Functional characterization of wild-type and FTDP-17 tau structural and regulatory mutations on in vitro microtubule dynamic instability

The microtubule-associated protein tau is implicated in the pathogenesis of many neurodegenerative diseases, including Alzheimer's disease and fronto-temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). In FTDP-17, both RNA-splicing regulatory and amino acid substitution and deletion structural mutations in tau cause dominantly-inherited dementia. RNA-splicing FTDP-17 mutations alter the wild-type ∼50:50 3-repeat (3R) to 4-repeat (4R) tau isoform ratio, usually resulting in an excess of 4R tau. Amino-acid substitution and deletion FTDP-17 mutations are localized primarily in the microtubule-binding region. Here, I examine the effects of wild-type and mutant tau protein on in vitro dynamic instability to test the model that FTDP-17 is caused by misregulated microtubule dynamics.; To test the hypothesis that FTDP-17 RNA splicing mutations in tau cause alterations in microtubule dynamics, I used video microscopy to determine the in vitro behavior of microtubules stabilized by varying amounts of human 4R and 3R tau. At low tau:tubulin ratios, all 3R tau isoforms reduced microtubule growth rates relative to the no-tau control, while all 4R tau isoforms increased them; however, at a high tau:tubulin ratio, both 4R and 3R tau increased the growth rates. Further, increasing concentrations of both 4R and 3R tau resulted in an increase in a subpopulation of faster growing microtubules; however, 4R tau caused a redistribution to the faster growing subpopulation at lower tau:tubulin ratios than 3R tau.; To more directly test the hypothesis that altered 4R:3R ratios found in FTDP-17 cause alterations in microtubule dynamics, I analyzed the dynamic instability of microtubules stabilized by 4R:3R tau ratios representative of wild-type (50:50) and RNA-splicing FTDP-17 (25:75 and 75:25) tau levels. In both in vitro microtubule dynamic instability and assembly assays, the 25:75 and 50:50 4R:3R conditions yielded nearly identical dynamic instability, while the 75:25 condition was significantly different from each of the other 4R:3R mixes.; Lastly, to test the hypothesis that FTDP-17 amino-acid substitution and deletion mutations in tau cause alterations in microtubule dynamics, I analyzed the dynamic instability of microtubules stabilized by wild-type and FTDP-17 mutant tau protein. FTDP-17 mutations generally resulted in alterations in tau regulatory activity. Tau protein harboring mutations in a repeated PGGG amino-acid sequence, G272V or P301L, regulated MT dynamics novel manner distinct from their respective wild-type tau isoforms.; Taken together, these observations provide strong support for the hypothesis that mutations in tau or altered 4R:3R tau ratios found in FTDP-17 cause neuronal cell death and dementia by virtue of aberrant regulation of MT dynamics.