Tumor-suppressor PTEN affects tau ... - The FASEB Journal

The FASEB Journal • FJ Express Summary

Tumor suppressor PTEN affects tau phosphorylation, aggregation, and binding to microtubules Xue Zhang, Feng Li, Ayelen Bulloj,†,1 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 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5721fje

SPECIFIC AIMS The abnormally hyperphosphorylated tau is the major component of the neurofibrillary tangles (NFTs) that are found in a number of neurodegenerative diseases. In this study, we investigate the effects of tumorsuppressor chromosome 10 (PTEN) on tau phosphorylation, aggregation and binding to microtubules, and consequentially neuronal morphology.

PRINCIPAL FINDINGS 1. Overexpression of PTEN affects tau phosphorylation In human tau and wild-type (WT) PTEN (PTEN–WT) cotransfected COS-7 cells, we examined 7 tau phosphorylation sites, including Akt targets Thr212 and Ser-214; GSK-3 targets Ser-199, Thr205, Thr212, and Ser-396; and Thr262 (Fig. 1A). The levels of Thr212 and Ser-214 phospho-tau were significantly decreased by ⬃30 and 50%, respectively, compared with those in plasmid construct DNA control cells. Quantification of phospho-tau at other examined sites revealed increased phosphorylation of tau at Ser-199, Ser-202, and Thr205, and the phosphorylation of tau at Thr262 was virtually unchanged by overexpression of PTEN. We also examined the tau phosphorylation at these 7 sites in the cells coexpressing human tau and a phosphatase activity-null mutant PTEN, PTEN-CG. Compared with the control, the mutant PTEN selectively increased phospho-tau at Ser-396, a major tau phosphorylation site in PHF and NFTs, while leaving the other 6 sites unaltered (Fig. 1B, C). 2. PTEN affects tau aggregation and microtubulebinding ability The effect of PTEN on tau solubility was examined using a filter-retardation assay designed to detect pro1272

tein (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 PTEN–WT 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). 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. We found that PTEN-WT has little effect on the soluble cytosolic tau, while PTEN-CG reduced this population of tau by ⬃30%. On the one hand, PTEN–WT increased the amount of microtubule-associated tau by 40%, concomitant with a 25% reduction in insoluble tau aggregation. 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. 3. The effects of PTEN on tau phosphorylation are mediated through PIP3 signaling pathway We examined tau phosphorylation in a PTEN knockout cell line that bears an inducible human Pten transgene with or without a PI3K inhibitor, LY294002 (LY). On induction of PTEN expression, tau phosphorylation at Ser214 was decreased by 40%, accompanied by reduced Akt activity. In the presence of LY, tau phosphorylation at Ser214 was further down-regulated (up to 80%) as well as Akt activity. In addition, Ser205 phospho-tau was increased by ⬃20 or ⬃40%, in the presence of induced PTEN or PI3K inhibitor, respectively. 1

Present address: Fundacion Instituto Leloir, Buenos Aires, Argentina 2 Correspondence: Center for Neuroscience and Aging, Burnham Institute for Medical Research, La Jolla 92037, CA, USA. E-mail: [email protected] doi: 10.1096/fj.06-5721fje 0892-6638/06/0020-1272 © FASEB

4. Phosphatase activity-null PTEN impairs neuronal tau binding to microtubules and alters neuronal morphology We infected rat cortical primary neurons with Sindbis viruses expressing enhanced GFP (EGFP) alone, WT PTEN and EGFP, or the mutant PTEN and EGFP (Fig. 2A). By staining endogenous tau and tubulin in the neurons, we found that tau and tubulin were completely overlapped in EGFP control or WT PTEN expressing neurons. However, in the neurons expressing the mutant PTEN, the majority of tau was concentrated in cell bodies and failed to colocalize with tubulin in dendrites (Fig. 2B). 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 (Fig. 2C). Only slight increases in the number and the length of dendrites were observed when WT PTEN was expressed. 5. A decreased concentration of PTEN and increased tau phosphorylation at Ser214 in AD brains

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 following, M, MAPK; G, GSK-3; C5, CDK5; A, PKA; C, PKC; PK, phosphorylase kinase. B) pcDNA, wildtype (PTEN-WT) 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 wild-type tau (T40) were further transfected with pcDNA vector, PTEN-WT, 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.

