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Published as doi: 10.1096/fj.08-109181.
 (The FASEB Journal. 2008;22:3186-3195.)
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Phosphorylation of tau regulates its axonal transport by controlling its binding to kinesin

Inmaculada Cuchillo-Ibanez*,1, Anjan Seereeram*, Helen L. Byers{dagger}, Kit-Yi Leung{dagger},2, Malcolm A. Ward{dagger}, Brian H. Anderton* and Diane P. Hanger*

* Medical Research Council Centre for Neurodegeneration Research and

{dagger} Proteome Sciences, King’s College London, Institute of Psychiatry, London, UK

1Correspondence: MRC Centre for Neurodegeneration Research, King’s College London, Institute of Psychiatry (P037), De Crespigny Park, SE5 8AF London, UK. E-mail: spneici{at}iop.kcl.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Defective axonal transport has been proposed as an underlying mechanism that may give rise to neurodegeneration. We investigated the effect of phosphorylation on the axonal transport of tau, a neuronal protein that stabilizes microtubules and is hyperphosphorylated and mislocalized in Alzheimer’s disease. We report here that specific inhibition of glycogen synthase kinase-3 (GSK-3) reduces tau phosphorylation and significantly decreases the overall rate of axonal transport of tau in rat cortical neurons. Tau mutants, with serine/threonine targets of GSK-3 mutated to glutamate to mimic a permanent state of phosphorylation, were transported at a significantly increased rate compared to wild-type tau. Conversely, tau mutants, in which alanine replaced serine/threonine to mimic permanent dephosphorylation, were transported at a decreased rate compared to wild-type tau. We also found that tau interacts with the light chain of kinesin-1 and that this is dependent on the phosphorylation state of tau. Tau phosphorylation by GSK-3 increased binding, and dephosphorylated tau exhibited a reduced association with kinesin-1. We conclude that GSK-3 phosphorylation of tau modulates its axonal transport by regulating binding to kinesin-1. Hyperphosphorylated tau in Alzheimer’s disease appearing first in distal portions of axons may result from aberrant axonal transport of phosphorylated tau reported here.—Cuchillo-Ibanez, I., Seereeram, A., Byers, H. L., Leung, K.-Y., Ward, M. A., Anderton, B. H., Hanger, D. P. Phosphorylation of tau regulates its axonal transport by controlling its binding to kinesin.


Key Words: GSK-3 • mass spectrometry • lithium


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TAU IS A NEURONAL CYTOSKELETAL PROTEIN that is expressed in the central nervous system (CNS) as six alternatively spliced isoforms generated from a single gene. Tau stabilizes microtubules and regulates neurite outgrowth. Aberrant forms of tau are implicated in the pathogenesis of several neurodegenerative diseases in which tau deposits are found, and these are collectively termed tauopathies (1) . One invariable feature of tauopathies is the presence of significant amounts of insoluble tau, present as either straight or paired helical filaments in intraneuronal or intraglial inclusions containing hyperphosphorylated tau that is unable to bind to microtubules. Recently, we identified 11 novel phosphorylated sites on insoluble tau isolated from Alzheimer brain (2) , bringing to 39 the number of directly identified phosphorylations on this form of tau (3 , 4) . An additional six phosphorylation sites have been suggested by immunological methods (5) . Identification of the protein kinases responsible for physiological and pathological tau phosphorylation has been complicated by the large number of protein kinases that phosphorylate tau in vitro. However, fewer kinases have been shown to phosphorylate tau in cells, and we found previously that glycogen synthase kinase-3β (GSK-3β) and GSK-3{alpha} are both good candidates for tau phosphorylation (2 , 6 , 7) . At least 28 GSK-3β sites have been identified in tau following in vitro phosphorylation and mass spectrometric analysis, and many of these sites are also found in hyperphosphorylated tau isolated from Alzheimer brain (2 3 4 , 8 9 10 11) . GSK-3β phosphorylation of tau decreases tau-induced microtubule assembly and stability as well as the microtubule-binding capacity of tau, and thus phosphorylation affects tau physiological function (7 , 12 , 13) .

