Tau phosphorylation by GSK-3β promotes tangle-like filament morphology
© Rankin et al; licensee BioMed Central Ltd. 2007
Received: 18 April 2007
Accepted: 28 June 2007
Published: 28 June 2007
Neurofibrillary tangles (NFTs) are intraneuronal aggregates associated with several neurodegenerative diseases including Alzheimer's disease. These abnormal accumulations are primarily comprised of fibrils of the microtubule-associated protein tau. During the progression of NFT formation, disperse and non-interacting tau fibrils become stable aggregates of tightly packed and intertwined filaments. Although the molecular mechanisms responsible for the conversion of disperse tau filaments into tangles of filaments are not known, it is believed that some of the associated changes in tau observed in Alzheimer's disease, such as phosphorylation, truncation, ubiquitination, glycosylation or nitration, may play a role.
We have investigated the effects of tau phosphorylation by glycogen synthase kinase-3β (GSK-3β) on tau filaments in an in vitro model system. We have found that phosphorylation by GSK-3β is sufficient to cause tau filaments to coalesce into tangle-like aggregates similar to those isolated from Alzheimer's disease brain.
These results suggest that phosphorylation of tau by GSK-3β promotes formation of tangle-like filament morphology. The in vitro cell-free experiments described here provide a new model system to study mechanisms of NFT development. Although the severity of dementia has been found to correlate with the presence of NFTs, there is some question as to the identity of the neurotoxic agents involved. This model system will be beneficial in identifying intermediates or side reaction products that might be neurotoxic.
Tau is a remarkably soluble neuronal microtubule-associated protein that normally functions to promote the assembly and stabilization of the microtubule cytoskeleton. In Alzheimer's disease (AD) and other related neurodegenerative disorders, tau aggregates into straight and paired helical filaments . As these diseases progress, the tau fibrils associate with one another to form large densely packed networks of interwined filaments termed neurofibrillary tangles (NFTs). Although the level of NFT formation has been shown to correlate with the severity of dementia [2, 3], it is unclear whether NFT formation is neurotoxic. There are examples of NFT formation that correlate with neurodegeneration [4–6] and also examples of neurodegeneration in the absence of NFT formation [7–9]. It has even been suggested that NFT formation is protective for neurons . However, NFTs are not inert pathological lesions, but rather follow a definite developmental progression (reviewed in ). It is possible that the neurotoxic agents in AD may be intermediates in the development of NFTs or even products of side reactions. Therefore, detailed studies of NFT formation would be beneficial to our understanding of toxic elements in AD.
Although polymerized tau is a major component of NFTs, numerous other molecules have also been found associated with NFTs. Some of these include ubiquitin [11, 12], RNA , α-synuclein , GSK-3β , microtubule affinity regulating kinase , and apolipoprotein E . One of these nonfibrillar components, GSK-3β, appears to have an active role in the pathological progression of neurodegeneration. A drosophila model expressing both human tau and the kinase GSK-3β exhibited enhanced neurodegeneration and neurofibrillary pathology  compared to expression of tau alone . A similar result was seen in a transgenic mouse model expressing a mutated tau and the GSK-3β kinase . To emphasize the role of phosphorylation, treatment with the GSK-3β inhibitor, lithium chloride, showed a reduction in neurodegeneration, tau phosphorylation, and tau pathology when administered at early stages of neuropathology [19, 20]. Further evidence that phosphorylation may play a role in NFT development was the discovery of highly phosphorylated paired helical filaments, the form of filamentous tau most prevalent in AD (reviewed in [1, 21, 22]). Since GSK-3β appears to phosphorylate tau in many of the same phosphorylation sites identified in paired helical filaments of AD [23–25], it would appear that phosphorylation by GSK-3β may play a role in formation of fibrils or NFTs.
