PTEN recruitment controls synaptic and cognitive function in Alzheimer s models

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1 PTEN recruitment controls synaptic and cognitive function in Alzheimer s models Shira Knafo 1 3, Cristina Sánchez-Puelles 1, Ernest Palomer 1, Igotz Delgado, Jonathan E Draffin 1, Janire Mingo, Tina Wahle 5,, Kanwardeep Kaleka 7, Liping Mou 8, Inmaculada Pereda-Perez 9, Edvin Klosi 1, Erik B Faber 1, Heidi M Chapman 1, Laura Lozano-Montes 1, Ana Ortega-Molina 11, Lara Ordóñez-Gutiérrez 1,1, Francisco Wandosell 1,1, Jose Viña 13, Carlos G Dotti 1, Randy A Hall 8, Rafael Pulido 3,, Nashaat Z Gerges 7, Andrew M Chan 1, Mark R Spaller 1, Manuel Serrano 11, César Venero 9 & José A Esteban 1 npg 1 Nature America, Inc. All rights reserved. Dyshomeostasis of amyloid-b peptide (Ab) is responsible for synaptic malfunctions leading to cognitive deficits ranging from mild impairment to full-blown dementia in Alzheimer s disease. Ab appears to skew synaptic plasticity events toward depression. We found that inhibition of PTEN, a lipid phosphatase that is essential to long-term depression, rescued normal synaptic function and cognition in cellular and animal models of Alzheimer s disease. Conversely, transgenic mice that overexpressed PTEN displayed synaptic depression that mimicked and occluded Ab-induced depression. Mechanistically, Ab triggers a PDZdependent recruitment of PTEN into the postsynaptic compartment. Using a PTEN knock-in mouse lacking the PDZ motif, and a cell-permeable interfering peptide, we found that this mechanism is crucial for Ab-induced synaptic toxicity and cognitive dysfunction. Our results provide fundamental information on the molecular mechanisms of Ab-induced synaptic malfunction and may offer new mechanism-based therapeutic targets to counteract downstream Ab signaling. Gradual accumulation of Aβ induces a series of synaptic and neuronal dysfunctions that appear to be responsible for cognitive deficits ranging in severity from mild-cognitive impairment (MCI) to dementia in Alzheimer s disease (AD) 1. Accumulating evidence indicates that soluble Aβ assemblies directly alter synaptic plasticity mechanisms by inhibiting long-term potentiation (LTP) and facilitating long-term depression (LTD) in hippocampal neurons Thus, it appears that Aβ shifts the synaptic plasticity balance toward a pathologically enhanced form of depression. PIP 3 signaling is an important regulator of AMPA receptor (AMPAR) function and synaptic plasticity 1 1, and we have recently shown that PI3K (the PIP 3 synthesizing enzyme) favors synaptic potentiation 1, whereas the lipid phosphatase PTEN (down-regulator of PIP 3 ) mediates depression 15. We investigated the role of PTEN in Aβ-induced synaptic failure and its cognitive consequences. Specifically, we manipulated PTEN function (in vivo and in vitro) as a general strategy to elucidate and potentially counteract the role of PTEN in the depressing effect of Aβ. We found that PTEN is a target of Aβ s action, which involves PDZ-dependent recruitment of PTEN to dendritic spines. Moreover, genetic and pharmacological blockade of these PDZ-dependent interactions protected neurons from Aβ-induced synaptic toxicity and prevented cognitive deterioration. These data offer fundamental information about the mechanisms by which Aβ perturbs synaptic function and reveal PTEN as a critical mediator and potential therapeutic target in AD. RESULTS PTEN inhibition rescues cognitive function in App/ mice We first reasoned that, if early Aβ-induced cognitive impairment results from a general synaptic bias toward LTD events, dampening this form of synaptic plasticity might prevent cognitive decay. We therefore examined whether in vivo inhibition of PTEN, a lipid phosphatase that is essential for LTD 15, would rescue cognitive deficits in App/ mice, as a model for AD 1 (Supplementary Fig. 1a). The specific PTEN inhibitor -OHpic 17 or vehicle (artificial cerebrospinal fluid, ACSF) were infused over a period of 3 weeks into the brain ventricles of -month-old transgenic mice and their wild-type () littermates using osmotic minipumps. Mice were tested on two hippocampal-dependent cognitive tasks: novel object location and contextual fear conditioning (these experiments were 1 Department of Molecular Neurobiology, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC) / Universidad Autónoma de Madrid, Madrid, Spain. Unidad de Biofísica CSIC-UPV/EHU, Campus Universidad del País Vasco, Leioa, Spain. 3 Ikerbasque, Basque Foundation for Science, Bilbao, Spain. Biocruces Health Research Institute, Barakaldo, Spain. 5 VIB Center for the Biology of Disease and Center for Human Genetics, University of Leuven (Katholieke University of Leuven), Leuven, Belgium. IUF - Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany. 7 Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, USA. 8 Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, USA. 9 Department of Psychobiology, Universidad Nacional de Educación a Distancia, Madrid, Spain. 1 Department of Pharmacology and Toxicology, Geisel School of Medicine at Dartmouth, and Norris Cotton Cancer Center, Lebanon, New Hampshire, USA. 11 Tumor Suppression Group, Spanish National Cancer Research Center (CNIO), Madrid, Spain. 1 Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED). 13 Department of Physiology, Faculty of Medicine, University of Valencia, Valencia, Spain. 1 School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong. Correspondence should be addressed to J.A.E. (jaesteban@cbm.csic.es), S.K. (s.knafo@ikerbasque.org) or C.V. (cvenero@psi.uned.es). Received 3 April 15; accepted 3 December 15; published online 18 January 1; doi:1.138/nn.5 nature NEUROSCIENCE LUME 19 NUMBER 3 MARCH 1 3

