A Complex Mechanism for Inducer Mediated Tau Polymerization

Abstract

The accumulation of polymers of the microtubule associated protein tau is correlative with increased neurodegeneration in Alzheimer's disease and other related tauopathies. In vitro models have been developed in order to investigate molecular mechanisms that regulate the polymerization of tau. Arachidonic acid and heparin have been proposed to induce tau polymerization via a ligand dependent nucleation-elongation mechanism. However, certain aspects of these in vitro results are inconsistent with a classic nucleation-elongation mechanism. Using steady state and kinetic analyses of tau polymerization at a variety of protein and inducer concentrations, we have found that the thermodynamic barrier for nucleation in the presence of inducers is negligible, which was manifested by increases in protein polymerization at low tau concentrations and very rapid kinetics of polymerization. However, the mechanism of polymerization is complicated by the observation that high concentrations of inducer molecules result in the inhibition of tau fibril formation through different mechanisms for arachidonic acid and heparin. These observations indicate that the molar ratio of inducer to protein is a greater determinant of the rate and extent of tau polymerization than the concentration of tau itself. Our results are therefore not consistent with a canonical nucleation-elongation reaction but rather are more consistent with an allosteric regulation model in which the presence of small molecules induce a conformational change in the protein that decreases the thermodynamic barrier for polymerization essentially to zero.

Sulfated Polysaccharides Promote the Assembly of Amyloid β1–42 Peptide into Stable Fibrils of Reduced Cytotoxicity

“…In the absence of Ca 2ϩ , Tau shows a tendency to form ϳ200-kDa species that might correspond to a trimer or tetramer of the protein. As previously described (48,49), GAGs such as heparan sulfate and especially CSB promote the formation of larger Tau aggregates that do not enter SDS-polyacrylamide gels. In the presence of 1.25 mM Ca 2ϩ Tau becomes aggregated into large structures excluded from the gels.…”

Understanding the Kinetic Roles of the Inducer Heparin and of Rod-like Protofibrils during Amyloid Fibril Formation by Tau Protein

“…Tau-Heparin has been used extensively as an inducer in studies of Tau aggregation (16, 17, 20, 23-25, 30 -32, 35-47), but only a few quantitative models (20,30,45) have been proposed to describe important aspects of amyloid fibril formation. However, even in these elegant models, the kinetic role of heparin either in activation, in nucleation, or in allosteric regulation was not quantitatively described as was not the unusual dependence of aggregation kinetics on both protein and heparin concentration (20,30,45).…”

“…Similarly, studies with other inducers have also suggested an NDP mechanism for Tau aggregation (28,29,62). But a more recent study has suggested that polyanion-induced (including heparin-induced) Tau aggregation occurs not through a NDP mechanism but through essentially a downhill polymerization mechanism (30) in which heparin allosterically regulates conformational change into aggregation-competent subunits. Clearly, fundamental aspects of the heparin-induced aggregation reaction remain unresolved.…”

Calpain-Mediated Tau Cleavage: A Mechanism Leading to Neurodegeneration Shared by Multiple Tauopathies

“…Full-length tau (4 μmol/L) was aggregated alone or in the presence of the 17-kDa tau fragment (4 μmol/L) by incubating these proteins with 150 μmol/L arachidonic acid (AA; Cayman Chemical, Ann Arbor, MI, USA) in polymerization buffer (5 mmol/L DTT, 100 mmol/L sodium chloride, 10 mmol/L HEPES, pH 7.6) for 5 h at room temperature as previously described (28,29). Polymerization was monitored by adding Thioflavine S (ThS) at a final concentration of 20 μmol/L and measuring fluorescence (excitation of 440 nm and emission of 520 nm) in the Infinite M200 microplate reader (Tecan, Männedorf, Switzerland) as previously described (29).…”