In another experiment, we cotransfected COS-7 cells with WT PTEN and human tau, and treated the cells with insulin-like growth factor (IGF-1), LY or DMSO (control). We observed ⬃30% more Ser214 phosphotau in the presence of IGF-1, but an approximate 70% decrease in the LY- treated cells, compared with that in the DMSO control. A ROLE OF PTEN IN TAUOPATHY

We detected PTEN in 3 cases of AD patients by immunohistochemistry and found an ⬃40% reduction of PTEN in the AD brains. The detection of Ser-214 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.

CONCLUSIONS AND SIGNIFICANCE Collectively, we found tumor suppressor PTEN affects tau phosphorylation at multiple sites to regulate tau’s aggregation and binding to microtubules. For the first time we provide evidence for involvement of the PTENmodulated PIP3 signaling pathway in the pathophysiological functions of tau. Our findings are in line with the previous studies showing that increased levels of active Akt are colocalized with NFTs in AD brain and in AD temporal cortex neurons, suggesting a molecular mechanism underlying tauopathy by defensive overactivation of the pro-survival Akt signaling through deregulation of PTEN. PTEN is one of the dual specificity phosphatases (DSPs), possessing both protein and lipid phosphatase activity that are speculated as potential therapeutic targets for the treatment of both cancer and AD due to their roles in multiple signaling pathways. Our data support this new concept by showing that PTEN has different effects on different tau phosphorylation sites, which may be attributed to PTEN⬘s role in modulating varied tau kinases in different signaling pathways. Although we suggest that Ser214 phosphorylation may be 1273

Figure 2. 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-wild-type 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-wild-type 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 ␮m.

Figure 3. PTEN regulates PIP3 signaling pathway to affect tau phosphorylation. The dashed lines indicate the putative mechanisms by which tau could be directly dephosphorylated by PTEN, and the feedback effects of cell death to provoke the Akt activity.

1274

Vol. 20

June 2006

a crucial major factor contributing to tau malfunction, the possible impacts of other PTEN-responsive phosphorylation sites on tau functions need further investigation. In addition, it is conceivable that PTEN may also regulate tau phosphorylation directly through its protein phosphatase activity. Therefore it remains to be further clarified whether tau is a direct substrate of PTEN. 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. These 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.

The FASEB Journal

ZHANG ET AL.

The FASEB Journal • FJ Express Full-Length Article

Tumor-suppressor PTEN affects tau phosphorylation, aggregation, and binding to microtubules Xue Zhang, Feng Li, Ayelen Bulloj†,1, 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 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 Ser214 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. FASEB J. 20, E605–E613 (2006)

ABSTRACT

Key Words: Alzheimer’s disease 䡠 tauopathy 䡠 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 0892-6638/06/0020-0605 © FASEB

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 FTDP17, which led to identification of tau mutations that cause the disease (3–5). 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 – 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 –17). PTEN contains a tyrosine phosphatase functional domain, exhibiting both protein and lipid phosphatase activity in vitro (18). The phosphatidylinositol (3–5)-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 1

Present address: Fundacion Instituto Leloir, Buenos Aires, Argentina 2 Correspondence: Center for Neuroscience and Aging, Burnham Institute for Medical Research, La Jolla 92037, CA, USA. E-mail: [email protected] doi: 10.1096/fj.06-5721fje E605

for its tumor-suppressor function (21). Pten-null mice die at early embryonic stages, and heterozygous knockout mice develop a number of tumors (22–25). 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 –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 microtubulebinding 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.

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 antiPTEN antibody (Ab) (Cell Signaling, CA; 1:1000). Tubulin was detected using anti-␣-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

MATERIALS AND METHODS

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 ␮M 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).

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 ballbearing 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 ␮M nocodazole, followed by ultracentrifugation for 1 h at 190,000 ⫻ 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.

Sindbis virus expression

Filter/trap assays for tau aggregates

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

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䡠HCl, pH 8.0/120 mM NaCl/protease inhibitors mixture. After brief sonication, cell lysates were passed through a cellulose acetate membrane (0.2 ␮m; Bio-Rad) using Bio-Dot Microfiltration Apparatus (Bio-Rad,

Cell cultures and transient transfection

E606

Vol. 20

June 2006

The FASEB Journal

ZHANG ET AL.

