GSK-3β phosphorylation of functionally distinct tau isoforms has differential, but mild effects

<p>Abstract</p> <p>Background</p> <p>Tau protein exists as six different isoforms that differ by the inclusion or exclusion of exons 2, 3 and 10. Exon 10 encodes a microtubule binding repeat, thereby resulting in three isoforms with three microtubule binding repeats (3R) and three isoforms that have four microtubule binding repeats (4R). In normal adult brain, the relative amounts of 3R tau and 4R tau are approximately equal. These relative protein levels are preserved in Alzheimer's disease, although in other neurodegenerative tauopathies such as progressive supranuclear palsy, corticobasal degeneration and Pick's disease, the ratio of 3R:4R is frequently altered. Because tau isoforms are not equally involved in these diseases, it is possible that they either have inherently unique characteristics owing to their primary structures or that post-translational modification, such as phosphorylation, differentially affects their properties.</p> <p>Results</p> <p>We have determined the effects of phosphorylation by a kinase widely believed to be involved in neurodegenerative processes, glycogen synthase kinase-3β (GSK-3β), on the microtubule binding and inducer-initiated polymerization of these isoforms in vitro. We have found that each isoform has a unique microtubule binding and polymerization profile that is altered by GSK-3β. GSK-3β phosphorylation had differential effects on the isoforms although there were similarities between isoforms and the effects were generally mild.</p> <p>Conclusion</p> <p>These results indicate that tau phosphorylation by a single kinase can have isoform specific outcomes. The mild nature of these changes, however, makes it unlikely that differential effects of GSK-3β phosphorylation on the isoforms are causative in neurodegenerative disease. Instead, the inherent differences in the isoform interactions themselves and local conditions in the diseased cells are likely the major determinant of isoform involvement in various neurodegenerative disorders.</p

Heat Shock Protein 70 Prevents both Tau Aggregation and the Inhibitory Effects of Preexisting Tau Aggregates on Fast Axonal Transport

Aggregation and accumulation of the microtubule-associated protein tau are associated with cognitive decline and neuronal degeneration in Alzheimer's disease and other tauopathies. Thus, preventing the transition of tau from a soluble state to insoluble aggregates and/or reversing the toxicity of existing aggregates would represent a reasonable therapeutic strategy for treating these neurodegenerative diseases. Here we demonstrate that molecular chaperones of the heat shock protein 70 (Hsp70) family are potent inhibitors of tau aggregation in vitro, preventing the formation of both mature fibrils and oligomeric intermediates. Remarkably, addition of Hsp70 to a mixture of oligomeric and fibrillar tau aggregates prevents the toxic effect of these tau species on fast axonal transport, a critical process for neuronal function. When incubated with preformed tau aggregates, Hsp70 preferentially associated with oligomeric over fibrillar tau, suggesting that prefibrillar oligomeric tau aggregates play a prominent role in tau toxicity. Taken together, our data provide a novel molecular basis for the protective effect of Hsp70 in tauopathies

Pseudohyperphosphorylation has differential effects on polymerization and function of tau isoforms

The microtubule-associated protein tau exists as six isoforms created through the splicing of the second, third, and tenth exons. The isoforms are classified by their number of N-terminal exons (0N, 1N or 2N) and by their number of microtubule-binding repeat regions (3R or 4R). Hyperphosphorylated isoforms accumulate in insoluble aggregates in Alzheimer’s disease and other tauopathies. These neurodegenerative diseases can be categorized based on the isoform content of the aggregates they contain. Hyperphosphorylated tau has the general characteristics of an upward electrophoretic shift, decreased microtubule binding, and an association with aggregation. Previously we have shown that a combination of seven pseudophosphorylation mutations at sites phosphorylated by GSK-3β, referred to as 7-Phos, induced several of these characteristics in full-length 2N4R tau and led to the formation of fewer but longer filaments. We sought to determine whether the same phosphorylation pattern could cause differential effects in the other tau isoforms, possibly through varied conformational effects. Using in vitro techniques, we examined the electrophoretic mobility, aggregation properties and microtubule stabilization of all isoforms and their pseudophosphorylated counterparts. We found that pseudophosphorylation affected each isoform, but in several cases certain isoforms were affected more than others. These results suggest that hyperphosphorylation of tau isoforms could play a major role in determining the isoform composition of tau aggregates in disease

Hsp70 Alters Tau Function and Aggregation in an Isoform Specific Manner

Tauopathies are characterized by abnormal aggregation of the microtubule associated protein tau. This aggregation is thought to occur when tau undergoes shifts from its native conformation to one that exposes hydrophobic areas on separate monomers, allowing contact and subsequent association into oligomers and filaments. Molecular chaperones normally function by binding to exposed hydrophobic stretches on proteins and assisting in their refolding. Chaperones of the heat shock protein 70 (Hsp70) family have been implicated in the prevention of abnormal tau aggregation in adult neurons. Tau exists as six alternatively spliced isoforms, and all six isoforms appear capable of forming the pathological aggregates seen in Alzheimer’s disease. Because tau isoforms differ in primary sequence, we sought to determine whether Hsp70 would differentially affect the aggregation and microtubule assembly characteristics of the various tau isoforms. We found that Hsp70 inhibits tau aggregation directly and not through inducer-mediated effects. We also determined that Hsp70 inhibits the aggregation of each individual tau isoform and was more effective at inhibiting the three repeat isoforms. Finally, all tau isoforms robustly induced microtubule formation while in the presence of Hsp70. The results presented herein indicate that Hsp70 affects tau isoform dysfunction while having very little impact on the normal function of tau to mediate microtubule assembly. This indicates that targeting Hsp70 to tau may provide a therapeutic approach for the treatment of tauopathies that avoids disruption of normal tau function