Defective axonal transport has been proposed by several laboratories as an underlying mechanism that may give rise to neurodegeneration (14 15 16 17 18 19 20 21) . Phosphorylation has been demonstrated to be a regulatory mechanism for the axonal transport of neurofilaments and MAP1B (22 23 24 25 26) . Up-regulation of GSK-3β in Alzheimer’s disease (27) may play a central role in generating the abnormally phosphorylated tau species reported to increase in pretangle neurons.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids
Plasmids expressing the longest isoform of wild-type human CNS tau containing both C-terminal repeats (2N) and all four microtubule-binding repeats (4R) h2N4Rtau, and two mutant forms of full-length human tau containing 18 (E18tau) or 27 residues (E27tau) mutated to glutamate were gifts from M. Goedert (Medical Research Council Laboratory of Molecular Biology, Cambridge, UK) (28) . These constructs were subcloned into pEGFP-C1 [enhanced green fluorescent protein (EGFP) fused to the amino terminus of tau; Clontech, Saint-Germain-en-Laye, France] to generate WTtau-EGFP, E18tau-EGFP, and E27tau-EGFP, respectively. The A18tau-EGFP construct was prepared from wild-type h2N4Rtau in pEGFP-C1. Codons were mutated to those encoding alanine residues by site-directed mutagenesis (QuikChange Multi Site-Directed Mutagenesis Kit; Stratagene, La Jolla, CA, USA). To verify the mutations, the A18tau-EGFP construct was sequenced using specific primers (MWG-Biotech, London, UK). pCMV-DsRed-Express was from Clontech.

Neuronal cell culture and transfection
Embryonic rat cortical neurons were cultured and transfected at 5 days in vitro (DIV) with tau constructs using calcium phosphate (Profection Kit; Promega, Southampton, UK), as described previously (29) . As appropriate, 10 mM NaCl, 10 mM LiCl, or 40 µM SB-415286 was added to the transfection medium and was present for the duration of the experiment. For immunoprecipitation experiments, neurons were transfected with tau constructs using Lipofectamine 2000 (Invitrogen, Paisley, UK), following the manufacturer’s instructions.

Fluorescence microscopy
Fluorescence microscopy was performed on an Axioskop microscope (Carl Zeiss, Welwyn Garden City, Hertfordshire, UK), equipped with a camera (CoolSnap HQ; Photometrics, Marlow, Buckinghamshire, UK) and with Plan-NeoFluor x20 0.50 NA or x40 0.75 NA objectives. In experiments to determine the overall rate of tau transport, neurons were fixed with 4% (w/v) paraformaldehyde at appropriate times post-transfection, and the distances traveled by tau were measured as described previously (29) . Cells (25–100) were measured at each time point in each independent experiment. The intensity of fluorescence in the soma was determined from the area defined by the perimeter of the neuronal cell body. Analysis of the images was performed after subtracting the background fluorescence from each measurement using MetaMorph (Universal Imaging Corporation, Marlow, Buckinghamshire, UK). Statistical analyses were performed using t test and Pearson correlation.

Mass spectrometric analysis of endogenous tau in rat cortical neurons
Endogenous heat-stable tau was extracted from rat cortical neurons at 7 DIV. Briefly, cells were harvested by scraping into phosphate buffered saline (PBS), pH 7.5, 4°C containing Complete protease inhibitor with ethylenediaminetetraacetic acid (EDTA) (Roche, Basel, Switzerland) and collected by centrifugation at 15,800 g (av) for 5 min at 4°C. The pellet was resuspended in Mes/NaCl buffer [100 mM Mes, pH 6.5; 1 M NaCl; 0.2 mM phenylmethanesulphonylfluoride (PMSF); 1 mM Na3VO4; 10 mM NaF] containing Complete protease inhibitor with EDTA. The suspension was heated at 100°C for 5 min and centrifuged at 15,800 g (av) for 20 min at 4°C. Tau in the supernatant was concentrated by adding an equal volume of 20% (w/v) trichloroacetic acid and incubating on ice for 15 min. After centrifugation at 15,800 g (av) for 5 min at 4°C, the pellet was washed with acetone and centrifuged as before. The final pellet was dissolved in Laemmli sample buffer; proteins were separated on 10% (w/v) polyacrylamide gels and visualized with Brilliant blue-G Coomassie stain (Sigma-Aldrich, Dorset, UK).

The extracted tau was digested with trypsin as described previously (30) . O-Methylation labeling of the peptides was undertaken using 2 M methanolic acid (31) , using methanol and deuterated methanol for light and heavy labeling, respectively. The reaction mix was lyophilized, and immediately prior to analysis by liquid chromatography/mass spectrometry (LC/MS, for peptide quantification) and LC/MS/MS (for peptide identification), the mixture of methylated tau peptides was resuspended in 0.05% (v/v) formic acid. LC/MS/MS analysis of methylated tau peptides was performed as described previously (30) . LC/MS was performed similarly, but without MS/MS analysis, to allow all the data acquisition time to be spent acquiring data in the MS mode for accurate peptide quantitation. The MS/MS data were searched against a custom-built database containing the different isoforms of rat CNS tau using the Mascot searching algorithm (Matrix Science, London, UK). Methylated phosphopeptides of tau (light and heavy pairs) were identified based on the following search criteria (set as variable modifications): carbamidomethylation of cysteines; oxidized methionine, tyrosine, serine, and threonine phosphorylation; and methylation of glutamic acid, aspartic acid, and the C-terminus. All peptides were tryptic with up to three missed cleavages. To calculate the light:heavy peptide ratio, the particular peptide ions were extracted from the raw LC/MS data and processed using the deisotoping algorithm MaxEnt3, provided within Masslynx (Waters, Watford, Hertfordshire, UK); this yielded the total ion intensity, and the light:heavy ratio was calculated for each peptide.

