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Published as doi: 10.1096/fj.06-5721fje.
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(The FASEB Journal. 2006;20:1272-1274.)
© 2006 FASEB

Tumor-suppressor PTEN affects tau phosphorylation, aggregation, and binding to microtubules

Xue Zhang, Feng Li, Ayelen Bulloj1, Yun-wu Zhang, Gang Tong, Zhuohua Zhang, Francesca-Fang Liao and Huaxi Xu2

Center for Neuroscience and Aging, Burnham Institute for Medical Research, La Jolla, California, USA

2Correspondence: Center for Neuroscience and Aging, Burnham Institute for Medical Research, La Jolla 92037, CA, USA. E-mail: xuh{at}burnham.org

ABSTRACT

Neurofibrillary tangles (NFTs), consisting of abnormally hyperphosphorylated tau, are implicated in the pathogenesis of several neurodegenerative diseases including Alzheimer’s disease (AD). The molecular mechanisms underlying the regulation of tau phosphorylation are largely unknown. While the PI3K/Akt pathway has been shown to regulate multiple cellular events pertinent to AD pathogenesis, potential functions of tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in AD pathogenesis have not been explored. Here, we examine the effects of PTEN on tau phosphorylation, its microtubule association and formation of aggregates, and consequentially neuronal morphology. In cultured cells, overexpression of wild-type (WT) PTEN alters tau phosphorylation at several sites, increases tau-microtubule association and decreases formation of tau aggregates. In addition, the phosphatase-null PTEN increases tau aggregation and impairs tau binding to microtubule and neurite outgrowth of neurons expressing the mutant PTEN. We also found a significant loss of PTEN in AD patient brains correlated with a dramatically increased concentration of phospho-tau at Ser-214 in NFTs. Together, our results demonstrate that PTEN regulates tau phosphorylation, binding to microtubules and formation of aggregates and neurite outgrowth. These findings suggest a link between malfunction of PTEN and tauopathy, and imply PTEN as a therapeutic target for tauopathy.—Zhang, X., Li, F., Bulloj, A., Zhang, Y.-w., Tong, G., Zhang, Z., Liao, F.-F., Xu, H. Tumor-suppressor PTEN affects tau phosphorylation, aggregation, and binding to microtubules.


Key Words: Alzheimer’s disease • tauopathy • PIP3/Akt

A KEY PATHOLOGICAL hallmark for Alzheimer’s disease (AD) is intracellular neurofibrillary tangles (NFTs), whose major component is bundles of paired helical filaments (PHF) of hyperphosphorylated tau proteins (1) . The biochemical study of neuropathological lesions has revealed that such intracellular tau filamentous deposits occur numerous other neurodegenerative diseases as well, including Pick’s disease (PiD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain disease and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) (2) . The evidence for a causal role of tau aggregation in neurodegenerative diseases was provided by the genetic analyses of the inherited FTDP-17, which led to identification of tau mutations that cause the disease (3 4 5) .

Tau is a class of microtubule-associated protein (MAP) in neuronal and glial cells, which exist in six tau splicing variants in human brain. The tau protein is normally expressed in cytoplasm including cell bodies, neurites, and axons, where it binds to and stabilizes microtubules (6 7 8) . Tau can be phosphorylated at several serine or threonine sites before proline. Numerous kinases, including CDK5 (cyclin-dependent kinase 5), GSK-3 (glycogen synthase kinase-3), MAPK (mitogen-activated protein kinase), protein kinase A (PKA), protein kinase (PKC), and Akt have been identified to phosphorylate tau in vitro (9) . Tau is phosphorylated under normal physiological conditions, carrying 2–3 phosphates per molecule. However, hyperphosphorylation of tau, which renders tau 3–4 times more phosphates (10 , 11) , will cause dysfunction of tau (12) , tau aggregation, and likely NFTs formation (13) .