2 npg 1 Nature America, Inc. All rights reserved. P =.3 a b P =.1 c d e f kda P =.37.8 P =.1 App 11 P =.5 P =. CC 5 P =.1 5 Actin P =.. LV P = 1. App/. 3 3 PS1 CTFβ. CTFα 1 1 Actin kda pakt 5 App/ App/ App/ App/ takt 5 P =.8 App/ g Recognition index N = 1 N = 13 N = 1 N = 15 Freezing (%) N = 18 N = 19 N = 17 N = carried out blind with respect to genotype and treatment). Vehicletreated App/ mice showed a significant impairment in both tasks as compared with vehicle-infused mice (P =. for novel object location, P =.1 for contextual fear conditioning; Fig. 1a,b). Notably, although the infusion of -OHpic did not affect the performance of mice, treated App/ mice showed a significant improvement in both spatial learning tasks (P =.37 for novel object location, P =.1 for contextual fear conditioning), to the extent that their performance was similar to that of mice. -OHpic treatment did not enhance learning in a hippocampal-independent task (auditory-cued fear conditioning) for either of the genotypes (Fig. 1c). In addition, -OHpic treatment did not alter exploratory activity (Supplementary Fig. 1d) or basal freezing behavior (Supplementary Fig. 1e) in animals of either genotype. Cannula placement was ascertained with Nissl staining at the end of the experiment (Fig. 1d). Only those mice with a cannula placement in the lateral ventricle were used for analysis. The effectiveness of PTEN inhibition in vivo was evidenced by a transient increase in phospho-akt (pakt) levels after acute injection of -OHpic in the lateral ventricle (Fig. 1d), although this was no longer observable after chronic infusion of -OHpic (Supplementary Fig. 1f). After the end of the last test, we prepared hippocampal extracts and measured the levels of App and its C-terminal fragments (CTFα and CTFβ, western blot; Fig. 1e,f) and Aβ monomers (ELISA; Fig. 1g). We did not detect any changes in these parameters following -OHpic treatment. Thus, in vivo PTEN inhibition rescues cognitive function in App/ mice in the absence of detectable changes in Aβ levels or App processing. Freezing (%) N = 18 N = 19 N = 17 N = Figure 1 -OHpic semi-chronic infusion rescues the learning deficits of App/ mice. (a) App/ and littermates (treated with -OHpic or with ACSF) were tested in the novel object location task. The recognition index was calculated as the time spent exploring the displaced object/time spent exploring both objects. Thus, a score of.5 would indicate no preference (F (3, 5) =.85, P =.15, Kruskal-Wallis ANOVA). (b) Percentage freezing to the context for vehicle-infused mice and mice infused with -OHpic (F (3, 7) = 1.3, P <.1, Kruskal-Wallis ANOVA). (c) Percentage freezing to the tone for vehicle-infused mice and mice infused with -OHpic (F (3, 7) =.55, P =., Kruskal-Wallis ANOVA, reflecting an overall effect of genotype and treatment on this behavior). (d) Top, Nissl-stained coronal section showing the track of the cannula to the lateral ventricle. LV, lateral ventricle, CC, corpus callosum. Scale bar represents 1 mm. Bottom, western blot analysis of total hippocampal extracts from and App/ animals injected with -OHpic or ACSF control. Cannula Aβ (normalized to vehicle-treated ) 3 1 P =.3 P =.1 P =.7 App/ App level (normalized to vehicle-treated ) Aβ (normalized to vehicle-treated ) P =.38 P =. PTEN activity mediates Ab induced synaptic dysfunction To evaluate the synaptic basis of the cognitive protection achieved by PTEN inhibition, we tested whether PTEN is required for Aβ-induced synaptic depression. We used three different experimental systems as sources of Aβ: water-soluble, oligomeric assemblies of synthetic Aβ; Aβ secreted from neurons following virally driven expression of a mutant form of App (human App with the Swedish/London double mutation, App swe/lnd ); and chronic accumulation of Aβ in App/ mice. The specificity of the PTEN inhibitor -OHpic was evidenced by the upregulation of the PIP 3 downstream effector pakt and the lack of effect on phospho-tyr levels in acute hippocampal slices (Supplementary Fig. a,b; as previously published 17 ). Water-soluble Aβ assemblies were prepared (Online Methods, protocol I) 18. We followed the kinetics of Aβ aggregation using thioflavin T fluorescence, western blot and electron microscopy (Supplementary Fig. c f). Synaptic function was assessed by electrophysiological recordings of field excitatory postsynaptic potential (fepsps) between hippocampal CA3 and CA1 cells in acute slices prepared from 5-month-old mice (Online Methods). We observed a % decrease in the slope of synaptic response (input-output curve) when slices were incubated for h in ACSF containing pathogenic Aβ assemblies (1 µm; Fig. a and Supplementary Fig. g). This decrease in basal synaptic transmission was prevented when Aβ was added to slices pre-incubated with the PTEN inhibitor (5 nm -OHpic, dissolved in water) for 1 h (-OHpic and Aβ were also present in the perfusion solution during electrophysiological recordings). -OHpic alone had no effect on basal synaptic transmission (Fig. a). Similarly, LTP was impaired by Aβ oligomers (as reported P =. P =. N = N = 5 P =. P =.7 P =.1 P =.3 N = N = App/ N = App/ Uncropped versions of the western blots are shown in Supplementary Figure 9. (e) App full-length (top) and App C-terminal products CTFα and CTFβ (bottom) were detected with App Ct antibody in hippocampal lysates from and App/ animals infused with -OHpic or ACSF control. App was present as a double band corresponding to the mature and immature protein. Actin served as a loading control. Uncropped versions of the western blots are shown in Supplementary Figure 9. (f) Quantification of App levels in App/ mice and in their littermates from experiments such as the one shown in e (F (3, 18) = 3.59, P =.3, Kruskal-Wallis ANOVA). (g) Monomeric Aβ was assayed in lysates of hippocampi using Aβ and Aβ ELISA kits (F (3, 3) =.7 for Aβ and F (3, 8) = 7.81 for Aβ, P <.1 Kruskal-Wallis ANOVA). Data are represented as mean ± s.e.m. (error bars). P values shown in all graphs were derived using Dunn s multiple post hoc test. LUME 19 NUMBER 3 MARCH 1 nature NEUROSCIENCE

3 fepsp slope a (mv ms 1 ) Stimulation intensity (µa) P =. P =. P =.7 Vehicle N = N = 17 Aβ N = 18 + Aβ N = 15 b fepsp fold potentiation TBS Vehicle Aβ + Aβ npg 1 Nature America, Inc. All rights reserved. Figure PTEN activity determines the synaptic response to synthetic Aβ. (a) Left, inputoutput curves of field excitatory postsynaptic potentials (fepsps) evoked by stimulation of Schaffer collaterals from slices treated with vehicle or Aβ assemblies (-h incubation), with or without 1 h pre-incubation with 5 nm -OHpic, as indicated. Right, superimposed representative fepsps at 1, 5, 1, 15 and fepsp fold potentiation previously 19 3 ), and this effect was also rescued with the PTEN inhibitor (Fig. b,c). Presynaptic short-term plasticity was not altered by Aβ under these conditions, as evaluated by paired-pulse facilitation (Supplementary Fig. h). Thus, -OHpic prevents Aβ-induced impairment in both basal synaptic transmission and LTP, suggesting that PTEN activity mediates these synaptic pathologies. These effects do not appear to be a result of changes in neurotrophic factor signaling, such as BDNF, as TrkB