Immunoprecipitation
Rat cortical neurons (5 DIV) were treated with either 10 mM LiCl or 10 mM NaCl (control) for 4 h, harvested, and lysed in ice-cold 50 mM Tris-HCl (pH 7.6) with 0.1% (v/v) Triton X-100 containing Complete protease inhibitor with EDTA. After incubation for 30 min on ice, lysates were centrifuged at 100,000 g (av) for 30 min at 4°C. Supernatants were precleared with protein A (Sigma-Aldrich) for 1 h at 4°C. The protein concentration of the precleared lysates was determined by Bradford assay (Bio-Rad, Hemel Hempstead, Hertfordshire, UK). The total protein content of the lysates was standardized before immunoprecipitation. Proteins were immunoprecipitated with either a polyclonal antibody to human tau (Dako, Ely, Cambridgeshire, UK) or a monoclonal antibody to kinesin-1 (MAB1614; Millipore, Watford, UK) overnight at 4°C with agitation. The immunoprecipitates were collected using protein A/G agarose beads (Autogen Bioclear UK Ltd, Calne, Wiltshire, UK), and the bound proteins were recovered by heating in Laemmli sample buffer. Proteins were detected by immunoblotting with both the polyclonal anti-tau antibody and the monoclonal anti-kinesin-1 antibody. Visualization of proteins separated by SDS-PAGE was determined using an Odyssey scanner (Li-Cor Biosciences, Cambridge, UK).

In vitro GSK-3β phosphorylation of tau and GST-binding assay
Purified recombinant h2N4R tau (10 µg) was incubated with 1 mM ATP and either 100 U of GSK-3β enzyme (New England Biolabs, Hitchin, Hertfordshire, UK; phosphorylated tau) or no GSK-3β (control tau) and incubated at 30°C for 16 h. Phosphorylation of tau was confirmed on Western blots probed with antibodies to tau (Dako).

Untreated or GSK-3-phosphorylated recombinant tau was dissolved (2.5 µg/ml) in modified RIPA buffer [20 mM Tris-HCl, pH 7.4; 150 mM NaCl; 10 mM NaF; 1 mM Na3PO4; 1 mM ETDA; 1 mM EGTA; 1% (v/v) Nonidet 40; and Complete protease inhibitor without EDTA] and centrifuged at 15,800 g (av) for 10 min at 4°C before incubating for 1 h with purified glutathione-S-transferase (GST) or GST-kinesin light chain 1 (GST-KLC1, prepared as described previously by Utton et al., ref. 44 ) immobilized on glutathione Sepharose 4B beads (GE HealthCare, Slough, Berkshire, UK). The bead-protein complexes were pelleted by centrifugation at 500 g (av) for 1 min, washed in PBS, and resuspended in Laemmli sample buffer. Bound proteins were analyzed on Western blots with a phosphorylation-independent antibody to tau (Dako) using an Odyssey scanner. Equal loading was confirmed by Coomassie stain of gels loaded in parallel.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibiting GSK-3 with lithium or SB415282 slows the rate of tau axonal transport
The rate of overall axonal transport of tau was measured in cortical neurons transfected with the longest human CNS isoform of tau, 2N4R, fused with EGFP (WTtau-EGFP). We have previously shown that expression of the EGFP tag does not affect the transport of tau in neurons and that the six CNS tau isoforms are transported at similar rates (29) .

The effect of GSK-3 phosphorylation on the overall rate of transport of tau in neurons was studied using lithium and SB-415286, both inhibitors of GSK-3 (32 33 34 35) , inhibiting both GSK-3{alpha} and GSK-3β (36 , 37) and reducing tau phosphorylation in vitro and in vivo (38 39 40) .

Neurons were transfected with WTtau-EGFP in the presence of either 10 mM NaCl (control) or 10 mM LiCl and imaged at time points from 2 h 20 min to 5 h post-transfection. The rate of transport of WTtau-EGFP was calculated as the distance traveled by the leading edge of EGFP fluorescence in the axon with respect to the first time point. The mean distance traveled by WTtau-EGFP at the longest time point (5 h) was ~200 µm.

Cotransfection of neurons with a plasmid expressing DsRed, a soluble fluorescent protein, together with WTtau-EGFP showed that the DsRed fluorescent front was always more distal in the axon compared to the front of WTtau-EGFP (Fig. 1 A). Thus, this confirmed that the transport of WTtau-EGFP within the neurites was not a reflection of the rate of neurite outgrowth.