We have tested the hypothesis that GSK-3β is involved in some aspect of NFT formation by using an in vitro cell-free model, where reagents can be clearly defined and controlled. Cell-free polymerization models of tau have been well established, generally using either arachidonic acid (ARA) or heparin to initiate polymerization (reviewed in ). However, use of these models to study effects of tau phosphorylation on levels of tau polymer have been inconclusive [27–32]. We have found that phosphorylation of tau by GSK-3β had little effect on polymerization levels, although it had a significant role in the formation of large localized accumulations of intertwined filaments. These aggregates of tau fibrils were stable through sucrose gradient centrifugation and migrated to the same region as NFT-like structures isolated from AD brain [33, 34]. Further investigation of the reaction conditions producing these NFT-like structures showed that the concentration and nature of inducer(s) were important factors in defining aggregation characteristics of size and density. This cell-free model of the formation of NFT-like accumulations provides a useful tool for future studies to understand how aggregation of tau polymer into NFT-like structures might occur and which steps in the process might potentially produce neurotoxic products.
Phosphorylation of monomeric tau by GSK-3β
The number of phosphates incorporated per mole of tau was quantified by utilizing [γ-32P] ATP as a substrate (Figure 1B). In comparing gel shift and radioactive phosphate incorporation data (Figure 1B) a similar increase in phosphorylation over time was observed. At 20 h incubation and a GSK-3β concentration of 0.018 U/pmol tau, approximately 3 moles of phosphate were incorporated per mole of tau, which is similar to the 2–4 mols phosphate incorporated/mol of tau in previously published reports of in vitro GSK-3β phosphorylation of tau .
Sites phosphorylated by GSK-3β in tau monomer
Tau sites phosphorylated by GSK-3β.
Kinetic analysis comparing polymerization of GSK-3β phosphorylated tau with polymerization of non-phosphorylated tau
Assaying by ThS intensity allows for observation of changes very early in the reaction and upon the addition of ARA inducer there was a rapid and dramatic increase in ThS fluorescence (Figure 3B), as has been previously described . This rapid early increase in ThS intensity apparently registers a change in molecular structure but does not measure filament formation per se . At both suboptimal and optimal inducer:protein ratios, the phosphorylated protein seemed to have a greater initial velocity of polymerization compared to non-phosphorylated tau (Figure 3B), suggesting that phosphorylated protein is either in an altered conformation or more rapidly adopts a ThS positive conformation in the presence of ARA.
TEM analysis of filaments from GSK-3β phosphorylated tau
The major advantage of an in vitro cell-free system as a research tool is the ease with which various reagents (by their addition/omission) can be examined for their effect on a specific result, in this case the clustering or aggregation of filaments. Conventional, control reactions with non-phosphorylated tau, performed under standard polymerization conditions with either the suboptimal or optimal ratios of ARA:tau protein (75 or 25 μM ARA, respectively:2 μM tau protein) did not show filament clustering (Figure 5A and 5B). Polymerization reaction conditions are detailed in Materials and Methods. Since the 75 μM ARA TEM sample was diluted by a factor of ten whereas the 25 μM ARA sample was diluted by a factor of five, the TEM analysis appeared similar to the kinetic study observations (Figure 3) in regard to filament mass.
Since GSK-3β is a component of NFTs in AD, it was possible that its primary effect on cluster formation came not from its role as a kinase but from a hypothetical role as NFT or cluster "glue". To determine the role of GSK-3β, we carried out two mock phosphorylation reactions, one in which the GSK-3β was omitted (Figure 5C, D), the other in which the ATP was omitted (Figure 5E, F). Omission of ATP mimics the GSK-3β inhibitors that act by competing for ATP. Without ATP in the phosphorylation reaction, no filament clustering occurred in the polymerization reaction (Figure 5E, F) and none of the mock phosphorylation reactions showed a band shift on SDS-PAGE (data not shown). Since GSK-3β did not support filament clustering without ATP it would seem that phosphorylation of tau is the primary role of GSK-3β in cluster formation. Although polymerization of the mock-phosphorylated tau induced by 25 μM ARA showed no filament clustering if either GSK-3β or ATP were omitted (Figure 5C and 5E), at high ARA concentration (75 μM) we did observe a few small filament clusters when ATP was present without GSK-3β, suggesting that high inducer concentration plus ATP might partially compensate for tau phosphorylation in cluster formation.