phosphorylation was not altered by Aβ exposure and/or PTEN inhibition (Supplementary Fig. i). Given that both PTEN 15 and Aβ assemblies produce synaptic depression (Fig. a), we tested whether they act via common mechanisms, using an occlusion strategy. We used transgenic mice designed to possess an increased gene dosage of PTEN while preserving the natural pattern of gene expression (Pten tg mice; Fig. d). Indeed, the slope of the synaptically evoked field potential was significantly decreased in acute hippocampal slices from Pten tg mice when compared with slices from littermates (P =.9; Fig. d). Nevertheless, no further depression was observed following Aβ incubation (Fig. d). This occlusion effect implies that PTEN and Aβ act in a common pathway. PTEN inhibition protects synapses from neuron-expressed Ab As a complementary approach, we expressed an App gene carrying the Swedish and London mutations (App swe/lnd ) together with EGFP in rat hippocampal slice neurons using a viral expression system 5 (Fig. 3a and Supplementary Fig. 3a). Expression of App swe/lnd in c N = 1 P =. Vehicle P =. P =. N = 11 N = 18 Aβ + Aβ d fepsp slope (mv ms 1 )... PTEN pakt takt PTEN tg 55 kda 5 kda 5 kda P =.9 P =.1 P =.1 CA1 neurons led to substantially higher expression of App (3.1 ±.-fold (s.e.m.) relative to uninfected slices, N = 1 slices, P =.5; Fig. 3b), App C-terminal fragments (Fig. 3c) and accumulation of Aβ, as determined by immunohistochemistry (Fig. 3a), ELISA (Fig. 3d) and western blot (Fig. 3e). PTEN inhibition did not alter recombinant App expression and formation of C-terminal App fragments (Fig. 3c), Aβ production (Fig. 3d) or assembly formation (1.7 ±.8-fold (s.e.m.) relative to untreated slices; P =.31, N = 5 slices per condition; Fig. 3e). We then compared the synaptic responses evoked onto adjacent pairs of simultaneously recorded CA1 pyramidal neurons in which only one cell expresses the recombinant protein. After 8 h expression, App swe/lnd -expressing neurons showed % depression of AMPAR synaptic responses relative to uninfected cells (Fig. 3f and Supplementary Fig. 3b). At these expression times, depression was specific for AMPAR, but not for NMDA receptor (NMDAR), responses (Fig. 3f and Supplementary Fig. 3b). These results do not imply that uninfected cells adjacent to App swe/lnd -expressing cells are not affected by Aβ secreted from the neighboring cell, but they do suggest that App swe/lnd -expressing cells are depressed to a larger extent than uninfected bystanders. The depression induced by App swe/lnd expression was not just a result of protein overexpression or virus infection, as overexpression of a mutant App with little susceptibility to β-secretase (BACE) cleavage (App M59V, App MV ) 7 did not produce synaptic depression (Supplementary Fig. 3c e; as previously published ). To evaluate the role of PTEN in this form of depression, we expressed App swe/lnd in the presence of either 15 nm bpv(hopic) or PTEN tg (vehicle), N = 5 (vehicle), N = 13 PTEN tg (Aβ), N = 1 (Aβ), N = 11 Stimulation intensity (µa) µa of stimulation intensity. P values were determined using two-way ANOVA with Bonferroni post-tests (df, group, intensity, interaction, residual: 3., 11., 33., 98.). N represents number of slices. Vehicle,.1% DMSO. (b) Left, LTP was induced by θ-burst stimulation and recorded for min post-induction, following at least min of stable baseline. Right, representative field recordings at baseline (thin line) and after LTP induction (thick line) from slices treated with vehicle or Aβ assemblies (-h incubation), with or without 1 h pre-incubation with 5 nm -OHpic, as indicated. (c) Quantification of average fepsp maximal slopes, at 5 min after induction (F (3, 3) =.88, P =.7, Kruskal-Wallis ANOVA). P values shown in the graphs were derived using Dunn s multiple post hoc test. N represents number of slices. (d) Left, input-output curves of fepsps in slices from and Pten tg mice. Right, superimposed representative traces for fepsps at 1, 5, 1, 15 and µa of stimulation intensity. P values were determined using two-way ANOVA with Bonferroni post-tests (df, group, intensity, interaction, residual: 3., 11., 33., 8.). N represents number of slices. The inset shows a western blot presenting higher levels of PTEN and lower levels of pakt (Thr38) in the hippocampi from Pten tg mice. Uncropped versions of the western blots are shown in Supplementary Figure 9. Scale bars for all panels:.5 mv, ms. Data are represented as mean ± s.e.m. (error bars). nature NEUROSCIENCE LUME 19 NUMBER 3 MARCH 1 5

4 npg 1 Nature America, Inc. All rights reserved. a b c d e NU-1 E1 EGFP Alexafluor 59 (Aβ) App EGFP Control kda 1 9 Control kda 5 nm -OHpic (two chemically related PTEN inhibitors 17 ; these drugs were added 5 h after virus injection and were also present in the perfusion solution during electrophysiological recordings). Under these conditions, AMPAR-mediated excitatory postsynaptic currents (EPSCs) were no longer depressed in App swe/lnd -expressing neurons (Fig. 3g). In fact, AMPAR responses were significantly increased compared with uninfected cells, without altering NMDAR responses (P =.1; Supplementary Fig. 3f). These experiments, using Aβ produced by neurons, further demonstrate that inhibition of PTEN phosphatase activity rescues synaptic depression. Morphological analysis of neurons filled with biocytin during recording (Online Methods and Fig. a) revealed that App swe/lnd expression was not accompanied by changes in spine density or size (this experiment was done blind with respect to App swe/lnd expression and treatment; Fig. b e). Other studies have reported a decrease in spine density after App overexpression,8, although this effect may depend on expression levels and the proximity to amyloid deposition Thus, our results imply that the observed depression in AMPAR transmission arose from functional rather than structural modifications. Notably, spines from App swe/lnd -infected neurons were larger after -OHpic treatment (Fig. e), implying a possible structural component for synaptic function recovery (Fig. 3g). PTEN blockade protects against secreted Ab To test the role of PTEN specifically in the effects of neuronally secreted Aβ, we monitored synaptic dysfunction in untransfected cells neighboring App swe/lnd -expressing neurons. Aβ produced by App swe/lnd -infected neurons was secreted into the slice culture medium, and this was not altered by PTEN inhibition with -OHpic (Fig. f). App CTFβ CTFα