Figure 1
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  		Figure 1. A) Tau-EGFP transport is not a reflection of neurite outgrowth. Fluorescent image of a cortical neuron cotransfected with WTtau-EGFP (green) and pCMV-DsRed Express (red). WTtau-EGFP traveled 167 µm by 4 h after transfection, whereas DsRed protein traveled 413 µm. WTtau-EGFP did not reach the growth cone of the axon at this time, indicating that the distance traveled by WTtau-EGFP is not a reflection of the length of the axon. Arrows indicate the front of fluorescence. Scale bar = 100 µm. B) Lithium slows the axonal transport of tau. Rat cortical neurons were transfected with WTtau-EGFP, and tau transport was measured in the presence of 10 mM NaCl (control), 10 mM LiCl, or 40 µM SB-415286 (inset). The distance traveled by WTtau, normalized with respect to the first time point (2 h, 20 min for LiCl, 3 h for SB-415286), is reduced 45% in the presence of GSK-3 inhibitors. Values are mean ± SE distance traveled.

An analysis of the distance traveled by WTtau-EGFP in the presence of 10 mM NaCl (control) revealed a mean rate of transport of 0.81 ± 0.04 mm/day (mean±SE, n=25–100 cells, from 4 independent experiments) (Fig. 1B ), similar to our previous report of 0.92 ± 0.08 mm/day (29) . In contrast, the mean rate of transport of WTtau-EGFP in neurons treated with 10 mM LiCl was reduced to 0.46 ± 0.01 mm/day (mean±SE, n=25–100 cells, from 4 independent experiments), a 44% decrease compared to that of tau in control neurons (t test; P<0.001).

We next tested the effects of the competitive GSK-3 inhibitor SB-415286. This compound exhibits greater selectivity toward GSK-3 and is reported to have fewer off-target effects compared to lithium (35) . We measured the distance traveled by WTtau-EGFP at 3 and 5 h post-transfection with or without treatment with 40 µM SB-415286 (Fig. 1B , inset). The rate of WTtau-EGFP transport was reduced by 45% on treatment compared to control (1.05±0.08 mm/day in control, 0.58±0.07 mm/day in SB-415286-treated neurons, mean±SE, n=25–100 cells, 3 independent experiments, t test; P<0.01). Taken together, the results from the lithium and the SB-415286 experiments strongly implicate GSK-3 activity as a modulator of tau axonal transport.

Lithium reduces the phosphorylation state of endogenous tau
To determine the complement of phosphorylation sites on endogenous neuronal tau that are affected by lithium treatment, we used a combination of MS and differential isotopic labeling with methanol or deuterated methanol, similar to the method described by Ficarro et al. (31) .

Endogenous tau extracted from rat cortical neurons contains no N-terminal repeats (0N) and only 3 of the 4 microtubule-binding domain repeats (3R) and is termed 0N3R tau. This tau was extracted from embryonic rat cortical neurons (7 DIV) treated with either 10 mM NaCl (control) or 10 mM LiCl for 4 h. Peptides from tryptic digests of tau were separated and sequenced by LC/MS/MS. A total of 11 defined phosphorylation sites were identified, and 4 phosphopeptides with up to 6 additional phosphorylated sites were detected in rat tau from control cultures (Table 1 ). These sites correspond to residues Thr-114, Ser-131, Ser-132, Ser-135, Ser-147, Thr-164, Ser-168, Ser-298, Ser-306, Ser-315, Ser-324, Ser-143/Thr-145 (1 site), Ser-302/Thr-305/Ser-306 (2 sites), Ser-315/Thr-316/Ser-318 (1 site), and Ser-311/Ser-314/Ser-315/Thr-316/Ser-318 (3 sites) in 0N3R rat tau. Following lithium treatment, the number of phosphorylation sites in tau identified by MS/MS decreased to 4 sites: Thr-114, Ser-132, Ser-135, and a single phosphopeptide containing one undefined phosphorylation site, Ser-315/Thr-316/Ser-318. The doubly phosphorylated peptide containing Ser-131 and Ser-135 was not detected after lithium treatment. The MS survey data from lithium-treated cells was scanned for evidence of phosphorylation sites in the other tau peptides detected in control neurons, but none were detected.