Morphological characteristics of filament clusters
To verify our TEM observations (and to assess the statistical significance of potential modifying reagents), the number of filaments and the average filament length in each cluster, as well as the area covered by each cluster were measured. These cluster properties, average filament length, filament number, area covered, and density allowed us to quantify cluster morphology and assess the modifying effects of various reagents.
Effect of ThS on filament formation
Comparison of filament clusters formed in vitro to NFT-like filament bundles isolated from AD brain
Due to the controversial role of neurofibrillary tangle (NFT) formation in the neurodegenerative process [2–4, 6–9, 43–47], a better understanding of the mechanisms leading to the formation of NFT would be beneficial to our understanding of AD.
In this report, we have demonstrated that GSK-3β phosphorylation of tau is sufficient to induce the clustering of ARA-induced filaments into structures similar to the NFT-like aggregates of tau filaments purified from AD brain [33, 34]. These results suggest that GSK-3β phosphorylation not only produces a small but significant increase in tau filament formation, but also shows that phosphorylation alters the nature of interactions between those filaments resulting in their clustering into NFT-like structures. Although in this report we address only the effects of tau phosphorylation by GSK-3β, ARA inducer concentration (the inducer:tau ratio in polymerization), and ThS on the clustering of tau filaments into NFT-like structures, we feel that this is an important first step in unraveling the molecular mechanisms of NFT formation through cell-free in vitro modeling.
The clusters of tau filaments formed by polymerization of GSK-3β phosphorylated tau are stable and their formation is readily reproducible, although various factors influence the size, mass and density of the clusters. Here we demonstrate the effects of inducer and the ratio of inducer:tau concentration on these properties. In general, we have found that phosphorylation by GSK-3β is sufficient for cluster formation. In addition, conditions that alter filament length modify both the density of the filaments in the cluster, and the size of the cluster. Increases in inducer concentration which result in a change from the suboptimal to optimal ratio of inducer:tau concentration in the polymerization reaction increase the filament length within clusters and the area covered by the clusters. This results in clustered filaments that are less densely packed. Conversely, conditions that decrease filament length produce smaller clusters that contain a higher density of filaments.
With this newly developed in vitro model, we can begin to dissect the molecular mechanisms that are involved in filament aggregations that form NFT-like structures. Further studies will be aimed at understanding whether GSK-3β phosphorylation unmasks regions of tau molecules that interact with one another in forming clusters or whether the GSK-3β phosphorylation sites are interacting directly. It is tempting to speculate a role for the former since GSK-3β phosphorylation results in an SDS-resistant conformational change as observed by an upward shift in mobility on SDS page analysis. The apparent increase in initial polymerization velocity as monitored by ThS fluorescence also suggests that the GSK-3β phosphorylated tau may be in a conformation that more readily interacts with the ARA inducer or with the ThS as used to detect amyloid-type interactions in tau kinetic analyses. Although the co-localization of GSK-3β with tau pathology in AD suggests that NFTs may form from the direct interaction of GSK-3β with tau filaments , our mock-phosphorylation results strongly suggest that phosphorylation is the primary role of GSK-3β in promoting cluster formation.
