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

Background: 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. Results: 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. Conclusion: 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

Modulation of Tau Dysfunction In Vitro

The microtubule associated protein tau is a causative factor in a class of neurodegenerative diseases termed tauopathies. Alzheimer's disease (AD) is the most prevalent tauopathy. In AD, natively unfolded, tau becomes hyperphosphorylated, and undergoes a conformational change allowing hexapeptide regions near microtubule binding repeat region 2 to aggregate into fibers. Tau exists in neurons as six alternatively spliced isoforms. The expression levels of tau are not changed in many tauopathies; however, various levels of each isoform can be included in aggregation depending on the disease. This indicates post-translational modifications or interactions with other proteins causes certain tau isoforms to be included in or excluded from these insoluble inclusions. Tau functions by assembling and stabilizing microtubules and is regulated by phosphorylation. In AD, the phosphorylation state of tau is altered so the affinity for microtubules is reduced; however, how changes in tau phosphorylation affect polymerization of tau isoforms is not fully understood. Abnormal aggregation of protein in a cell is mediated by molecular chaperones. A class of molecular chaperones, heat shock proteins, is upregulated in response to cellular stress. The most widely involved is heat shock protein 70 (Hsp70), which has upregulated and inversely proportional protein levels to tau aggregation in hippocampal neurons. Hsp70 can increase tau solubility in cells; however, it is unknown if Hsp70 can act directly on tau to prevent its dysfunction. This dissertation explores how phosphorylation with GSK-3β, the major kinase believed to be involved in tau hyperphosphorylation, and interactions with Hsp70, affect tau dysfunction (polymerization) and function (microtubule binding and assembly). Tau isoforms were found to be differentially affected by phosphorylation, as indicated by differences in their overall polymerization and various morphologies. Likewise, tau phosphorylation differentially affected their affinity for stabilized microtubules. Tau isoform polymerization was inhibited by Hsp70, with various concentrations of Hsp70 needed for complete inhibition. Finally, while Hsp70 altered the microtubule assembly properties of tau isoforms, each individual isoform was able to assemble microtubules robustly. Taken together, this dissertation shows tau isoforms respond differently to modifications and interaction with Hsp70, indicating each isoform could play a specific role in the progression of tauopathies

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

Secondary nucleating sequences affect kinetics and thermodynamics of tau aggregation

Tau protein was scanned for highly amyloidogenic sequences in amphiphilic motifs (X)nZ, Z(X)nZ (n≥2) or (XZ)n (n≥2), where X is a hydrophobic residue and Z is a charged or polar residue. N-acetyl peptides homologous to these sequences were used to study aggregation. Transmission electron microscopy (TEM) showed 7 peptides, in addition to well known primary nucleating sequences c275VQIINK (AcPHF6*) and Ac306VQIVYK (AcPHF6), formed fibers, tubes, ribbons or rolled sheets. Of the peptides shown by TEM to form amyloid, Ac10VME, AcPHF6*, Ac375KLTFR, and Ac393VYK were found to enhance the fraction of β-structure of AcPHF6 formed at equilibrium, and Ac375KLTFR was found to inhibit AcPHF6 and AcPHF6* aggregation kinetics in a dose-dependent manner, consistent with its participation in a hybrid steric zipper model. Single site mutants were generated which transformed predicted amyloidogenic sequences in tau into non-amyloidogenic ones. A M11K mutant had fewer filaments and showed a decrease in aggregation kinetics and an increased lag time compared to wild type tau, while a F378K mutant showed significantly more filaments. Our results infer that sequences throughout tau, in addition to PHF6 and PHF6*, can seed amyloid formation or affect aggregation kinetics or thermodynamics

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

Identification and Genome-Wide Prediction of DNA Binding Specificities for the ApiAP2 Family of Regulators from the Malaria Parasite

The molecular mechanisms underlying transcriptional regulation in apicomplexan parasites remain poorly understood. Recently, the Apicomplexan AP2 (ApiAP2) family of DNA binding proteins was identified as a major class of transcriptional regulators that are found across all Apicomplexa. To gain insight into the regulatory role of these proteins in the malaria parasite, we have comprehensively surveyed the DNA-binding specificities of all 27 members of the ApiAP2 protein family from Plasmodium falciparum revealing unique binding preferences for the majority of these DNA binding proteins. In addition to high affinity primary motif interactions, we also observe interactions with secondary motifs. The ability of a number of ApiAP2 proteins to bind multiple, distinct motifs significantly increases the potential complexity of the transcriptional regulatory networks governed by the ApiAP2 family. Using these newly identified sequence motifs, we infer the trans-factors associated with previously reported plasmodial cis-elements and provide evidence that ApiAP2 proteins modulate key regulatory decisions at all stages of parasite development. Our results offer a detailed view of ApiAP2 DNA binding specificity and take the first step toward inferring comprehensive gene regulatory networks for P. falciparum

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