The tumor suppressor gene Pten (phosphatase and tensin homologue deleted on chromosome 10), also known as MMAC1 and TEP1, is the second most frequently mutated gene after p53 in many human sporadic and hereditary cancers (14 15 16 17) . PTEN contains a tyrosine phosphatase functional domain, exhibiting both protein and lipid phosphatase activity in vitro (18) . The phosphatidylinositol (3 4 5) -triphosphate (PIP3) has been identified as a major lipid substrate for PTEN (15 , 16) , but the putative substrates of PTEN with proteinaceous nature are unknown. PTEN antagonizes the phosphoinositide 3-kinase (PI3K) signaling to govern a variety of crucial cellular functions, including cell proliferation, migration, and apoptosis (19 , 20) . Therefore, the lipid phosphatase activity of PTEN is critical for its tumor-suppressor function (21) . Pten-null mice die at early embryonic stages, and heterozygous knockout mice develop a number of tumors (22 23 24 25) . Mouse brains with conditionally inactivated Pten showed an increased soma size of neurons without altering proliferation (26 , 27) . A recent study showed decreased levels and altered distribution of PTEN along with elevated PI3K signaling in AD patient brains, suggesting that a loss of PTEN contributes to neurodegeneration in AD (28) .

Akt and GSK-3 are two major downstream effectors of the PIP3 pathway. PTEN down-regulates Akt, which in turn activates GSK-3. GSK-3 has been shown to phosphorylate tau at multiple sites in vitro and in vivo (29 30 31 32 33) . The observation that Akt/GSK-3 can affect tau phosphorylation raises a possibility that PTEN may also modulate tau phosphorylation. In the present study, we have analyzed tau phosphorylation and investigated whether the altered phosphorylation of tau by PTEN leads to changes in tau aggregation and microtubule-binding ability in cultured cells and primary neurons. We demonstrate that WT PTEN modulates tau phosphorylation at certain residues reducing tau aggregation and promoting its microtubule binding, while lipid phosphatase-null PTEN has opposite effects. In addition, we observe a decreased concentration of PTEN accompanied by increased phospho-tau at Ser-214 (a major Akt site) in AD brains.

MATERIALS AND METHODS

Cell cultures and transient transfection
COS-7 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS and antibiotics. Primary neurons derived from the cerebral cortices of day 17 (E17) embryos of timed pregnant Sprague Dawley rats were cultured for 2 wk in Neurobasal medium (Invitrogen, Carlsbad, CA) supplemented with B27 and 0.5 mM glutamine as described (34) . Mouse Pten knockout fibroblasts with inducible human Pten transgene (a gift from Dr. Hong Wu, UCLA) were cultured in DMEM supplemented with 10% tetracycline-free FBS (BD Biosciences Clontech, Palo Alto, CA) and antibiotics. The expression of human PTEN was induced by addition of 2 ng/ml doxycycline (Sigma, St. Louis, MO), and cells were cultured continuously for 48 h before the experiment. In some experiments, cells were treated with 50 µM LY294002 (Biosource, CA) or 100 ng/ml insulin-like growth factor-1 (IGF-1) (PeproTech, Inc., Rocky Hill, NJ) for 5 h. Cells were first transfected with WT tau (T40) and equally split, followed by a second transfection with either WT PTEN (PTEN–wild-type) or the lipid phosphatase null mutant PTEN (PTEN–CG), using Lipofectamine (Invitrogen).

Sindbis virus expression
Human WT and mutant Pten cDNAs were subcloned into pIRES-enhanced GFP (Invitrogen) using EcoRI/BamHI sites to generate pIRES-Pten. The 2.5 kb DNA fragment, including Pten cDNA followed by IRES and enhanced GFP (EGFP) sequences, was digested by EcoRI/NotI from pIRES-Pten. After Klenow treatment, the fragment was ligated into pSin-Rep5 (Invitrogen), which was digested by XbaI and filled with Klenow. Viral particles were then generated by in vitro transcription and transfected into baby hamster kidney cells according to the manufacturer’s protocol. Virus particles were collected from media 24–48 h posttransfection, and frozen at –80°C until use. Primary neurons were infected by the virus in conditioned medium for 1 h.