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  		Table 1. Phosphorylation sites determined by mass spectrometric analysis of endogenous tau extracted from rat cortical neurons o:p

To determine whether phosphorylated residues that persisted after 10 mM LiCl treatment were quantitatively altered, the carboxylic groups (C termini of all peptides and side chains of glutamic acid and aspartic acid) of tau peptides from neurons treated with NaCl (control), or LiCl were methylated with either methanol or deuterated methanol, respectively, and the peptides were mixed before MS/MS analysis to obtain quantification of their relative abundance. Methylation results in a 3 Da difference for each carboxyl group between tau peptides from control and lithium-treated neurons. The introduction of an isotopic label into the tau peptides allows the direct comparison of the amount of a particular phosphopeptide in control and lithium-treated neurons. In corroboration of the MS data obtained from unmethylated tau phosphopeptides from control and lithium-treated neurons, we observed qualitative decreases in phosphopeptide ions present in control tau and apparently absent in tau from lithium-treated neurons. Figure 2 A shows an example of a tau peptide containing phosphorylated Ser-306 (corresponding to Ser-404 in human 2N4R tau) in control neurons that was not detected after lithium treatment. Phosphopeptides containing the phosphorylation sites Thr-114, Ser-132, Ser-135, and Ser-315/Thr-316/Ser-318 (1 site) were all present but at significantly reduced levels following lithium treatment. Figure 2B shows differential phosphorylation of a peptide spanning residues 128–142 (corresponding to residues 195–209 in 2N4R human tau). This peptide was present as two singly phosphorylated forms, the site of phosphorylation being either Ser-132 or Ser-135 (Ser-199 or Ser-202 in 2N4R human tau), and this was confirmed from the MS/MS spectra of a single precursor ion. These results show that phosphorylation at all the sites detected in endogenous tau from rat cortical neurons was either reduced or abolished by lithium treatment, suggesting that GSK-3 activity is a significant factor in the physiological phosphorylation of tau.


Figure 2
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  		Figure 2. Relative quantitation of tau phosphopeptides in cortical neurons treated with lithium. The MaxEnt algorithm was used to process mass spectra containing molecular ions for pairs of phosphopeptides isolated from cells treated for 4 h with 10 mM NaCl (control) or 10 mM LiCl. A) Data relating to a methylated phosphopeptide SPVVSGDTpSPR (Mr 1209.2 Da). Lithium treatment reduced the phosphorylation at this site such that no phosphopeptide is detected. B) Methylated phosphopeptides SGYSSPGpSPGTPGSR and SGYSpSPGSPGTPGSR (Mr 1487.0 Da), in which some phosphorylation remains after lithium treatment. Exact sites of phosphorylation were unambiguously determined from MS/MS data.

Phosphomimic tau mutants are transported faster than wild-type tau
To investigate the effect of GSK-3 phosphorylation on the rate of the axonal transport of tau, we subcloned two different pseudophosphorylated mutants of tau (E18tau and E27tau) into pEGFP-C1 to allow visualization of their transport in neurons (Fig. 3 ). These two phosphomimics, both human 2N4R tau, simulate permanent phosphorylation at 18 and 27 phosphorylation sites, respectively, by mutation of serine or threonine residues to glutamate, which mimics the presence of a phosphate group (28) . All the mutated sites are known to be present in PHF tau, and the majority are phosphorylated by GSK-3 in vitro (2 , 3 , 41) . E18tau and E27tau mimic functional aspects of GSK-3-phosphorylated tau protein, including a reduced ability to promote microtubule assembly (28 , 42 , 43) . These phosphomimics are good approximations of high but physiological tau phosphorylation in vivo and allowed us to study the axonal transport of tau in cells unencumbered by the effects of cellular protein kinases or phosphatases.


Figure 3
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  		Figure 3. Phosphomimic tau constructs. The diagram shows the positions in the longest human tau isoform (2N4R, 441 residues) of the serine or threonine residues that are mutated to glutamate in the E18tau and E27tau constructs or alanine in the A18tau construct. R1, R2, R3, and R4 are the repeat regions of the microtubule binding domain (MBD) in tau, Nt inserts are two amino-terminal inserts of 29 amino acids close to the amino terminus of tau.

Following transfection of E18tau-EGFP, E27tau-EGFP, or WTtau-EGFP into rat cortical neurons, we observed no overt differences in the somal distribution of either of the tau phosphorylation mutants compared to WT-tau (Fig. 4A). However, the fluorescent front of the two phosphomimic tau mutants extended further along the axon compared to transfected WTtau-EGFP examined at identical time points (Fig. 4B ). We also confirmed by cotransfecting E18tau-EGFP or E27tau-EGFP with DsRed that neither of the mutant forms of tau extended to the terminals of the axon during our time course (data not shown).


Figure 4
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  		Figure 4. Axonal transport of E18tau and E27tau is faster and A18 is slower than that of WTtau. A) Neurons expressing WTtau-EGFP, A18tau-EGFP, E18tau-EGFP, and E27tau-EGFP at 5 h post-transfection show no overt changes in subcellular expression pattern of the mutants compared to WT. Scale bar = 50 µm. B) The distance traveled by each form of tau was normalized with respect to the first time point (2 h 30 min), showing an increase in the rate of transport of E18tau and E27tau vs. WTtau and a decrease in the rate of transport of A18tau vs. WTtau. Values are mean ± SE distance traveled. Arrows indicate the front of the fluorescence.

