Promotion of Hyperphosphorylation by Frontotemporal Dementia Tau Mutations

Abstract

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 4L and 4L more than 3L. In general, at all the sites studied, mutant tau proteins were phosphorylated faster and to a higher extent than 4L and 4L > 3L. The most dramatic difference found was in the rate and level of phosphorylation of 4L 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 4L. The mutated tau proteins polymerized into filaments when 4 -6 mol of phosphate per mol of tau were incorporated, whereas wild-type tau required ϳ10 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 ϳ4 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.

Polymerization of hyperphosphorylated tau into filaments eliminates its inhibitory activity

“…The binding between AD P-tau and MAPs seemed to be of hydrophobic nature because it is stimulated at 200 mM NaCl and inhibited at 2 M NaCl; also, it is completely inhibited by Ϸ0.5% Triton X-100 (15,18). AD P-tau can sequester the six isoforms of tau with different affinity; fetal tau binds the least of the six isoforms (19). AD P-tau is able to destroy the microtubules formed with all of the tau isoforms (19).…”

“…Recombinant human brain tau on in vitro phosphorylation to a stoichiometry of 4-6 mol sequesters N-tau and disassembles microtubules. On further hyperphosphorylation to Ϸ10 mol of phosphate per 1 mol of protein, tau self-assembles into bundles of PHF͞SF (17,20). Dephosphorylation of AD P-tau as well as PHF converts them into normal-like protein, which promotes assembly and stabilizes microtubules (14,21,22).…”

“…AD P-tau can sequester the six isoforms of tau with different affinity; fetal tau binds the least of the six isoforms (19). AD P-tau is able to destroy the microtubules formed with all of the tau isoforms (19). Recombinant human brain tau on in vitro phosphorylation to a stoichiometry of 4-6 mol sequesters N-tau and disassembles microtubules.…”

“…Dephosphorylation of AD P-tau as well as PHF converts them into normal-like protein, which promotes assembly and stabilizes microtubules (14,21,22). The FTDP-17 mutated taus behave as more favorable substrates than N-tau for hyperphosphorylation by brain protein kinases and self-assemble into tangles of PHF͞SF (20). Because of the presence of P-tau in different aggregation states, it was not known whether the toxic entity was the misfolded modified protein, the oligomer, or the filaments.…”

“…(D) N-tau was hyperphosphorylated in vitro, as described in ref. 19; one aliquot was probe-sonicated for 1 min to disrupt the tau filaments formed during the incubation period (19). The sonicated sample was centrifuged at 55,000 ϫ g for 20 min, and the pellet was discarded.…”