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 agematched 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 ␮m) 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-A␤42 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-A␤ 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-3indolyl 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-␣-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. 1A). To study whether PTEN affects tau A ROLE OF PTEN IN TAUOPATHY

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–wildtype) 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.

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 E607

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. 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.

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 in-

PTEN affects tau aggregation and microtubulebinding 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. E608

Vol. 20

June 2006

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 FASEB Journal

ZHANG ET AL.

creased 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. 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 3A). 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).

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 microtubulebinding 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. 4A). We stained endogenous tau and tubulin in the

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 ␮M 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 ␮M 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.

A ROLE OF PTEN IN TAUOPATHY

E609

neurons and showed that tau and tubulin were completely overlapped in EGFP control or WT PTEN expressing neurons (Fig. 5B, 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). 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

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 GFPIRES-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. E610

Vol. 20

June 2006

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

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 ␮m.

The FASEB Journal

ZHANG ET AL.

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 ␮m. 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.

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 (51–53), including the locus of Pten, chromosome 10q region, despite that A ROLE OF PTEN IN TAUOPATHY

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 ␤-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 E611

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. 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.).

17.

18.

19. 20. 21.

REFERENCES 1.

2. 3.

4.

5.

6. 7.

8. 9.

10.

11. 12.

13.

14. 15.

16.

E612

Grundke-Iqbal, I., Iqbal, K., Tung, Y. C., Quinlan, M., Wisniewski, H. M., and Binder, L. I. (1986) Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology. Proc. Natl. Acad. Sci. USA 83, 4913– 4917 Lee, V. M., Goedert, M., and Trojanowski, J. Q. (2001) Neurodegenerative tauopathies. Annu. Rev. Neurosci. 24, 1121–1159 Hutton, M., Lendon, C. L., Rizzu, P., Baker, M., Froelich, S., Houlden, H., Pickering-Brown, S., Chakraverty, S., Isaacs, A., Grover, A., et al. (1998) Association of missense and 5⬘-splicesite mutations in tau with the inherited dementia FTDP-17. Nature 393, 702–705 Poorkaj, P., Bird, T. D., Wijsman, E., Nemens, E., Garruto, R. M., Anderson, L., Andreadis, A., Wiederholt, W. C., Raskind, M., and Schellenberg, G. D. (1998) Tau is a candidate gene for chromosome 17 frontotemporal dementia. Ann Neurol 43, 815– 825 Spillantini, M. G., Murrell, J. R., Goedert, M., Farlow, M. R., Klug, A., and Ghetti, B. (1998) Mutation in the tau gene in familial multiple system tauopathy with presenile dementia. Proc. Natl. Acad. Sci. USA 95, 7737–7741 Weingarten, M. D., Lockwood, A. H., Hwo, S. Y., and Kirschner, M. W. (1975) A protein factor essential for microtubule assembly. Proc. Natl. Acad. Sci. USA 72, 1858 –1862 Cleveland, D. W., Hwo, S. Y., and Kirschner, M. W. (1977) Purification of tau, a microtubule-associated protein that induces assembly of microtubules from purified tubulin. J. Mol. Biol. 116, 207–225 Cleveland, D. W., Hwo, S. Y., and Kirschner, M. W. (1977) Physical and chemical properties of purified tau factor and the role of tau in microtubule assembly. J. Mol. Biol. 116, 227–247 Ksiezak-Reding, H., Pyo, H. K., Feinstein, B., and Pasinetti, G. M. (2003) Akt/PKB kinase phosphorylates separately Thr212 and Ser214 of tau protein in vitro. Biochim Biophys Acta 1639, 159 –168 Kopke, E., Tung, Y. C., Shaikh, S., Alonso, A. C., Iqbal, K., and Grundke-Iqbal, I. (1993) Microtubule-associated protein tau. Abnormal phosphorylation of a non-paired helical filament pool in Alzheimer disease. J. Biol. Chem. 268, 24374 –24384 Kenessey, A., and Yen, S. H. (1993) The extent of phosphorylation of fetal tau is comparable to that of PHF-tau from Alzheimer paired helical filaments. Brain Res. 629, 40 – 46 Alonso, A. C., Zaidi, T., Grundke-Iqbal, I., and Iqbal, K. (1994) Role of abnormally phosphorylated tau in the breakdown of microtubules in Alzheimer disease. Proc. Natl. Acad. Sci. USA 91, 5562–5566 Alonso, A., Zaidi, T., Novak, M., Grundke-Iqbal, I., and Iqbal, K. (2001) Hyperphosphorylation induces self-assembly of tau into tangles of paired helical filaments/straight filaments. Proc. Natl. Acad. Sci. USA 98, 6923– 6928 Stokoe, D. (2001) Pten. Curr. Biol. 11, R502 Li, D. M., and Sun, H. (1997) TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor beta. Cancer Res. 57, 2124 –2129 Li, J., Yen, C., Liaw, D., Podsypanina, K., Bose, S., Wang, S. I., Puc, J., Miliaresis, C., Rodgers, L., McCombie, R., et al. (1997)