To ensure that the apparent increase in the rates of transport of E18tau-EGFP and E27tau-EGFP compared to WTtau-EGFP were not due to the effect of variable tau concentration, we measured the intensity of EGFP fluorescence in the soma of individual transfected neurons. We observed no statistically significant differences in the amounts of fluorescence expressed by any of the three different tau constructs at 2 h 20 min, 3 h, and 5 h post-transfection, (t test; P>0.05) (Supplemental Fig. S1). Treating the transfected neurons with 10 mM LiCl did not affect the expression of the WTtau-EGFP construct compared to control cells. In contrast, a small but nonsignificant decrease was found in the expression of both the E18tau-EGFP and E27tau-EGFP constructs. Furthermore we could find no correlation between the level of expression of any of the three tau constructs with the distance achieved by the fluorescent front along the axons of individual transfected neurons at 3 or 5 h post-transfection (Pearson correlation, r2=0.00–0.15). The data remained uncorrelated in neurons treated with 10 mM LiCl (Supplemental Fig. S2).

We also investigated the possibility that the time of onset of expression of exogenous wild-type and mutant forms of tau might differ. At 30, 60, 90, and 120 min post-transfection, we found no statistically significant differences in either the number of neurons expressing any of the three tau constructs or the pattern of distribution of wild-type and mutant forms of tau in the cell (t test; P>0.05) (data not shown).

Therefore, we analyzed the overall rates of axonal transport of WTtau-EGFP, E18tau-EGFP, and E27tau-EGFP and determined their rates to be 0.9 ± 0.1 mm/day, 2.4 ± 0.30 mm/day, and 1.9 ± 0.1 mm/day, respectively (mean±SE, n=25–100 cells from 6 independent experiments). This represents a 165% increase in the net transport rate of E18tau-EGFP (t test; P=0.001) and a 111% increase in the net transport rate of E27tau-EGFP compared to WTtau-EGFP (t test; P<0.001).

A nonphosphorylatable mutant of tau A18tau-EGFP was constructed. In this mutant, 18 serine/threonine residues, corresponding to the same sites as those mutated in E18tau-EGFP, were mutated to alanine to mimic dephosphorylation (Fig. 3) . In contrast to the results obtained with the pseudophosphorylated mutants of tau, the transport rate of A18tau-EGFP was reduced by 40% compared to that of WTtau-EGFP (WTtau-EGFP; 1.2±0.1 mm/day, A18tau-EGFP; 0.8±0.1 mm/day, mean±SE, n=25–100 cells, from 3 independent experiments, t test; P=0.001) (Fig. 4) . These results indicate that mutant tau constructs that mimic GSK-3 phosphorylated tau directly increase the rate of tau transport in neurons, whereas elimination of endogenous phosphorylation at the same GSK-3 sites reduces the rate of tau transport.

We further investigated whether inhibition of GSK-3 affected the transport of E18tau and E27tau. The transport rate of E18tau-EGFP in these experiments was equivalent to 1.65 ± 0.23 mm/day, 1.60 ± 0.25 mm/day in the presence of 10 mM LiCl, and 1.20 ± 0.14 mm/day in the presence of 40µM SB-415286 (mean±SE, n=25–100 cells from 5 independent experiments with lithium and 3 independent experiments with SB-415286). Despite the tendency of SB-415286 to reduce the rate of transport of E18tau, the differences were not significant (t test; P>0.05 vs. control for each inhibitor). The transport rate of E27tau-EGFP in these experiments was 1.85 ± 0.32 mm/day, 1.77 ± 0.23 mm/day in the presence of 10 mM LiCl, and 1.65 ± 0.28 mm/day in the presence of 40µM SB-415286 (mean±SE, n=25–100 cells from 5 (LiCl) and 3 (SB-415286) independent experiments), which were not significantly different (t test; P>0.05 vs. control for each inhibitor). These results corroborate our finding that GSK-3 regulates tau transport principally via a direct effect on tau phosphorylation, and GSK-3 phosphorylation of other components of the transport machinery is not required to mediate this effect.

Dephosphorylation of tau decreases complex formation with kinesin-1 in vivo
We have previously reported that both rat brain tau and recombinant human tau bind to kinesin-1 (44) . We hypothesized that the phosphorylation state of tau may influence its interaction with kinesin-1. We investigated this by immunoprecipitating tau-kinesin-1 complexes from rat cortical neurons treated with 10 mM LiCl to inhibit GSK-3 phosphorylation and comparing these complexes to those from neurons treated with 10 mM NaCl (control).

