we saw fundamental differences in total aggregation, aggregation kinetics, immunoreactivity, and filament numbers, length, and width. These data are consistent with the possibility that different tau aggregation inducer molecules make different structural polymorphs, although this possibility would need to be confirmed by high-resolution techniques such as cryo-electron microscopy. The data also show that disease-associated missense mutations in tau impact tau aggregation differently depending on the mechanism of aggregation induction.

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The microtubule-associated protein tau exists in six different isoforms that accumulate as filamentous aggregates in a wide spectrum of neurodegenerative diseases classified as tauopathies. One potential source of heterogeneity between these diseases could arise from differential tau isoform aggregation. in vitro assays employing arachidonic acid as an inducer of aggregation have been pivotal in gaining an understanding of the longest four repeat tau isoform (2N4R). These approaches have been less successful for modeling the shorter 1N4R and 0N4R tau isoforms in vitro. Through a careful analysis of in vitro conditions for aggregation, we found that the differences in the acidity of tau isoform N-terminal projection domains determine whether tau filaments cluster into larger assemblies in solution. Beyond the potential biological implications of filament clustering, we provide optimized conditions for the arachidonic acid induction of shorter 4R tau isoforms aggregation in vitro that greatly reduce filament clustering and improved modeling results.

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BACKGROUND: Neurofibrillary tangles (NFTs) are intraneuronal aggregates associated with several neurodegenerative diseases including Alzheimer's disease. These abnormal accumulations are primarily comprised of fibrils of the microtubule-associated protein tau. During the progression of NFT formation, disperse and non-interacting tau fibrils become stable aggregates of tightly packed and intertwined filaments. Although the molecular mechanisms responsible for the conversion of disperse tau filaments into tangles of filaments are not known, it is believed that some of the associated changes in tau observed in Alzheimer's disease, such as phosphorylation, truncation, ubiquitination, glycosylation or nitration, may play a role. RESULTS: We have investigated the effects of tau phosphorylation by glycogen synthase kinase-3beta (GSK-3beta) on tau filaments in an in vitro model system. We have found that phosphorylation by GSK-3beta is sufficient to cause tau filaments to coalesce into tangle-like aggregates similar to those isolated from Alzheimer's disease brain. CONCLUSION: These results suggest that phosphorylation of tau by GSK-3beta promotes formation of tangle-like filament morphology. The in vitro cell-free experiments described here provide a new model system to study mechanisms of NFT development. Although the severity of dementia has been found to correlate with the presence of NFTs, there is some question as to the identity of the neurotoxic agents involved. This model system will be beneficial in identifying intermediates or side reaction products that might be neurotoxic.