Western blotting
To analyze phospho-tau, cells were homogenized in a lysis buffer containing 10 mM Tris/Cl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 5 mM EGTA; 50 mM NaF; 1 mM Na3VOF3; 5 mM DTT; 1% Nonidet P-40; and a cocktail of protease inhibitors. Cell lysates were collected after brief sonication and centrifugation at 18,000 g. The equal amounts of lysates were then subjected to SDS-PAGE. Proteins were transferred to PVDF membranes and probed with antitau antibodies: H150 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), pS214 (1:1000; Biosource), pS199 (1:1000; Biosource), pT212 (1:1000; Biosource), pS396 (1:1000; Biosource), pS202 (1:1000; Biosource), pS262 (1:1000; Biosource), and pT205 (1:500; Biosource). PTEN proteins were detected using mouse anti-PTEN antibody (Ab) (Cell Signaling, CA; 1:1000). Tubulin was detected using anti-{alpha}-tubulin Ab (1:10000; Sigma). Phospho-Akt (Ser-473 and total Akt were detected using antiphospho-Akt (Ser-473 (1:500; Cell Signaling, Danvers, MA) and anti-Akt (1:500; Cell Signaling) antibodies, respectively. The membranes were incubated with peroxidase-labeled secondary antibodies, and signals were visualized by using enhanced chemiluminescence. In some experiments, Western blots were scanned and protein bands were quantified using Scion Image.

Fractionation of transfected COS-7 cells
COS-7 cells were cotransfected with human tau and either WT, the mutant human Pten, or plasmid construct DNA (pcDNA) control. Cells were fractionated as described previously with modification (35) . Specifically, cells were harvested 48 h after transfection and homogenized in breaking buffer (0.25 M sucrose/10 mM HEPES, pH 7.2/1, mM MgOAc2/protease inhibitors mixture) by using a stainless steel ball-bearing homogenizer. Cytosol was prepared from postnuclear supernatant by ultracentrifugation for 1 h at 190,000 g. The resulting membrane pellet was resuspended and incubated on ice for 30 min with 5 µM nocodazole, followed by ultracentrifugation for 1 h at 190,000 x g to produce postnocodazole supernatants containing the microtubule-associated tau. The generated pellets containing both membrane-associated and aggregated tau were further extracted using 100 mM sodium carbonate buffer (pH 11.5) at 4°C for 30 min. The post-Na2CO3 pellets were prepared by ultracentrifugation at 190,000 g for 1 h and washed with 1% SDS to produce a fraction containing tau aggregates. Aliquots containing equal amounts of protein were analyzed by SDS/PAGE–Western blotting for tau using H150. Western blotting results were quantified by densitometry to determine the tau concentration in each fraction.

Filter/trap assays for tau aggregates
COS-7 cells expressing human tau were transfected with human WT Pten, the mutant Pten, or pcDNA control. Cells were lysed in a buffer containing 0.5% Nonidet P-40/1 mM EDTA/50 mM Tris·HCl, pH 8.0/120 mM NaCl/protease inhibitors mixture. After brief sonication, cell lysates were passed through a cellulose acetate membrane (0.2 µm; Bio-Rad) using Bio-Dot Microfiltration Apparatus (Bio-Rad, Hercules, CA) and washed three times with 1% SDS followed by immunoblotting using H150 Ab. Quantitative Western blot analyses were used to determine levels of tau aggregates in each sample.