This in vitro model for NFT formation requires the induction of tau polymerization via the addition of ARA, which may lead to the questioning of its physiological relevance. An inducer of tau polymerization in AD and other neurodegenerative disorders has not been identified, but that does not dampen our enthusiasm for the use of ARA as an inducer in our cell-free in vitro model system. This is due to ample evidence that ARA induced tau filaments are structurally similar to filaments from AD [42, 48–51]. Additionally, there is growing evidence that ARA or its metabolites could be involved in the neurodegenerative process in AD (reviewed in ). While a direct connection between tau polymerization and ARA remains to be made in AD, the structural similarity between ARA induced filaments and AD filaments, plus the similarity between GSK-3β induced NFT-like clusters of tau filaments and those found in AD provide a strong argument for the physiological relevance of this model. In addition to the characterization of the GSK-3β induced clustering of filaments, this in vitro model provides a tool for investigating whether other kinases such as cyclin dependent kinase 5 or microtubule affinity regulating kinase have similar properties to induce the formation of NFT-like filament bundles. Likewise, other modifications found in association with AD NFTs, such as truncation, ubiquitination, nitration and glycation (reviewed in [1, 10]) could also be tested. Our hope is that these ongoing studies will isolate factors contributing not only to the formation of NFT-like clusters, but also to identify the conditions that could lead to potentially toxic tau aggregates in cell and animal culture models.
Reagents and Supplies
Arachidonic acid was obtained from Cayman Chemicals (Ann Arbor, MI), thioflavine S and recombinant glycogen synthase kinase 3β (GSK-3β) from Sigma (St. Louis, MO), and uranyl acetate and formvar carbon coated grids from Electron Microscopy Sciences (Hatfield, PA). SDS-PAGE markers are Precision Plus Protein Standards from Bio-Rad (Hercules, CA).
Tau protein (441 amino acids containing exons 2, 3 and 10) was expressed in and purified from BL21 E. Coli as described previously . Protein concentration was determined using the Pierce BCA assay (Pierce Biotechnology, Rockford, IL). The purity of the protein was assessed by SDS-PAGE electrophoresis.
Phosphorylation of tau by GSK-3β
Determination of optimal phosphate incorporation
Tau protein at a final concentration of 16 μM was incubated with either 0.006 or 0.018 U GSK-3β per pmol tau in buffer containing 40 mM HEPES, pH 7.64, 5 mM EGTA, 3 mM MgCl2, and 2 mM ATP for 20 h at 30°C. One unit of GSK-3β is defined as the amount of enzyme that will transfer one pmol phosphate from ATP to phosphatase inhibitor 2 per min at pH 7.5 at 30°C. Samples were removed at various times from the phosphorylation reaction, then boiled in Laemmli sample buffer  for 5 minutes to stop phosphorylation. One microgram of tau protein from the reaction time points was analyzed by SDS-PAGE.
Determination of gel band shift
SDS-PAGE gels from posphorylation reactions were converted to digital images using an HP Scanjet 7400c scanner (Hewlett-Packard company, Palo Alto, CA). The gels were converted to grayscale and inverted using Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA). Two fixed size marquees were used to determine the average intensity of pixels from the entire sample in a lane or only the area corresponding to the shifted band. The average intensity of the gel background was subtracted. The percentage of tau in the shifted band was determined by dividing the value for the shifted band by the value for the total tau in the lane.
Determination of phosphate incorporation
Tau protein at a final concentration of 16 μM was incubated with 0.018 U GSK-3β per pmol tau in buffer containing 40 mM HEPES, pH 7.64, 5 mM EGTA, 3 mM MgCl2, and 2 mM ATP containing 10 μCi [γ-32P] labeled ATP (Specific activity: 3000 Ci/mmol) (Perkin-Elmer, Boston, MA) for 20 h at 30°C. Samples were removed at various times from the phosphorylation reaction, diluted and filtered through a Millipore ULTRAFREE 10,000 molecular weight cut off filter (Millipore, Billerica, MA). Samples were washed with two-250 μl volumes buffer containing 5 mM DTT, 100 mM NaCl, 10 mM HEPES pH 7.64, and 0.1 mM EDTA. γ-32P incorporation in tau was measured using a Packard 1600TR liquid scintillation counter to measure the radioactivity in the retentate and filter. The protein content of the flow-through filtrate was assayed by the BCA microplate protocol (Pierce Biotechnology, Rockford, IL) and was below the minimum detectable amount (20 μg/ml), allowing the assumption that no protein was lost during the filtration process. The amount of phosphate per protein molecule was calculated using the specific activity of γ-32P and the molar concentration of tau.