Vol. 20

June 2006

22. 23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33. 34.

PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275, 1943– 1947 Steck, P. A., Pershouse, M. A., Jasser, S. A., Yung, W. K., Lin, H., Ligon, A. H., Langford, L. A., Baumgard, M. L., Hattier, T., Davis, T., et al. (1997) Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat. Genet. 15, 356 –362 Maehama, T., and Dixon, J. E. (1998) The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. 273, 13375–13378 Stiles, B., Groszer, M., Wang, S., Jiao, J., and Wu, H. (2004) PTENless means more. Dev. Biol. 273, 175–184 Sulis, M. L., and Parsons, R. (2003) PTEN: from pathology to biology. Trends Cell Biol. 13, 478 – 483 Myers, M. P., Pass, I., Batty, I. H., Van der Kaay, J., Stolarov, J. P., Hemmings, B. A., Wigler, M. H., Downes, C. P., and Tonks, N. K. (1998) The lipid phosphatase activity of PTEN is critical for its tumor supressor function. Proc. Natl. Acad. Sci. USA 95, 13513– 13518 Di Cristofano, A., Pesce, B., Cordon-Cardo, C., and Pandolfi, P. P. (1998) Pten is essential for embryonic development and tumour suppression. Nat. Genet. 19, 348 –355 Podsypanina, K., Ellenson, L. H., Nemes, A., Gu, J., Tamura, M., Yamada, K. M., Cordon-Cardo, C., Catoretti, G., Fisher, P. E., and Parsons, R. (1999) Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems. Proc. Natl. Acad. Sci. USA 96, 1563–1568 Stambolic, V., Suzuki, A., de la Pompa, J. L., Brothers, G. M., Mirtsos, C., Sasaki, T., Ruland, J., Penninger, J. M., Siderovski, D. P., et al. (1998) Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 95, 29 –39 Suzuki, A., de la Pompa, J. L., Stambolic, V., Elia, A. J., Sasaki, T., del Barco Barrantes, I., Ho, A., Wakeham, A., Itie, A., Khoo, W., et al. (1998) High cancer susceptibility and embryonic lethality associated with mutation of the PTEN tumor suppressor gene in mice. Curr. Biol. 8, 1169 –1178 Kwon, C. H., Zhu, X., Zhang, J., Knoop, L. L., Tharp, R., Smeyne, R. J., Eberhart, C. G., Burger, P. C., and Baker, S. J. (2001) Pten regulates neuronal soma size: a mouse model of Lhermitte-Duclos disease. Nat. Genet. 29, 404 – 411 Fraser, M. M., Zhu, X., Kwon, C. H., Uhlmann, E. J., Gutmann, D. H., and Baker, S. J. (2004) Pten loss causes hypertrophy and increased proliferation of astrocytes in vivo. Cancer Res. 64, 7773–7779 Griffin, R. J., Moloney, A., Kelliher, M., Johnston, J. A., Ravid, R., Dockery, P., O’Connor, R., and O’Neill, C. (2005) Activation of Akt/PKB, increased phosphorylation of Akt substrates and loss and altered distribution of Akt and PTEN are features of Alzheimer’s disease pathology. J. Neurochem. 93, 105–117 Hanger, D. P., Hughes, K., Woodgett, J. R., Brion, J. P., and Anderton, B. H. (1992) Glycogen synthase kinase-3 induces Alzheimer’s disease-like phosphorylation of tau: generation of paired helical filament epitopes and neuronal localisation of the kinase. Neurosci. Lett. 147, 58 – 62 Mandelkow, E. M., Drewes, G., Biernat, J., Gustke, N., Van Lint, J., Vandenheede, J. R., and Mandelkow, E. (1992) Glycogen synthase kinase-3 and the Alzheimer-like state of microtubuleassociated protein tau. FEBS Lett. 314, 315–321 Lovestone, S., Reynolds, C. H., Latimer, D., Davis, D. R., Anderton, B. H., Gallo, J. M., Hanger, D., Mulot, S., Marquardt, B., Stabel, S., et al. (1994) Alzheimer’s disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells. Curr. Biol. 4, 1077–1086 Moreno, F. J., Medina, M., Perez, M., Montejo de Garcini, E., and Avila, J. (1995) Glycogen synthase kinase 3 phosphorylates recombinant human tau protein at serine-262 in the presence of heparin (or tubulin). FEBS Lett. 372, 65– 68 Sperber, B. R., Leight, S., Goedert, M., and Lee, V. M. (1995) Glycogen synthase kinase-3 beta phosphorylates tau protein at multiple sites in intact cells. Neurosci. Lett. 197, 149 –153 Gouras, G. K., Xu, H., Jovanovic, J. N., Buxbaum, J. D., Wang, R., Greengard, P., Relkin, N. R., and Gandy, S. (1998) Generation and regulation of beta-amyloid peptide variants by neurons. J. Neurochem. 71, 1920 –1925