Immunohistochemistry and immunocytochemistry studies
Human brain tissues from AD (n=3) and normal age-matched controls (n=3) were obtained from The Alzheimer’s Disease Research Center of the University of California, San Diego (UCSD). This study was conducted according to approved guidelines of the UCSD Human Research Protection Program. For immunohistochemistry, 10% formalin-fixed, paraffin-embedded brain sections (8 µm) were deparaffinized, washed in PBS, quenched for endogenous peroxidase with 3% hydrogen peroxide for 30 min, and preincubated in 0.5% BSA/PBS for 30 min to prevent nonspecific staining. Slices were then incubated with the appropriate primary Ab in 0.1% BSA/PBS overnight at 4°C. Slides were washed with PBS and incubated with secondary Ab (antiprimary Ab species Ab) (Vectastain avidin-biotin complex (ABC) kit; Vector, Burlingame, CA) in 0.1% BSA/PBS at room temperature for 1 h. Samples were incubated with ABC kit, followed by development for 5 min with diaminobenzidine (Dako, 3,3'-diaminobenzidine kit). The primary antibodies used are anti-PTEN (1:50, Cell signaling), anti-Aß42 Ab (1:500, Chemicon, Temecula, CA); and antitau antibodies (pS214, 1:200, Biosource; AT8, 1:500, Innogenetics, Ghent, Belgium). For double immunolabeling, sections were stained sequentially first with polyclonal anti-Aß Ab and the Dako Fuchsin substrate-chromogen system (red) and then with either monoclonal anti-PTEN (1:50, Cell signaling) or AT8 (1:500, Innogenetics, Ghent, Belgium) and the Dako 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT) substrate system (blue). To compare the levels of PTEN in AD and non-AD brains, the intensity of PTEN immunostaining signal in neurons (20 neurons for each experimental group) was quantified by densitometry. The intensity was then subtracted by the average background density of each sample.

For immunocytochemistry on cultured primary neurons, cells on coverslips were fixed in 4% paraformaldehyde (PFA)/PBS for 15 min followed by 5 washes with PBS 5 min each time. Cells were then permeabilized with 0.1% Triton X-100 in PBS for 10 min before blocking with 5% BSA/PBS for 30 min. To stain tau and tubulin, the cells were incubated with antitau Ab, H150 (1:200) and anti-{alpha}-tubulin Ab (1:2000; Sigma) in 5% BSA/PBS for 2 h. Cells were then washed and incubated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA)-conjugated antimouse IgG (1: 300, Invitrogen) and Alexa Fluor 594-conjugated anti-rabbit IgG (1:300, Invitrogen) for 1 h. The coverslips were then washed and mounted on slides. All procedures were performed at room temperature. Fluorescent images were visualized and captured using a deconvolution microscope (Zeiss Axiovert 100M). To quantify the number and the length of dendrites, GFP-positive neurons were visualized and images were taken using a fluorescence microscope (Olympus IX71). For each experimental group, four fields containing 10 neurons were randomly selected. The numbers and length of dendrites were counted without knowledge of the transgenes.

RESULTS

Overexpression of PTEN affects tau phosphorylation
Tau can be phosphorylated at multiple sites by various kinases (Fig. 1 A). To study whether PTEN affects tau phosphorylation, we cotransfected the longest human tau splicing variant (T40) and human WT PTEN or a mutant PTEN lacking the lipid phosphatase activity (PTEN–CG) into COS-7 cells. We focused on the sites that are targets for GSK-3, Akt, and those that are prominent in PHF tau aggregates. Among the 7 phosphorylation sites examined, Thr212 and Ser214 are targets for Akt; Ser199, Thr205, Thr212, and Ser396 can be phosphorylated by GSK-3. By using phospho-tau specific antibodies (36) that are widely used for studies of tau phosphorylation, we determined and compared the amounts of different phospho-tau proteins in the transfected cells. In the WT PTEN transfected cells, the levels of Thr212 and Ser214 phospho-tau were significantly decreased by ~30 and 50%, respectively, compared with those in pcDNA control cells (Fig. 1B, C ). Since Akt preferably phosphorylates Ser214 (37) , the greater change in tau phosphorylation at Ser214 than at Thr212 is likely due to the fact that Ser214 is a more favorable target than Thr212 by Akt. However, the caution should be exercised when attributing this effect solely to inactivation of Akt, because Ser214 may also be phosphorylated by other kinases, such as PKA. Quantification of phospho-tau at other examined sites revealed increased phosphorylation of tau at Ser-199, Ser202, and Thr205, which are substrates for GSK-3, suggesting elevated GSK-3 activity on PTEN overexpression. The phosphorylation of tau at Thr262 was virtually unchanged by overexpression of PTEN.