Actin 15 n.s. App/actin 1 5 N = 1 N = 1 N = N = Aβ (normalized to vehicle-treated ) Figure 3 PTEN inhibition protects App swe/lnd -expressing neurons. (a) Confocal projection image of neurons in organotypic hippocampal slices co-expressing App swe/lnd and EGFP. Slices were fixed, permeabilized and immunostained for Aβ (NU-1, E1; Online Methods). Scale bar represents µm. (b) Representative western blot of App overexpression from organotypic slice cultures coexpressing App swe/lnd and EGFP. (c) Comparison of App and C-terminal fragments (CTFs) α and β, detected using App Ct antibody, from control and App swe/lnd -expressing slices, treated or not with 5 nm -OHpic. Actin served as loading control. Quantification is presented for App levels, relative 1 8 P =.7 P =.3 P =. N = 13 N = 13 P =.1 Aβ (normalized to vehicle-treated ) Control Control f 15 1 Uninf. + Normalized EPSC (%) P =. P =.3 P =.3 P =. N = 13 N = 13 P =.3 P =.9 N = 3 N = 3 AMPA N = 11 N = 11 NMDA Uninf P =.1 LTP was efficiently induced in neurons in control slices that were not injected with App swe/lnd virus (Fig. g,h and Supplementary Fig. a). In contrast, App swe/lnd -infected cells did not show any potentiation (Fig. g,h). Notably, uninfected neurons adjacent to App swe/lnd - infected cells (<3 µm) did not show any LTP either (Fig. g,h), probably as a result of their exposure to extracellular Aβ secreted from neighboring infected cells,3. As a control, viral infection to express EGFP (without App) did not impair LTP, suggesting that virus infection or recombinant protein expression do not cause impairment in synaptic plasticity (Supplementary Fig. b). Injected and control slices (not exposed to App swe/lnd virus) were incubated with the PTEN inhibitor -OHpic for 8 h during App swe/lnd expression. We found that, although -OHpic did not affect the magnitude of LTP in control slices, App swe/lnd -infected neurons and neighboring uninfected neurons recovered the ability to express significant LTP (P =.1; Fig. i,j and Supplementary Fig. c). Thus, PTEN inhibition rescues LTP in neurons exposed to extracellular Aβ secreted from App swe/lnd -expressing neurons. Finally, we also tested whether synaptic function can be rescued in conditions of chronic exposure to Aβ. To this end, we infused -OHpic or vehicle over a period of 3 weeks into the brain ventricles of -month-old App/ mice and their littermates using osmotic minipumps (similar to the behavioral experiment shown in Fig. 1). Acute hippocampal slices were then prepared and fepsps were recorded to yield input-output curves and LTP time courses. Basal synaptic transmission was lower in slices from App/ mice than animals, and this effect was not rescued by -OHpic treatment (Fig. 5a). In contrast, semi-chronic treatment with -OHpic fully rescued LTP levels in slices from App/ animals, and did not affect LTP from mice (Fig. 5b,c). These results suggest that PTEN g Normalized EPSC (%) N = N = App * Aβ N = 1 N = 1 AMPA NMDA to the corresponding untreated slices (F (3, 8) = 1., non-significant (n.s.), Kruskal-Wallis ANOVA). N represents number of slices. (d) Monomeric Aβ was assayed using Aβ and ELISA kit (Wako/Invitrogen) from control and App swe/lnd -expressing slices, treated or not with 5 nm -OHpic (P =.5 for Aβ (F (3, 38) = 8.) and.1 for Aβ (F (3, 38) = 13.3, Kruskal-Wallis ANOVA)). P values shown in the graphs were derived using Dunn s multiple post hoc test, N is the number of inserts. (e) Western blot analysis of SDS-stable Aβ species probed with the N-terminal Aβ antibody E1 from slices infected with App swe/lnd -EGFP and slices infected with EGFP only, with or without treatment with -OHpic. Bands corresponding to App and Aβ assemblies are indicated. Two nonspecific bands are indicated with asterisks. (f,g) Left, example traces from uninfected and App swe/lnd -expressing neurons recorded at mv (AMPAR EPSCs) and + mv (NMDAR EPSCs) without (f) or with (g) PTEN inhibition. Scale bars represent pa, 1 ms. Right, average relative EPSC in App swe/lnd -expressing neurons compared to neighboring uninfected neurons (Supplementary Fig. 3b,f). N represents number of cell pairs. Statistical significance was calculated using the Wilcoxon test for paired data (individual pairs of App swe/lnd cells versus uninfected cells; t =.3, df = for f, and t =.113, df = 17 for g). Data are represented as mean ± s.e.m. (error bars). Uncropped versions of the western blots in b, c and e are shown in Supplementary Figure 9. LUME 19 NUMBER 3 MARCH 1 nature neuroscience

5 a b c d e f 5 µm 5 µm Control App/PS Spine density (spines per µm).8... N = 8 N = 18 n.s. N = 3 N = Control App/PS1 Control 8 Uninf. P =.8 P =.3 N = 11 Control Uninf. 8 µm Head volume (µm 3 ) N = 1 P =.3 P =.7 P = Control Control ().. 1 App swe/ind() Control App/PS1 Control Head volume (µm 3 ) P =.1 N = 31 P =.1 N = 8 P =.1 N = 13 N = 377 Cumulative frequency g h i j Control Uninf. Control Uninf. Aβ level (pg ml 1 ) P =.3 N = N = Aβ level (pg ml 1 ) 8 P =.3 n.s. Control Uninf. P =.3 P =.7 P = Control 8 P =.7 N = N = N = 11 Control Uninf. npg 1 Nature America, Inc. All rights reserved. Figure Blockade of PTEN activity protects neurons from extracellular Aβ. (a) App swe/lnd -expressing CA1 neurons filled with biocytin and revealed using streptavidin-alexa Fluor 555 (green channel). (b) Projection images of representative dendrites from the apical tree of biocytin-filled neurons. (c) Quantification of dendritic spine density in App swe/lnd -expressing neurons and uninfected neurons with or without treatment with PTEN inhibitor. N represents number of dendrites (F (3, 91) =.3195). n.s., non-significant, Kruskal-Wallis ANOVA. (d) High-magnification projection image of a dendrite with spines (left) demonstrating the detection of spine head used for volume measurements (right, blue). (e) Left, quantification of spine head volume in dendrites of different conditions (P <.1 among groups, F (3, 155) = 13.7, Kruskal-Wallis ANOVA). P values shown in the graphs were calculated using Dunn s multiple post hoc test, N is the number of spines. Right, cumulative frequency of spine head volume from the same data. (f) Quantification of monomeric Aβ secretion. Levels of Aβ and Aβ were measured with ELISA in media of infected and control slices (7 h post infection) (P =.3 for Aβ (F (3, ) = 15.5) and. for Aβ (F (3, ) = 5, Kruskal-Wallis ANOVA)). P values shown in the graphs were calculated by Dunn s multiple post hoc test, N is the number of inserts. (g,i) Time course of