The FASEB Journal

ZHANG ET AL.

35.

36.

37.

38.

39.

40.

41. 42. 43.

44. 45.

46.

47.

Dou, F., Netzer, W. J., Tanemura, K., Li, F., Hartl, F. U., Takashima, A., Gouras, G. K., Greengard, P., and Xu, H. (2003) Chaperones increase association of tau protein with microtubules. Proc. Natl. Acad. Sci. U. S. A. 100, 721–726 Liu, F., Grundke-Iqbal, I., Iqbal, K., and Gong, C. X. (2005) Contributions of protein phosphatases PP1, PP2A, PP2B and PP5 to the regulation of tau phosphorylation. Eur. J. Neurosci. 22, 1942–1950 Kyoung Pyo, H., Lovati, E., Pasinetti, G. M., and Ksiezak-Reding, H. (2004) Phosphorylation of tau at THR212 and SER214 in human neuronal and glial cultures: the role of AKT. Neuroscience 127, 649 – 658 Mahimainathan, L., and Choudhury, G. G. (2004) Inactivation of platelet-derived growth factor receptor by the tumor suppressor PTEN provides a novel mechanism of action of the phosphatase. J. Biol. Chem. 279, 15258 –15268 Imahori, K., Hoshi, M., Ishiguro, K., Sato, K., Takahashi, M., Shiurba, R., Yamaguchi, H., Takashima, A., and Uchida, T. (1998) Possible role of tau protein kinases in pathogenesis of Alzheimer’s disease. Neurobiol. Aging 19, S93–98 Sun, H., Lesche, R., Li, D. M., Liliental, J., Zhang, H., Gao, J., Gavrilova, N., Mueller, B., Liu, X., and Wu, H. (1999) PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway. Proc. Natl. Acad. Sci. USA 96, 6199 – 6204 Bondy, C. A., and Cheng, C. M. (2004) Signaling by insulin-like growth factor 1 in brain. Eur. J. Pharmacol. 490, 25–31 Gasparini, L., and Xu, H. (2003) Potential roles of insulin and IGF-1 in Alzheimer’s disease. Trends Neurosci. 26, 404 – 406 Carro, E., and Torres-Aleman, I. (2004) The role of insulin and insulin-like growth factor I in the molecular and cellular mechanisms underlying the pathology of Alzheimer’s disease. Eur. J. Pharmacol. 490, 127–133 Hong, M., and Lee, V. M. (1997) Insulin and insulin-like growth factor-1 regulate tau phosphorylation in cultured human neurons. J. Biol. Chem. 272, 19547–19553 Schubert, M., Brazil, D. P., Burks, D. J., Kushner, J. A., Ye, J., Flint, C. L., Farhang-Fallah, J., Dikkes, P., Warot, X. M., Rio, C., et al. (2003) Insulin receptor substrate-2 deficiency impairs brain growth and promotes tau phosphorylation. J. Neurosci. 23, 7084 –7092 Lesort, M., Jope, R. S., and Johnson, G. V. (1999) Insulin transiently increases tau phosphorylation: involvement of glycogen synthase kinase-3beta and Fyn tyrosine kinase. J. Neurochem. 72, 576 –584 Lesort, M., and Johnson, G. V. (2000) Insulin-like growth factor-1 and insulin mediate transient site-selective increases in