Figure 1
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  		Figure 1. The effects of overexpression of PTEN proteins on tau phosphorylation and aggregation. A) Schematic drawing of human tau and major phosphorylation sites by various kinases. The sites examined are in bold. Kinases are abbreviated as follows: M, MAPK; G, GSK-3; C5, CDK5; A, PKA; C, PKC; PK, phosphorylase kinase. B) pcDNA, WT (PTEN–wild-type) or lipid phosphatase null mutant PTEN (PTEN-CG) transfected COS-7 cells were analyzed by Western blots using phospho-tau specific antibodies as indicated. C) Levels of phospho-tau were quantified and normalized to total tau levels. Error bars indicate means ± SE; n = 4, *P < 0.01 and **P < 0.02 compared with the respective controls (vector alone). D) COS-7 cells transiently overexpressing WT tau (T40) were further transfected with pcDNA vector, WT PTEN, or PTEN-CG. Insoluble aggregated tau proteins were isolated and detected by using a filter-trap/immunoblotting assay (upper panels). Transfected PTEN and total tau proteins (as indicated in middle and lower panels) were detected by Western blots from same amounts of lysates as in filter assays. E) The amounts of aggregated tau protein were quantified by densitometry and normalized using the amounts of total tau. Error bars indicate means ± SE; n = 3, *P < 0.01 compared with the vector transfection control.

Interestingly, the mutant PTEN selectively increased phospho-tau at Ser396, a major tau phosphorylation site in PHF and NFTs, while leaving the other 6 sites unaltered, which suggests that this PTEN mutant mediates PHF-prone tau phosphorylation via a lipid phosphatase-independent mechanism (Fig. 1B, C ). It has been shown that PTEN can down-regulate MAPK activity through attenuating platelet-derived growth factor receptor (PDGFR) (38) . Since Ser396 can be phosphorylated by both GSK-3 and MAPK (39) , it is likely that the elevated phosphorylation at this site is due to a higher activity of MAPK in the presence of the mutant PTEN.

PTEN affects tau aggregation and microtubule-binding ability
The opposite regulatory effects of PTEN on tau phosphorylation at GSK-3 and Akt sites led us to investigate how these changes may affect tau aggregation and the microtubule-binding function. The effect of PTEN on tau solubility was examined using a filter-retardation assay designed to detect protein (tau) aggregates. Insoluble tau molecules in cell lysates were retained in the filter after SDS washes, and the amount of insoluble tau was significantly decreased by 50% on overexpression of WT PTEN as measured by immunoblotting of the filters. The aggregation of tau was increased 1.8-fold when the mutant PTEN was overexpressed (Fig. 1D, E ). It is likely that the reduction of tau aggregation by WT PTEN is primarily due to the reduced phosphorylation at Ser214 and/or Thr212, despite the increased phosphorylation at GSK-3 sites, whereas the significant increase in tau aggregation by PTEN-CG probably resulted from the hyperphosphorylation of tau at Ser396. While we cannot rule out the possibility that tau phosphorylation at sites other than those examined may also individually contribute to PTEN's effect on tau aggregation, it is evident that PTEN can reduce tau aggregation as a net effect of modulation at various phosphorylation sites.

Our findings on PTEN's effects on tau phosphorylation and aggregation strongly suggested that PTEN plays an important role in tau’s physiological function in microtubule binding. In addition, part of the pathogenic effect of tau in AD is hypothesized to result from an inability of hyperphosphorylated tau to bind efficiently to microtubules. To test the effect of PTEN on tau’s partitioning into microtubules, we fractionated COS-7 cells cotransfected with human tau and either WT or the mutant PTEN to separate transfected tau proteins according to their intracellular localization (Fig. 2 ). We found that PTEN-WT has little effect on the soluble cytosolic tau, while PTEN-CG reduced this population of tau by ~30%. Consistent with the data obtained using filter-retardation assay, PTEN-WT increased the amount of microtubule-associated tau by 40%, concomitant with a 25% reduction in insoluble tau aggregation (Fig. 2B, C ). On the other hand, expression of PTEN-CG resulted in a 40% increase in the insoluble membrane-bound tau and an ~1.5-fold increase in aggregated tau. The mutant PTEN significantly reduced microtubule-bound and soluble free tau (Fig. 2B, C ). Taken together, our data demonstrate that WT PTEN promotes tau binding to microtubules, whereas mutant PTEN deteriorates tau’s microtubule association property.