Generation of GSK-3β phosphorylated tau for further analysis
Based on the results above, we determined that the optimal phosphate incorporation was achieved by incubating 16 μM tau with 0.018 U GSK-3β per pmol of tau in buffer containing 40 mM HEPES, pH 7.64, 5 mM EGTA, 3 mM MgCl2, and 2 mM ATP for 20 h at 30°C. These conditions were used to generate GSK-3β phosphorylated tau for use in Figures 2, 3, 4, 6, 7, and 9.
Two separate conditions were used to generate "mock" phosphorylated tau analyzed in Figure 5. The first was by eliminating GSK-3β from the reaction such that the phosphorylation reaction consisted of 16 μM tau in buffer containing 40 mM HEPES, pH 7.64, 5 mM EGTA, 3 mM MgCl2, and 2 mM ATP for 20 h at 30°C and was also used as a control in Figure 9. The second was by eliminating ATP from the reaction such that the phosphorylation reaction consisted of 16 μM tau with 0.018 U GSK-3β per pmol of tau in buffer containing 40 mM HEPES, pH 7.64, 5 mM EGTA, and 3 mM MgCl2, and then incubated for 20 h at 30°C.
Dot blots of phosphorylated tau protein
Phosphorylated tau was diluted in TBS (20 mM Tris pH 7.5, 150 mM NaCl) such that 3 μl of the dilution contained the desired amount of protein. The protein (3 μl) was spotted onto Immobilon P membrane which was prepared according to manufacturer's instructions (Millipore, Billerica, MA). Blots were blocked for 1 h in TBS containing 1% BSA and 2% normal goat serum. The primary, tau phosphorylation-specific antibodies (Biosource International, Camarillo, CA) were diluted 1:1000 in blocking solution and blots were rotated overnight at 4°C. Blots were washed with TBS/Tween 20 (0.1%)/NP40 (0.05%), blocked with blocking buffer and incubated with an alkaline phosphatase conjugated anti-rabbit IgG secondary antibody (Sigma, St. Louis, MO). Blots were developed with chemiluminescence reagent, CDP-Star (PerkinElmer Life Sciences, Boston, MA). Images were captured on a Kodak Image Station 4000R (Eastman Kodak Co, Molecular Imaging Systems, Rochester, NY), and analyzed using the Array Analysis feature of the ImageQuantTL v2003.03 software that accompanies the Typhoon Trio, Variable Mode Imager (Amersham Biosciences, Piscataway, NJ). After subtracting non-specific binding data (density of the non-phosphorylated tau dots for each concentration), the density data was plotted using GraphPad Prism 4 GraphPad Software Inc., San Diego, CA).
Tau polymerization reactions
Standard polymerization reactions
Tau was diluted to a final concentration of 2 μM into buffer containing 10 mM HEPES, pH 7.64, 100 mM NaCl, 0.1 mM EDTA, and 5 mM DTT. Arachidonic acid (ARA) was added to a final concentration of either 25 or 75 μM to induce polymerization. Standard polymerization reaction conditions were used as controls for Figures 3, 5 and 8.
Polymerization of phosphorylated tau
In polymerization reactions using phosphorylated tau, the phosphorylation reaction was diluted to a final concentration of 2 μM into polymerization buffer containing 10 mM HEPES, pH 7.64, 100 mM NaCl, 0.1 mM EDTA, and 5 mM DTT. This resulted in some minor buffer additions: 0.625 mM EGTA, 0.375 mM MgCl2, 0.25 mM ATP and increased the final HEPES concentration to 15 mM. These buffer additions did not appear to affect polymerization. The GSK-3β that carried over from the phosphorylation reaction was not de-activated. Phosphorylation reaction products were analyzed by coomassie blue staining on 10–15% SDS-PAGE prior to use in polymerization reactions.