AMPAR-mediated synaptic responses before and after LTP induction (black arrow), without (g) and with (i) PTEN inhibition (5 nm -OHpic, 5 h after virus injection and during recordings) from neurons in control (uninjected) slices, App swe/lnd -infected neurons or neighboring uninfected neurons. Representative traces are shown on the right, before (thin lines) and after (thick lines) LTP induction (Supplementary Fig. a,c). Scale bars represent pa, 1 ms. (h,j) Quantification of average synaptic potentiation for paired (LTP) and unpaired (control) pathways from the last 5 min of the time course. Kruskal-Wallis ANOVA was used to determine the global effect (F (, ) =.978, P =.1 for h, and P =. for j). Statistical significance between conditions was determined with Dunn s multiple comparison post hoc test. Wilcoxon paired test was used to determine significance with respect to the baseline (for h, P control =.3, P uninf =.1, P Appswe/lnd =.; for j, P control =., P uninf =.1, P Appswe/lnd =.1). N represents number of cells. In all panels, data are represented as mean ± s.e.m. (error bars). inhibition can preserve synaptic plasticity in neurons chronically exposed to Aβ, although depression of basal synaptic transmission appears to be mediated by separate mechanisms (possibly unrecoverable synaptic loss). Synaptic protection by PTEN inhibition is cell-autonomous To test the role of PTEN as a cell-autonomous factor in Aβ synaptic malfunction, we expressed in CA1 neurons a catalytically dead form of PTEN that acts as a dominant-negative mutant (PTEN-C1S) 3. Neurons were then exposed to extracellular Aβ in two different manners: incubation with 1 µm synthetic Aβ assemblies (Online Methods, protocol II, and Supplementary Fig. d) or expression of App swe/lnd in neighboring neurons (see Fig. 5d,e and Supplementary Fig. e). Notably, both PTEN-C1S and control (uninfected) neurons are exposed to extracellular Aβ (either synthetic or secreted from neighboring App swe/lnd -expressing neurons) under these conditions. We found that AMPAR-mediated synaptic responses were significantly increased in PTEN-C1S expressing neurons as compared with uninfected cells (1.9 ±.3-fold (s.e.m.) potentiation relative to uninfected cells, P =., N = 18 cell pairs; Supplementary Fig. f). Given that PTEN inhibition did not alter basal synaptic transmission on its own (Figs. a and 5a) 15, these findings indicate that suppression of PTEN activity in individual postsynaptic neurons protects them from Aβ-induced synaptic depression (resulting in apparent potentiation with respect to uninfected neurons exposed to Aβ). We found that PTEN-C1S infected neurons exposed to 1 µm synthetic Aβ also exhibited significant LTP (P =.1; Fig. 5f,g and Supplementary Fig. g), in contrast with uninfected cells exposed to Aβ from neighboring Aβ-producing neurons (Fig. g,h), in which LTP was not expressed. Thus, suppression of PTEN phosphatase activity in a cell-autonomous manner in postsynaptic neurons protects from the synaptic plasticity impairment produced by either synthetic or neuronally secreted Aβ. As a general control, none of these viral and pharmacological manipulations altered basal ionic conductance in the cell, as evaluated from the holding current during the recordings (Supplementary Fig. h). NMDAR-dependent recruitment of PTEN to spines induced by Ab We previously showed that NMDAR-dependent LTD results in PTEN anchoring to the postsynaptic terminal 15. To test whether Aβ induces a similar redistribution of PTEN, we expressed EGFP-tagged PTEN (EGFP is fused to the N terminus of PTEN) in primary hippocampal neurons for h. Time-lapse imaging of infected hippocampal neurons before Aβ application revealed widespread and homogenous distribution of EGFP-PTEN, including in distal dendrites and spines (Fig. a and Supplementary Fig. 5a). Following the addition of synthetic Aβ assemblies ( µm), EGFP-PTEN rapidly accumulated in spine heads and remained there for at least 1 h (Fig. a,b and Supplementary Fig. 5a). This effect was to some extent dose dependent, as a lower concentration of synthetic Aβ assemblies (1 µm) nature NEUROSCIENCE LUME 19 NUMBER 3 MARCH 1 7

6 npg 1 Nature America, Inc. All rights reserved. Figure 5 Inhibition of PTEN activity in vivo and in vitro rescues synaptic plasticity in conditions of high Aβ levels. (a) Left, superimposed representative fepsps at 1, 5, 1, 15 and µa of stimulation intensity, recorded from acute slices taken from and App/ mice treated for 3 weeks with -OHpic or vehicle, as indicated. Scale bars represent.5 mv, 1 ms. N represents number of slices. Right, input-output curves of fepsps evoked by stimulation of Schaffer Collaterals, from recordings such as the ones shown on the left. P values were determined with two-way ANOVA with Bonferroni post-tests (df, group, intensity, interaction, residual: 3., 11., 33., 98.). Vehicle,.1% DMSO. (b) Left, LTP was induced by θ-burst stimulation and recorded for min post-induction, following at least min of stable baseline. Right, representative field recordings at baseline (thin line) and after LTP induction (thick line) from slices taken from mice treated for 3 weeks with -OHpic or vehicle, as indicated. Scale bars represent. mv, 1 ms. (c) Quantification of average fepsp maximal slopes at 5 min after induction (F (3, 35) = 5., P =., Kruskal-Wallis ANOVA). P values shown in the graphs were calculated using Dunn s multiple post hoc test. N represents number of slices. Data are represented as mean ± s.e.m. (d) Schematic diagram of the two experimental configurations used in this experiment. Synthetic Aβ (left): some neurons expressed PTEN-C1S, whereas others remained uninfected; they were all exposed to synthetic Aβ assemblies added to the medium (5 h after infection with PTEN-C1S virus). Secreted Aβ (right): some neurons expressed EGFP-PTEN-C1S (green fluorescence relatively excluded from the nucleus), others coexpressed App swe/lnd and EGFP (green fluorescence relatively concentrated in the nucleus), and others remained uninfected (light gray); they were all exposed to extracellular Aβ from App swe/lnd -infected cells. b fepsp fold potentiation a. 1. (Veh), N = 5 (), N = 19 App/PS1 (Veh), N = App/ PS1 (), N = f d Synthetic Aβ (Veh) App/PS1 (Veh) fepsp slope (mv ms 1 ) Secreted Aβ P = Stimulation intensity (ma) () App/PS1 () PTEN-C1S App swe/lnd Uninf. Aβ aggregates PTEN-C1S PTEN-C1S (Aβ) fepsp fold potentiation P =. N = N = 11 P =. P =.1 P =.1 P =.3 P =. + + P =.1 N = Paired pathway N = 1 N = 1 App/PS1 Unpaired pathway P =.1 PTEN-C1S PTEN-C1S (Aβ) In both configurations, recordings were performed simultaneously from uninfected neurons (devoid of any fluorescence) and neurons expressing PTEN-C1S (displaying brighter fluorescence in the cytoplasm than in the nucleus). (e) Representative confocal projection image of neurons in organotypic hippocampal neurons expressing either the PTEN dominant-negative mutant (EGFP-PTEN-C1S, white arrows, note that EGFP was relatively excluded from the nuclei) or coexpressing App swe/lnd and EGFP (orange arrows, GFP was relatively concentrated in the nuclei) (Supplementary Fig. e). Scale bar represents µm. (f) Left, time course of AMPAR-mediated synaptic responses before and after LTP induction (black arrow), in neurons expressing dominant-negative mutant of PTEN and exposed to synthetic Aβ (PTEN-C1S + Aβ) or not (PTEN-C1S). Right, representative traces from baseline (thin lines) and after LTP induction (thick lines). Scale bars represent pa, 1 ms. (g) Quantification of average synaptic potentiation from paired (LTP) and unpaired (control) pathways from the last 5 min of the time course shown in f (Supplementary Fig. g). Statistical significance was calculated according to the Wilcoxon test relative to baseline (t =.11, df = 5; t = 3.91 df = 5). N represents number of cells. Data are represented as mean ± s.e.m. (error bars). e c g N = produced a qualitatively similar result, although with a smaller and shorter-lived effect on PTEN recruitment (Supplementary Fig. 5b). The accumulation of PTEN in synaptic compartments was also observed for the endogenous protein using synaptosomal preparations from hippocampal slices treated with Aβ (Supplementary Fig. 5c). To test whether Aβ utilizes similar mechanisms for PTEN engagement as those operating during LTD 15, we evaluated its dependence on NMDAR activation and PDZ interactions. The NMDAR blocker AP5 completely abolished the movement of PTEN into dendritic spines (Fig. a,b), indicating that NMDARs are required for this engagement. To test the requirement for PDZ interactions, we carried out time-lapse imaging with a truncated form of PTEN lacking its PDZ-binding motif (EGFP-Pten- PDZ) 15. We found that compared with the full-length PTEN, Pten- PDZ concentrates in spines more slowly and to a lesser extent (Fig. a,b). Thus, Aβ triggers the delivery of PTEN to spines in a process requiring NMDAR activation and facilitated by PDZ-dependent interactions. Dephosphorylation of Akt and GSK3β in synaptic compartments Given that PTEN appears to be a regulated target in Aβ synaptic toxicity, we tested whether Aβ exposure can globally alter the PIP 3 pathway. This was evaluated by monitoring PTEN levels and the downstream effectors Akt and GSK3β, whose activity is regulated by phosphorylation. We found no change in these effectors in App swe/lnd -EGFP 8 LUME 19 NUMBER 3 MARCH 1 nature neuroscience

7 npg 1 Nature America, Inc. All rights reserved. Figure Aβ-induced local regulation of the PIP 3 pathway. (a) High-magnification images of dendritic spines from neurons expressing EGFP-PTEN (top and bottom) or truncated PTEN lacking the PDZ binding motif (EGFP-Pten- PDZ, middle panel) following Aβ application. Some EGFP-PTEN slices were treated with Aβ in the presence of AP5 (bottom). (b) Quantification of timelapse imaging of the spine/dendrite ratio of EGFP-PTEN (with or without AP5) and EGFP-PTEN- PDZ, as indicated, up to min after Aβ application. Data are represented as mean ± s.e.m. (error bars). N represents number of cells analyzed from independent experiments (total number of spines analyzed is presented in parenthesis). P values were determined with two-way ANOVA followed by Bonferroni s multiple comparisons test. Accumulation of GFP-PTEN was significantly different (df, group, intensity, interaction, residual:., 1., 3., 1.; P <.1) across the different conditions (wt-pten, PDZ-PTEN, and wt-pten+ap5). (c) Representative confocal images (, oil) of dendrites of primary neurons immunostained against phospho-akt (red signal) and total Akt (blue signal) and expressing EGFP-actin for spine identification (yellow signal). Antibodies for phospho- and total Akt were not compatible, and therefore immunostaining was carried out separately on different preparations. (d) Similar immunostaining for phospho-gsk3β (red) and total GSK3β (blue). Antibodies for phospho- and total GSK3β were compatible and immunostaining was carried out simultaneously on the same preparation. (e,f) Quantification of intensity of pakt, takt, pgsk3β, tgsk3β and the corresponding ratios (pakt/takt, pgsk3β/tgsk3β) in spines at different time points after addition of synthetic Aβ ( µm). Values were normalized to time (control). Signal intensity was measured (after background e Normalized intensity a c PTEN 3 PTEN (AP5) P =. pakt takt pakt/takt P =.1 P = pakt takt GFP pgsk3β tgsk3β GFP P =.1 P =.1 Spine/dendrite ratio (normalized to baseline) 1 µm P =.1 P = Normalized intensity 3 1 -PTEN, N = 3 cells (15 spines) Aβ PTEN- -PDZ, N = 3 cells (1 spines) -PTEN (AP5), N = 3 cells (117 spines) P =.1 pgsk3β tgsk3β pgsk3β/tgsk3β P =.1 P =.1 P =.1 P =.1 P = P = subtraction, Online Methods) with Imaris 7. software. Data are represented as mean ± s.e.m. (error bars) for pgsk3β/tgsk3β (f). In the case of AKT (e), as phospho and total values came from different experiments, the mean pakt/takt ratio was calculated from the mean phospho and total values (without error bars). N represents number of spines. In the case of the GSK3β immunostaining, N was 78, 3, 155, 8 and 1, for the time points of, 5, 1, and s, respectively (t =.918, df = 3; t = 7., df = 31; t = 8, df = 1; t =., df = 17, for pgsk3β; t =.8, df = 3; t = 5.8, df = 31; t = 7.98, df = 1, for tgsk3β). In the case of takt, N was 188, 19, 177, 9 and 18 (t = 1., df = ; t = 5.8, df = 15; t = 5.1, df = 33) for the same time points. In the case of pakt, N was 7, 13, 38, 8 and 178 (t =.89, df = 8; t = 7.937, df = 53; t =.5, df = 59; t =.371, df = 3) for the same time points. µm b f d P =.1 P =.1 infected slices 7 h post infection (Supplementary Fig. a c), or in App/ mice (Supplementary Fig. d f), in which high levels of Aβ are present in a chronic manner. These combined findings suggest that Aβ overexpression does not globally alter the PIP 3 pathway. To determine whether Aβ-induced translocation of PTEN to spines alters PIP 3 signaling locally, we immunostained primary hippocampal neurons for pakt (T38), pgsk3β (S9) and total