Examination of rat cortical neuronal lysates demonstrated that 10 mM LiCl increased the electrophoretic mobility of tau (Fig. 5 A, lysates), indicating partial dephosphorylation of tau; in contrast, the mobility of kinesin-1 was unaffected by 10 mM LiCl treatment (Fig. 5B , lysates). A polyclonal antibody to tau coimmunoprecipitated both tau and kinesin-1. This confirmed their interaction in cortical neurons. The amount of kinesin-1 pulled down in the complex with tau antibody was reduced with 10 mM LiCl treatment with respect to control (Fig. 5B , IP tau). Similarly, immunoprecipitated kinesin-1 pulled down less tau in the presence of 10 mM LiCl than in control neurons (Fig. 5A , IP kinesin).


Figure 5
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  		Figure 5. Dephosphorylated tau binds less to kinesin-1. A) Western blots of neuronal lysates and kinesin immunoprecipitates (IP kinesin) from 2 independent experiments probed with antibodies against tau from neurons treated with either 10 mM NaCl (Ctrl) or 10 mM LiCl. B) Western blots of neuronal lysates and tau immunoprecipitates (IP Tau) from 2 independent experiments probed with antibodies against kinesin-1 from neurons treated with either 10 mM NaCl (Ctrl) or 10 mM LiCl. "IP no Ab" shows nonspecific binding to protein-A beads in the absence of antibody. C) In both experiments, the interaction between tau and kinesin-1 is reduced on treatment with lithium.

The measurement of the intensity of gel bands of each of these proteins revealed that in tau immunoprecipitates, lithium reduced the kinesin:tau ratio to 47% (t test; P<0.01) of that in complexes from control neurons; in kinesin immunoprecipitates, lithium reduced the tau:kinesin ratio to 72% (t test; P<0.05) of that in complexes from control neurons (Fig. 5C ).

These results indicate that the phosphorylation state of tau is an important regulator of its binding to kinesin-1. Endogenous neuronal tau dephosphorylated by lithium treatment is less able to bind to kinesin-1.

The binding of tau to kinesin-1 is affected by GSK-3 phosphorylation of tau
To determine whether GSK-3 phosphorylation of tau alone is sufficient to decrease its interaction with kinesin-1, we performed in vitro phosphorylation of recombinant tau with GSK-3 and measured its interaction with GST-KLC1.

As expected, the electrophoretic mobility of tau after in vitro phosphorylation by GSK-3 was reduced with respect to that of untreated recombinant tau (Fig. 6 ). We observed an increased binding between GSK-3-phosphorylated tau and GST-KLC1 compared to unphosphorylated recombinant tau (Fig. 6) . This result suggests that GSK-3 phosphorylation of tau increases its binding to kinesin-1.


Figure 6
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  		Figure 6. GSK-3 phosphorylation of tau enhances its binding to GST-KLC1. Western blot of the binding of full-length hr2N4R tau (control) or GSK-3-phosphorylated tau (tau+GSK3) to GST-KLC1. An increase in the binding to KLC1 is seen on GSK-3 phosphorylation. The nonspecific binding to GST beads alone is negligible (beads only).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have found that GSK-3 phosphorylation of tau modulates its axonal transport by regulating its binding to kinesin. Thus, reducing tau phosphorylation by treating neurons with GSK-3 inhibitors decreases the overall rate of tau transport by 55%. Dephosphorylated tau from lithium-treated neurons binds significantly less to kinesin-1 than tau from untreated neurons. Consistent with these results, we have shown by two different approaches that phosphorylated tau, and particularly GSK-3, binds more to kinesin-1 and also shows an increased overall rate of axonal transport, by 140%. The overall rate of transport of the nonphosphorylatable A18tau mutant was transported more slowly, at approximately half the rate of wild-type tau.

We have shown previously that the axonal transport of tau is energy dependent (29) , indicating that tau axonal transport under these conditions is not due to diffusion, as suggested by Konzack et al. (45) .

Evidence indicates that GSK-3 activity reduces the fast anterograde axonal transport of membrane-bound organelles, leading to the detachment of kinesin from its cargo by phosphorylating the light chain of kinesin-1 (21 , 46 47 48) . Thus, a possibility exists that lithium may affect anterograde axonal transport in our study by dephosphorylating kinesin-1 light chain, by increasing the axonal transport rate of tau. Conversely, lithium and the more specific GSK-3 inhibitor SB-415286 decreased anterograde axonal transport of tau in our study, and furthermore, neither compound affected the axonal transport rate of the phosphomimic mutants of tau. This finding indicates that, under our experimental conditions, GSK-3 inhibitors did not affect axonal transport of tau via phosphorylation changes in kinesin, implying a different regulatory mechanism compared to transport of membrane-bound organelles.

Taken together, these results support the notion that it is the direct phosphorylation of tau that regulates tau transport. These results are in agreement with published data indicating that tau binds directly to complexes containing either kinesin-1 light chain 1 or 2 (44 , 49 , 50) .