Figure 2
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  		Figure 2. PTEN affects tau solubility and binding to microtubule. A) Fractionation scheme used to resolve different cellular pools of tau. Total membrane and cytosolic fractions were prepared. The tau fraction sedimenting with total membranes was treated with nocodazole to solubilize microtubule-associated tau (Soluble cytosolic tau). The remaining membrane-bound/aggregated tau (Pellet 1) was extracted with Na2CO3 to solubilize membrane-associated tau (Supernatant 2), thus separating it from aggregated tau (Pellet 2). B) Western blot analysis of the effects of WT or the mutant PTEN on tau pools generated from fractionation scheme of a. C) Data represent mean ± SE; n = 3; P < 0.02.

The effects of PTEN on tau phosphorylation are mediated through PIP3 signaling pathway
Since overexpression of PTEN had different effects on tau phosphorylation at Akt and GSK-3 sites, which are dependent on the lipid phosphatase activity of PTEN, we hypothesized that PTEN regulates tau phosphorylation through the PIP3 signaling pathway. We utilized a PTEN knockout fibroblast cell line derived from PTEN knockout mice (40) that bears an inducible human Pten transgene, which can be induced by doxycycline (DOX) (Fig 3 A). As expected, on induction of PTEN expression, tau phosphorylation at Ser214 was decreased by 40%, accompanied by reduced Akt activity (as manifested by Ser473 phospho-Akt) (Fig. 3A ). The PI3K inhibitor LY294002 (LY) further down-regulated tau phosphorylation at Ser214 (up to 80%) as well as Akt activity. In addition, phosphorylation of tau at Ser205 was increased by ~20 or ~40% in the presence of induced PTEN or PI3K inhibitor, respectively. This is likely due to the increase of GSK-3 activity, since the concentration of phospho-tau at Ser396 (a non-GSK-3/Akt site) was virtually unchanged (Fig. 3A, B ).


Figure 3
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  		Figure 3. The effects of PTEN on tau phosphorylation involve PIP3 signal pathways. A, B) Pten knockout cells expressing inducible human PTEN were pretreated with vehicle DMSO or 2 ng/ml doxycycline (DOX) for 48 h followed by 5 h treatment with or without 50 µM LY294002 (LY). Tau phosphorylation at Ser214, Thr205, and Ser396, as well as the active form of Akt, were examined by Western analysis using specific antibodies as indicated. Arrowhead indicates total Akt. Levels of phospho-tau were quantified by densitometry and normalized to total tau. C, D) COS-7 cells transiently overexpressing both WT tau (T40) and WT PTEN were treated with 100 ng/ml IGF-1, vehicle DMSO, or 50 µM LY294002 (LY) for 5 h. Cell lysates were analyzed for phospho-tau S214, total tau, and PTEN. Levels of phospho-tau S214 were quantified by densitometry and normalized to total tau. Error bars indicate means ± SE., n = 3. *P < 0.05, **P < 0.01.

Since IGF-1 can activate the PIP3 signaling pathway (41) , we expected that the exposure to IGF-1 would cause hyperphosphorylation of tau at Ser214. We then cotransfected COS-7 cells with WT PTEN and human tau and treated the cells with IGF-1, LY, or DMSO as control. Indeed, we observed ~30% more Ser214 phospho-tau in the presence of IGF-1, but an approximate 70% decrease in the LY-treated cells, compared with that in the DMSO control (Fig. 3C, D ). These results indicate that the effects of PTEN on tau phosphorylation are mediated through the PIP3 signaling pathway. A potential role of insulin and IGF-1 in preventing AD pathogenesis has been proposed (42 , 43) . Insulin and IGF-1 may deregulate tau phosphorylation through down-regulation of GSK3 activity (44) and/or up-regulation of protein phosphatase 2 (45) . However, it has also been indicated that insulin and IGF-1 can increase tau phosphorylation at specific sites (46 ,47) . Our findings showed that IGF-1 can increase tau phosphorylation at Ser214 through regulation of the PIP3 signaling pathway. Therefore, cautions must be exercised when considering the insulin/IGF-1 signaling pathway as potential AD therapeutic targets.