A ROLE OF PTEN IN TAUOPATHY

48.

49.

50. 51. 52.

53.

54.

55.

56.

57.

58.

tau phosphorylation in primary cortical neurons. Neuroscience 99, 305–316 Illenberger, S., Zheng-Fischhofer, Q., Preuss, U., Stamer, K., Baumann, K., Trinczek, B., Biernat, J., Godemann, R., Mandelkow, E. M., et al. (1998) The endogenous and cell cycledependent phosphorylation of tau protein in living cells: implications for Alzheimer’s disease. Mol. Biol. Cell 9, 1495–1512 Weng, L. P., Brown, J. L., Baker, K. M., Ostrowski, M. C., and Eng, C. (2002) PTEN blocks insulin-mediated ETS-2 phosphorylation through MAP kinase, independently of the phosphoinositide 3-kinase pathway. Hum. Mol. Genet. 11, 1687–1696 Michikawa, M. (2003) The role of cholesterol in pathogenesis of Alzheimer’s disease: dual metabolic interaction between amyloid beta-protein and cholesterol. Mol. Neurobiol. 27, 1–12 Tanzi, R. E., and Bertram, L. (2001) New frontiers in Alzheimer’s disease genetics. Neuron 32, 181–184 Kehoe, P., Wavrant-De Vrieze, F., Crook, R., Wu, W. S., Holmans, P., Fenton, I., Spurlock, G., Norton, N., Williams, H., Williams, N., et al. (1999) A full genome scan for late onset Alzheimer’s disease. Hum. Mol. Genet. 8, 237–245 Bertram, L., Blacker, D., Mullin, K., Keeney, D., Jones, J., Basu, S., Yhu, S., McInnis, M. G., Go, R. C., Vekrellis, K., et al. (2000) Evidence for genetic linkage of Alzheimer’s disease to chromosome 10q. Science 290, 2302–2303 Myers, M. P., Stolarov, J. P., Eng, C., Li, J., Wang, S. I., Wigler, M. H., Parsons, R., and Tonks, N. K. (1997) P-TEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase. Proc. Natl. Acad. Sci. USA 94, 9052–9057 Derkinderen, P., Scales, T. M., Hanger, D. P., Leung, K. Y., Byers, H. L., Ward, M. A., Lenz, C., Price, C., Bird, I. N., Perera, T., et al. (2005) Tyrosine 394 is phosphorylated in Alzheimer’s paired helical filament tau and in fetal tau with c-Abl as the candidate tyrosine kinase. J. Neurosci. 25, 6584 – 6593 Ducruet, A. P., Vogt, A., Wipf, P., and Lazo, J. S. (2005) Dual specificity protein phosphatases: therapeutic targets for cancer and Alzheimer’s disease. Annu. Rev. Pharmacol. Toxicol. 45, 725–750 Pei, J. J., Khatoon, S., An, W. L., Nordlinder, M., Tanaka, T., Braak, H., Tsujio, I., Takeda, M., Alafuzoff, I., Winblad, B., et al. (2003) Role of protein kinase B in Alzheimer’s neurofibrillary pathology. Acta Neuropathol. (Berl) 105, 381–392 Rickle, A., Bogdanovic, N., Volkman, I., Winblad, B., Ravid, R., and Cowburn, R. F. (2004) Akt activity in Alzheimer’s disease and other neurodegenerative disorders. Neuroreport 15, 955–959 Received for publication January 18, 2006 Accepted for publication February 12, 2006

E613