Polymerization of mock phosphorylated tau
The mock phosphorylation reaction was diluted into polymerization buffer to a final concentration of 2 μM tau as above, except that there was no carry over of GSK-3β or of ATP if those reagents were omitted from the mock phosphorylation reaction.
ThS containing reactions
Polymerization reactions with ThS contained 2 μM non-phosphorylated tau in buffer containing 10 mM HEPES, pH 7.64, 100 mM NaCl, 0.1 mM EDTA, 5 mM DTT and 20 μM ThS. ARA was added to a final concentration of 25 or 75 μM to induce polymerization. Reactions with phosphorylated or mock phosphorylated tau resulted in minor buffer additions (described above). ThS containing reactions were used in Figure 3 to compare levels of polymer for the kinetics study and also for Figures 4, and 6, 7, 8, 9.
The kinetics of polymerization reactions were assayed by ThS fluorescence, utilizing a FlexStation II fluorometer microplate reader (Molecular Devices Corporation, Sunnyvale, CA). Settings included: Excitation λ = 440, Emission λ = 520, PMT = high. Polymerization reactions containing all reagents except ARA were prepared. The FlexStation II automatically added the ARA and began fluorescence intensity readings thirteen seconds later. Readings were taken every 1.5 seconds for the first 30 minutes, then once every 5 minutes for 20 hours.
Electron microscopy of tau filaments and filament clusters
Samples were diluted either by a factor of five (25 μM ARA reactions) or ten (75 μM ARA reactions) in polymerization buffer, then applied to the grids, allowing one minute for filaments and/or clusters to attach. The edge of the grid was then touched to filter paper to blot away excess liquid. Grids were stained with 1% uranyl acetate for one minute then blotted as above. Grids were viewed with a JEOL 1200 EXII electron microscope and images were captured with the MegaViewII imaging system (Soft Imaging System, GmbH Münster, Germany).
Measurements of tau filaments and filament clusters
The area, mass and density of localized accumulations of tau filaments on electron microscopy grids were measured using the Optimas analytical imaging software (Media Cybernetics, Silver Spring, MD) and GraphPad Prism software (Graphpad Software, San Diego, CA). Digital electron micrographs were collected at 20,000 × magnification. For measurements of individual filaments, the entire field was selected using the "threshold tool" and "auto find lines" feature of the software. For clusters of filaments, the region of interest tool was used to outline the clusters of filaments in Optimas. Filaments within the clusters were selected using the "threshold tool" and the "auto find lines". For a field of individual filaments or for filaments in the selected "area of interest" cluster, the number and average length of filaments was determined and multiplied to obtain an estimate of filament mass in the field or region of interest. For clusters, the "draw area tool" was used to outline the outer boundary of the filaments in order to obtain the area occupied. The total sum length of filaments was divided by the area occupied by those filaments to obtain measurements for the density of the filament clusters.
Discontinuous Sucrose Gradients
Centrifugation of tau polymerization reactions through discontinuous gradients consisting of 1, 1.5 and 2 M sucrose were performed as previously described , except that sucrose was dissolved in buffer containing 5 mM DTT, 100 mM NaCl, 10 mM HEPES, and 0.1 mM EDTA. Polymerization reactions were overlaid on the gradient and centrifuged at 100,000 × g for 2 h in a TLA 100.3 rotor (Beckman-Coulter, Fullerton, CA). To compensate for any reduction of filament adherence due to sucrose in the samples, gradient fraction samples were applied 5 times to the formvar carbon coated electron microscopy grids, allowing 1 minute each time for filaments to attach. Grids were rinsed five times with polymerization buffer and stained with 0.5% uranyl acetate for 1 minute. Therefore, the filaments viewed by electron microscopy were concentrated five fold in addition to any concentration of filaments produced by the centrifugation through the gradient.
List of abbreviations
glycogen synthase kinase 3β
transmission electron microscopy
We thank Mike Branden for assistance with protein purification. We thank Dr. Richard Himes and Dr. Lester Binder for suggestions during the preparation of the manuscript. This work was supported by AG022428 (TCG).
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