Phosphatase activity-null PTEN impairs neuronal tau binding to microtubule and alters neuronal morphology
To corroborate PTEN's effects on tau’s microtubule-binding function in primary neurons, we infected rat cortical primary neurons with Sindbis viruses expressing EGFP alone, WT PTEN and EGFP, or the mutant PTEN and EGFP. The expression of the exogenous PTEN proteins was confirmed by Western blots (Fig. 4 A). We stained endogenous tau and tubulin in the neurons and showed that tau and tubulin were completely overlapped in EGFP control or WT PTEN expressing neurons (Fig. 5 B, a–h). However, in the mutant PTEN expressing neurons, the majority of tau was concentrated in cell bodies and failed to colocalize with tubulin in dendrites. (Fig. 4B, j-l ). In addition, the morphology of the neurons expressing the mutant PTEN was significantly altered with marked (60–70%) decreases in both the number and the length of dendrites, suggesting that the dissociation of tau from microtubule by the mutant PTEN in the dendrites acutely impairs neurite outgrowth. Only slight increases in the number and the length of dendrites were observed when WT PTEN was expressed (Fig. 4C ).


Figure 4
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  		Figure 4. The lipid phosphatase null mutant PTEN impairs neuronal structure/neurite outgrowth. Rat cortical primary neurons were cultured for 2 wk and infected with sindbis viruses expressing GFP alone, GFP-IRES–WT PTEN or GFP-IRES-mutant PTEN. A) Expression of exogenous PTEN in infected primary neurons was confirmed by Western blots. Endogenous total tau and tubulin were also detected. B) Neurons expressing GFP alone (a–d), GFP-IRES–WT PTEN (e–h), or GFP-IRES-mutant PTEN (i–l) were visualized based on the GFP fluorescence (representing the expression of the transgenes), and further immunostained to detect tubulin (b, f, j) and tau (c, g, k). Fluorescence micrographs were visualized and recorded using a deconvolution microscope; d, h, and i are digitally merged images of tubulin and tau staining. C) Images of GFP-positive neurons were taken by fluorescence microscope (inset). The length and number of the neurons were counted. Results represent mean ± SE from at least 40 neurons of each experimental group. P < 0.05. Scale bars indicate 20 µm.


Figure 5
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  		Figure 5. Reduced PTEN and increased phospho-tau Ser-214 were observed in AD patient brains. Fronto cortex brain slices of AD patients (B, D, and F) and non-AD age control (A, C, and E) were immunostained to detect amyloid plaques (A and B, in red), NFTs (A and B, in black), PTEN (C–F, in red), and phospho-tau Ser-214 (E and F, in black). Arrows point to NFTs. Scale bars indicate 20 µm. Immunostaining signal was quantified by densitometry from 4 random fields (30 cells each field) and presented as the fold increase over the non-AD controls. *P < 0.05.

A decreased concentration of PTEN and increased tau phosphorylation at Ser214 in AD brains
A recent study reported a decrease in the concentration of PTEN in AD brains (28) . Consistent with this observation, we also detected PTEN in three cases of AD patients by immunohistochemistry and found an ~40% reduction of PTEN in the AD brains. The detection of Ser214 epitope in AD but not the non-AD, age-control brains was evident and found to be localized within NFTs in the fronto-forebrain neurons in all three cases of AD patient brains; a representative illustration was shown in Fig. 5 . These observations support the notion that PTEN plays a potential role in tauopathies, especially in the context of modulating tau phosphorylation at the Ser214 site.

DISCUSSION

We found that tumor suppressor PTEN regulates tau phosphorylation at multiple sites, providing evidence for involvement of the PTEN-modulated PIP3 signaling pathway in the physio- and pathological functions of tau. Among the examined phosphorylation sites, the Ser-214, which is mainly phosphorylated by Akt in vitro and in vivo (9 , 37) , exhibits the most significant changes in response to the concentration of PTEN. Given that Ser214 is one of the major tau phosphorylation sites in NFTs and phosphorylation of this residue interferes with the tau-microtubule interaction in vitro (48) and our observation demonstrating a decrease in the levels of PTEN in AD brains, we speculate that Ser214 phosphorylation may be a crucial major factor contributing to tau malfunctioning. However, the possibility that other PTEN-responsive phosphorylation sites may also impact tau functions cannot be excluded. It remains to be further investigated whether the effects of PTEN on tau aggregation and microtubule binding, and neurite outgrowth, can be suppressed by specific inhibition of phosphorylation at Ser214.