Lithium does not affect axonal growth
It has been reported that neurons exposed to GSK-3 inhibitors show a reduction of neurite growth from undifferentiated neurons lacking neurites of significant length after long exposure to GSK-3 inhibitors (51 52 53) . In our study, neurons were incubated for 5 days in vitro prior to treatment, thus extending long neurites before transfection, and neurons were exposed to lithium for a maximum of 5 h. Under these conditions, our data demonstrate that GSK-3 inhibitors do not affect axonal growth, because they would decrease the distance traveled by all of the tau constructs as a consequence of a reduction of the length of the axons (by 50%, according to Owen et al.; ref. 53 ). Clearly, this is not the case, because neither lithium nor SB-415286 affected the distance traveled by E18tau and E27tau, whereas both of these GSK-3 inhibitors affected the transport of wild-type tau.

GSK-3 is the major kinase acting on endogenous rat tau
Ten of the 11 phosphorylation sites we unambiguously identified by mass spectrometric sequencing in endogenous tau from cultured rat cortical neurons have been reported previously as sites of GSK-3 phosphorylation (2 , 10 , 11) .

Lithium treatment of rat fetal neurons abolished phosphorylation at 7 sites completely and greatly reduced phosphorylation at the remaining 3 sites (equivalent to Thr-181, Ser-199, and Ser-202 in human 2N4R tau). Residual low-level phosphorylation at one ambiguous site was retained in a peptide containing 3 potential sites of phosphorylation, namely, Ser-315, Thr-316, or Ser-318 (equivalent to Ser-413, Thr-414, or Ser-416 in human 2N4R tau). These results show that lithium completely ablates phosphorylation at the majority (>80%) of sites phosphorylated in endogenous tau and thus positions GSK-3 as a major physiological tau kinase in fetal rat neurons.

The proline-rich region of tau interacts with kinesin
Previous studies have shown that phosphorylation mimics of sites in the proline-rich region and N terminus of tau reduce both its interaction with microtubules and its aggregation into filaments in vitro (43 , 54 , 55) . Here we show that glutamates located in the common mutated region of E18tau and E27tau, that is, the proline-rich region (Ser-198 to Ser-235), exon 7 (Thr-111 to Thr-181), and the first N-terminal repeat (Ser-46 to Thr-69), may be involved in the regulation of axonal transport of tau and binding to kinesin, because the transport rates of E18tau and E27tau were similar. Furthermore, because lithium and SB-415286 decreased the rate of transport of WTtau-EGFP, and lithium also reduced binding of endogenous tau to kinesin, this finding indicates that the GSK-3 sites located in the proline-rich region (Ser-198, Ser-202, Ser-214, Thr-231, Ser-235, and one of Ser-210/Thr-212) may be the major region in tau that interacts with kinesin.

Phosphorylation of tau regulates its binding to kinesin
Here we show that binding of tau to kinesin-1 is dependent on its phosphorylation by GSK-3. However, axonal transport of CFP-tau in murine Nb2a/d1 cells cotransfected with cyclin-dependent kinase 5 (Cdk5) is reduced by 20% (25) . This effect on axonal transport of tau is opposite to our results and is a small effect in comparison with those we observe with lithium, SB-415286, or the phosphomimic mutants of tau, and suggests that Cdk5 may have a secondary role in regulation of axonal transport of tau. Alternatively, because cdk5 has been shown to phosphorylate GSK-3, with the result that GSK-3 is less active (56) , the report that cdk5 reduced tau transport rate would be consistent with our finding if the mechanism was via GSK-3 inhibition.

Finally, our findings showing that GSK-3 phosphorylation increases the rate of tau axonal transport are supported by another study showing that mice lacking the regulatory subunit p35 of Cdk5 exhibited a shift in the distribution of PHF-1 positive tau from the soma to distal sites of the neuropil. In these mice, GSK-3 activity was increased ~70% with respect to wild-type mice (57) . Taken together with our data, these results may account for the observation that tangle pathology in Alzheimer’s disease appears to commence distally and then spreads in a retrograde fashion to the perikaryon (58 , 59) . This may be caused by hyperphosphorylated tau accumulating distally due to alterations in axonal transport in disease, and the progressive and retrograde accumulation of hyperphosphorylated aggregates may ultimately produce perikaryal neurofibrillary tangles. This process may be a mechanism to protect the stability of the microtubules by transporting hyperphosphorylated tau more rapidly to other cellular locations where tau can form aggregates.


   ACKNOWLEDGMENTS
 
This work was supported by the Wellcome Trust, the Alzheimer’s Society, and the UK Medical Research Council. Plasmids expressing 2N4R tau, E18tau, and E27tau were kind gifts from M. Goedert (Medical Research Council Laboratory of Molecular Biology, Cambridge, UK).


   FOOTNOTES
 
2 Current address: William Harvey Research Institute, Barts and the London, Charterhouse Sq., EC1M 6BQ London, UK.

Received for publication March 20, 2008. Accepted for publication April 24, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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