levels of these kinases after treatment with Aβ ( µm) for different time periods (5, 1, and min) (these neurons also expressed GFP-actin to facilitate spine identification). We found that by 5 min after Aβ application Akt and GSK3β gradually accumulated in spines (Fig. c f). The migration of GSK3β into spines in response to Aβ could also be visualized by monitoring the distribution of GFP-fused GSK3β in real time (Supplementary Fig. g). Notably, the movement of Akt and GSK3β did not result in a net accumulation of their phosphorylated forms in spines, but there was a net dephosphorylation of these proteins by the end of the time course, as reflected in the pakt/takt and pgsk3β/tgsk3β ratios (Fig. e,f). This effect was also detectable in synaptosomal preparations from hippocampal slices treated with Aβ (Supplementary Fig. h). These findings imply that Aβ recruits multiple components of the PIP 3 pathway to the postsynaptic terminals, in addition to PTEN. But the net effect of this recruitment is downregulation of the PIP 3 pathway in these sites, possibly because of the dominant effect of PTEN accumulation. PTEN-PDZ interaction is essential to Ab synaptic toxicity After observing the involvement of PDZ interactions for the Aβinduced recruitment of PTEN to spines, we asked whether this mechanism was relevant for Aβ-induced synaptic toxicity. To this end, we developed a new knock-in mouse model in which PTEN lacks the last five amino acids (-QITKV), thereby removing the C-terminal PDZ binding motif (Pten PDZ mice; Fig. 7a). Total levels of PTEN nature NEUROSCIENCE LUME 19 NUMBER 3 MARCH 1 9

8 npg 1 Nature America, Inc. All rights reserved. a b c d P KO P P Normalized EPSC (%) P/P P P/P P P/P P H Exon H H P =.3 N = 1 N = 1 AMPA 7 S S S N = 1 N = 1 NMDA P E B P/S/P/S * 8 (B) P E * Neo loxp loxp P E B P/S/P/S * AMPA R1 1 kb (B) P/S/P/S NMDA PTEN pakt fepsp fold potentiation fepsp fold depression takt Homogenates Synaptosomes TBS pakt/takt N = 5 Homogenates Pten PDZ n.s. 1 Hz, 9 pulses fepsp slope (mv ms 1 ) Stimulation intensity (µa) e f g Figure 7 Aβ-induced synaptic depression depends on PTEN-PDZ interaction. (a) The Pten PDZ knock-in mouse strain was generated by homologous recombination in itl1 19S/SvEvBrdTac(19Sv)-derived embryonic stem cells. The PDZ-binding motif was deleted by substituting glutamine codon 399 (CAA) with a stop codon (TAA). Top, schematic representation of the Pten tm(q399stop)amc targeting allele (KO) highlighting exon 8, which harbors the PDZ-binding motif. Bottom, the amino acid and nucleotide sequences of the knock-in region with the substitution of codon 399 (CAA, glutamine) with TAA (Stop codon). The PDZ-binding motif is underlined. B, BsiWI; h N = 5 N = 5 N = 5 Synaptosomes (Vehicle) (Aβ) (Aβ), AP5, AP5 + Aβ PTEN- PDZ, AP5 P =.1 i PTEN- PDZ, AP5 + Aβ fepsp fold potentiation fepsp fold depression P = P =.1 P =.1 P =. N = 11 Aβ + + P =.3 P =.1 P =.1 P =.1 N = 31 (vehicle) N = 3 N = 1 (Aβ) N = 17 (Aβ) Aβ + + E, EcoRI; H, HindIII; P, PstI; S, SphI; Neo, neomycin; arrow, transcriptional start site; loxp, locus of X-over P1. (b) Western blot showing PTEN, pakt (Thr38) and takt in the hippocampal homogenates (left) and synaptosomes (right) of Pten PDZ mice and their littermates. Note the lower position of PTEN lacking the PDZ-binding motif. Uncropped versions of the western blots are shown in Supplementary Figure 9. (c) Quantification of pakt/takt ratio in homogenates and synaptosomes from Pten PDZ mice and their littermates. N represents number of mice (t =.5, df = 8; t =.783, df = 8; n.s. indicates non-significant). (d) Input-output curves of fepsps (left) in slices from Pten PDZ mice and their littermates with or without Aβ treatment, as indicated, and superimposed representative fepsps at 1, 5, 1, 15 and µa of stimulation intensity (right). P values were determined with two-way ANOVA with Bonferroni post-tests (df, group, intensity, interaction, residual: 3., 11., 33., 99.). N represents number of slices. Scale bars represent.5 mv, ms. (e) Left, average relative EPSC in App swe/lnd -EGFP expressing neurons compared to neighboring uninfected neurons. N represents number of cell pairs. Statistical significance was calculated according to the Wilcoxon test for paired data (individual pairs of App swe/lnd -EGFP cells versus uninfected cells; t =.8, df = ). Scale bars represent pa, 1 ms. Right, example voltage clamp traces from uninfected and App swe/lnd -EGFP expressing neurons recorded at mv (AMPAR EPSCs) and + mv (NMDAR EPSCs) in Pten PDZ mice or in their littermates. (f) Left, LTP was induced by θ-burst stimulation and recorded for min post-induction following at least min of stable baseline in acute hippocampal slices from Pten PDZ mice and their littermates treated with vehicle or Aβ assemblies ( h incubation). Right, representative field recordings at baseline (thin line) and after LTP induction (thick line). Scale bars represent.5 mv, 1 ms. (g) Quantification of average fepsp maximal slopes at 5 min after induction (P =., Kruskal-Wallis ANOVA). P values shown in the graphs are according to Dunn s multiple post hoc test (F (3, 9) =.755). N represents number of slices. (h) Left, NMDARdependent LTD was induced in acute hippocampal slices (9 pulses at 1 Hz) under partial NMDARs blockade (3 µm AP5). Right, representative fepsps before (thin line) and after (thick line) LTD induction. Scale bars represent.5 mv, ms. (i) Quantification of the average fepsp maximal slopes at 5 min after induction (P =.5, Kruskal-Wallis ANOVA). P values shown in the graphs are according to Dunn s multiple post hoc test (F (3, 31) = 1.1). N represents number of slices. Depression values were also compared to baseline with the Wilcoxon test and were significantly different only for slices treated with Aβ (P =.5). Data are represented as mean ± s.e.m. (error bars). protein and global activation of the PIP 3 pathway (as reported by phospho-akt levels) are normal in both hippocampal homogenates and synaptosomes from these animals (Fig. 7b,c). Acute hippocampal slices from these mice and from their age-matched mice (5 months old) were treated with 1 µm synthetic Aβ assemblies for h and fepsps were recorded to yield input-output curves. Basal synaptic transmission was similar in and Pten PDZ animals (Fig. 7d). However, although incubation with Aβ significantly depressed the input-output curve in slices (P =.), slices taken from Pten PDZ mice were completely resistant to Aβ treatment, and basal synaptic transmission remained similar to that of untreated slices (Fig. 7d). These results suggest that Aβ-triggered synaptic depression relies on PTEN interactions with PDZ proteins. We also tested whether this was the case using neuronally produced Aβ, via App swe/lnd expression. Using organotypic slice cultures from Pten PDZ mice and from their littermates, we compared synaptic responses evoked onto adjacent pairs of simultaneously recorded neurons in which only one neuron expresses App swe/lnd. As expected, App swe/lnd -expressing neurons showed significant depression of AMPAR-mediated synaptic responses relative to uninfected cells 5 LUME 19 NUMBER 3 MARCH 1 nature neuroscience