The effect of lipid phosphatase null PTEN on tau phosphorylation also suggested that PTEN can regulate tau phosphorylation by multiple mechanisms. Indeed, it has been shown that PTEN inhibits phosphorylation of transcription factor ETS-2 through MAPK, independent of the PIP3 signaling pathway (49) . Therefore, it is possible that PTEN targets downstream effectors of multiple signaling pathways through both lipid and perhaps protein phosphatase activity to affect tau phosphorylation. The protein substrate(s) of PTEN needs to be identified to fully understand the mechanisms by which PTEN regulates tau phosphorylation. In addition, given that tau interacts with lipid (50) and PI3K/Akt signaling inevitably changes the levels of PIP2 and PIP3, it is possible that PTEN can also regulate tau phosphorylation, aggregation, and association with microtubules through altering the lipid composition of the membrane. The observed changes in tau phosphorylation at different sites are likely the combined effect of PTEN on several targets/pathways.

Our data demonstrate that PTEN regulates tau phosphorylation to modulate its function, suggesting PTEN may be an upstream regulator in the pathophysiological cascades leading to tauopathy in neurodegenerative diseases, including AD. Interestingly, genetic studies have predicted genes that are related to AD located at chromosomes 9, 10, and 12 (51 52 53) , including the locus of Pten, chromosome 10q region, despite that further genetic studies, especially analyses of mutations in Pten in AD patients, are required. PTEN can dephosphorylate tyrosine-, serine-, and threonine-phosphorylated peptides in vitro (54) . Derkinderen et al. have shown tyrosine phosphorylated tau in PHF (55) . It is hence conceivable that, in addition to its regulatory effects through lipid phosphatase activity, PTEN may regulate tau phosphorylation directly through its protein phosphatase activity. In addition, PTEN is one of the dual specificity protein phosphatases (DSPs) that are speculated as potential therapeutic targets for the treatment of both cancer and AD due to their roles in multiple signaling pathways (56) . Therefore, mutations of PTEN resulting from genetic defects or environmental stress increase not only the risk of tumorigenesis, but also of AD genesis. In fact, our data support this novel concept by showing that the phosphatase-deficient mutant PTEN increases tau aggregation and impairs the neuronal structure/morphology. Furthermore, previous studies reported that increased levels of active Akt are colocalized with NFTs in AD brain (57) and in AD temporal cortex neurons (58) . The pathogenic factors such as oxidative insults, neural toxic ß-amyloid, and PHF-tau/NFTs could lead to defensive overactivation of the pro-survival Akt signaling through deregulation of PTEN, which in turn further worsens the tau pathology.

In summary, our data demonstrate for the first time that tumor-suppressor PTEN can affect tau phosphorylation at different important PHF sites to regulate tau’s microtubule-binding function and aggregation. Mutations in Pten or deficiency in its phosphatase activity can lead to malfunction of tau. The findings delineate the link between the PIP3 pro-survival signaling pathway and tauopathy in neurodegeneration, and potentially assign PTEN as a potential therapeutic target for AD.

ACKNOWLEDGMENTS

We thank Dr. Chengxin Gong for technical assistance, Drs. Xin Liu and Hong Wu for Pten knockout/inducible cell lines. This work was supported by National Institutes of Health grants (R01 NS046673 to H.X., R01 AG024895 to H.X., R01 DC006497 to Z.Z., AG05131 and K12-AG00975 to G.T.), and by grants from the Alzheimer’s Association (to H.X., and Z.Z.) and American Health Assistance Foundation (to H.X.).

FOOTNOTES

1 Present address: Fundacion Instituto Leloir, Buenos Aires, Argentina

Received for publication January 18, 2006. Accepted for publication February 12, 2006.

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