9 npg 1 Nature America, Inc. All rights reserved. a b c d N-myr-QHSQITKV (PTEN-PDZ) PTEN-PDZ Scrambled N-myr-SVHTIQKQ (scrambled) in slices from mice (Fig. 7e). In contrast, App swe/lnd -expression in neurons from Pten PDZ mice did not depress AMPAR-mediated synaptic transmission (Fig. 7e), similar to the results with acute slices and synthetic Aβ. Notably, Pten PDZ mice presented normal LTP expression, even in the presence of Aβ assemblies (Fig. 7f,g). Thus, PTEN-PDZ interaction is essential to Aβ-induced impairments in both basal synaptic transmission and LTP. Besides depression of basal synaptic transmission and impairment of LTP, Aβ has been shown to enhance LTD 5. Thus, we tested whether PTEN-PDZ interactions also mediate this altered form of synaptic plasticity. To this end, we induced LTD in acute hippocampal slices with an NMDAR-dependent protocol (9 pulses at 1 Hz), under partial NMDAR blockade (3 µm AP5) 5. In agreement with previously published observations 5, Aβ facilitated LTD in slices (Fig. 7h,i and Supplementary Fig. 7). In contrast, slices from Pten PDZ mice failed to show LTD, and there was no facilitation of LTD following Aβ treatment (Fig. 7h,i). Thus, enhancement of NMDAR-dependent LTD by Aβ requires PTEN-PDZ interactions. All together, these experiments support the notion that removal of PTEN-PDZ interactions renders neurons resistant to Aβ-induced synaptic alterations. PTEN-PDZ peptide protects against Ab synaptic depression These results offer a new potential target amenable for pharmacological intervention: PDZ-dependent interactions of PTEN. To this end, we designed a peptide corresponding to the last eight amino acids of rat and mouse PTEN with the addition of myristic acid to augment cell permeability (N-myristoyl-QHSQITKV, PTEN-PDZ; Fig. 8a). A scrambled control peptide with the same amino acid composition µm 1 µm fepsp fold depression Hz, 9 pulses Recognition index Vehicle PTEN-PDZ Scrambled e f g Vehicle N = 17 Aβ N = 5 PTEN-PDZ N = 15 PTEN-PDZ + Aβ, N = 1 fepsp slope (mv ms 1 ).8... P = Stimulation intensity (µa) P =. P =.9 was also synthesized (N-myristoyl-SVHTIQKQ, scrambled; Fig. 8a and Supplementary Fig. 8a). Time-lapse imaging of the fluoresceintagged peptides revealed their rapid (few minutes) diffusion into neurons, including dendritic spines (Fig. 8b and Supplementary Fig. 8b). To assess the specificity of the PTEN PDZ peptide, we overlaid a biotinylated version on a PDZ domain array 33. The peptide exhibited specific binding to a handful of PDZ domains (Supplementary Fig. 8c), which corresponded to proteins that have been previously described as binding partners for PTEN 15,3 3. Finally, using a GST pulldown assay, we found that the PTEN-PDZ peptide could effectively compete with PTEN for binding to a PDZ domain partner, whereas the scrambled peptide could not (Supplementary Fig. 8d). Based on these binding assays, we hypothesized that, when incubated with hippocampal slices, the PTEN-PDZ peptide would compete for PDZ interaction sites of PTEN, consequently preventing anchoring of PTEN to PDZ proteins, and thereby preventing LTD. Indeed, incubation of acute mouse slices in PTEN-PDZ peptide (1 µm, 1.5 h) completely blocked LTD, whereas the scrambled peptide did not have any effect (Fig. 8c,d). We then tested the effect of these peptides on Aβ-treated neurons. Pre-incubation of slices with the PTEN-PDZ peptide for.5 h before the addition of synthetic Aβ prevented synaptic depression, whereas the control-scrambled peptide did not (Fig. 8e and Supplementary Fig. 8e). In addition, neither peptide altered basal synaptic transmission in the absence of Aβ treatment (Fig. 8e and Supplementary Fig. 8e). Finally, to test whether blocking PTEN-PDZ interaction can prevent cognitive impairment in the AD mouse model, we infused PTEN-PDZ peptide or vehicle over a period of 3 weeks into the fepsp fold depression P =..8 P =.17 P =.5... PTEN-PDZ N = 1 N = 1 Freezing (%) 1. P =.5 P = N = 1 N = 3 N = 13 N = Vehicle PTEN-PDZ Scrambled P =.1 P =. P =. N = PTEN-PDZ + + App/ App/ Figure 8 Synthetic peptide corresponding to the PTEN-PDZ binding motif prevents Aβ-induced synaptic and cognitive deficits. (a) Chemical structure of the N-myristoylated octapeptides corresponding to the PTEN C terminus (top) and the scrambled control with the same amino acid composition (bottom). (b) Representative confocal image of primary hippocampal neurons (1 d in vitro) after 1-h incubation with fluorescein-labeled PTEN-PDZ or scrambled peptides (Online Methods). (c) Left, NMDAR-dependent LTD was induced in acute hippocampal slices (9 pulses at 1 Hz) pre-incubated (.5 h) with the PTEN-PDZ peptide, the scrambled peptide or with vehicle (.1% DMSO). Right, representative traces of fepsps before (thin line) and after LTD induction (thick line). Scale bars represent.5 mv, ms. (d) Quantification of the average fepsp maximal slopes at 5 min after induction. P value was determined with Mann-Whitney test. N represents the number of slices. (e) Left, representative fepsps at 1, 5, 1, 15 and µa of stimulation intensity. Right, input-output curves of fepsps evoked by stimulation of Schaffer Collaterals after -h incubation in Aβ assemblies (or vehicle control) with or without pre-incubation (.5 h) with 1 µm PTEN-PDZ peptide. P values were determined with two-way ANOVA with Bonferroni post-tests. Scale bars represent.5 mv, 1 ms. (f) App/ and littermates (treated with PTEN-PDZ or with vehicle) were tested in the novel object location task (P =.1, Kruskal-Wallis ANOVA). N represents the number of mice. (g) Percentage freezing to the context for vehicle-infused mice and mice infused with PTEN-PDZ (P <.1, Kruskal-Wallis ANOVA). N represents the number of mice. Data are represented as mean ± s.e.m. (error bars). nature NEUROSCIENCE LUME 19 NUMBER 3 MARCH 1 51