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PTEN recruitment controls synaptic and cognitive function in Alzheimer's models

Nature Neuroscience volume 19, pages 443–453 (2016)Cite this article

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Abstract

Dyshomeostasis of amyloid-β peptide (Aβ) is responsible for synaptic malfunctions leading to cognitive deficits ranging from mild impairment to full-blown dementia in Alzheimer's disease. Aβ 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 Aβ-induced depression. Mechanistically, Aβ triggers a PDZ-dependent 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 Aβ-induced synaptic toxicity and cognitive dysfunction. Our results provide fundamental information on the molecular mechanisms of Aβ-induced synaptic malfunction and may offer new mechanism-based therapeutic targets to counteract downstream Aβ signaling.

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Figure 1: VO-OHpic semi-chronic infusion rescues the learning deficits of App/Psen1 mice.
Figure 2: PTEN activity determines the synaptic response to synthetic Aβ.
Figure 3: PTEN inhibition protects Appswe/lnd-expressing neurons.
Figure 4: Blockade of PTEN activity protects neurons from extracellular Aβ.
Figure 5: Inhibition of PTEN activity in vivo and in vitro rescues synaptic plasticity in conditions of high Aβ levels.
Figure 6: Aβ-induced local regulation of the PIP3 pathway.
Figure 7: Aβ-induced synaptic depression depends on PTEN-PDZ interaction.
Figure 8: Synthetic peptide corresponding to the PTEN-PDZ binding motif prevents Aβ-induced synaptic and cognitive deficits.

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Acknowledgements

We thank F. Valdivieso (Centro de Biología Molecular “Severo Ochoa”) for the Appswe/lnd construct, W. Klein (Northwestern University) for the Aβ antibody (NU-1), D. Walsh (Harvard Institutes of Medicine) for expert advice and providing protocols, and members of the Esteban laboratory for critical reading of this manuscript. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (CSD-2010-00045, SAF-2011-24730 and SAF2014-57233-R to J.A.E.; SAF2010-15676, SAF2013-43902-R and SAF2015-62540-ERC to S.K.; SAF-2009-09129 to C.V.; SAF2009-12249-C02-01 to F.W.; CSD2010-00045 and SAF2010-14906 to C.G.D.; SAF2013-48812-R to R.P.). The laboratory of S.K. is supported by a grant from Alzheimer's Association (NIRG-13-279533), from the Basque Ministry of Health (2013111138), from the University of the Basque Country (EHUrOPE14/03) and from Ikerbasque foundation. The laboratory of F.W. is also supported by CIBERNED (an initiative of ISCIII) and by EU-FP7-2009-CT222887 grant. M.S. is funded by grants from the MINECO, European Union (ERC Advanced Grant), Regional Government of Madrid, Botín Foundation, Ramón Areces Foundation, and AXA Foundation. A.M.C. is funded by grants R01CA095063 and R21CA133669 from the US National Institutes of Health. T.W. is funded from the Federal Office for Scientific Affairs (IUAP P6/43) and Flemish Government's Methusalem Grant. N.Z.G. is funded from the US National Institute on Aging (AG032320), Alzheimer's Association and American Health Assistance Foundation (AHAF). S.K. was the recipient of a “Ramón y Cajal” contract from the Spanish Ministry of Science and Innovation and is now an IkerBasque Research Professor. C.S.-P. is a recipient of an FPI scholarship from the Spanish Ministry of Economy and Competitiveness (BES-2011-043464). J.M. is the recipient of a predoctoral fellowship (PRE_2014_1_285) from Gobierno Vasco, Departamento de Educación (Basque Country, Spain).

Author information

Authors and Affiliations

  1. Department of Molecular Neurobiology, Centro de Biología Molecular “Severo Ochoa”, Consejo Superior de Investigaciones Científicas (CSIC) / Universidad Autónoma de Madrid, Madrid, Spain

    Shira Knafo, Cristina Sánchez-Puelles, Ernest Palomer, Jonathan E Draffin, Laura Lozano-Montes, Lara Ordóñez-Gutiérrez, Francisco Wandosell, Carlos G Dotti & José A Esteban

  2. Unidad de Biofísica CSIC-UPV/EHU, Campus Universidad del País Vasco, Leioa, Spain

    Shira Knafo & Igotz Delgado

  3. Ikerbasque, Basque Foundation for Science, Bilbao, Spain

    Shira Knafo & Rafael Pulido

  4. Biocruces Health Research Institute, Barakaldo, Spain

    Janire Mingo & Rafael Pulido

  5. VIB Center for the Biology of Disease and Center for Human Genetics, University of Leuven (Katholieke University of Leuven), Leuven, Belgium

    Tina Wahle

  6. IUF - Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany

    Tina Wahle

  7. Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, USA

    Kanwardeep Kaleka & Nashaat Z Gerges

  8. Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, USA

    Liping Mou & Randy A Hall

  9. Department of Psychobiology, Universidad Nacional de Educación a Distancia, Madrid, Spain

    Inmaculada Pereda-Perez & César Venero

  10. Department of Pharmacology and Toxicology, Geisel School of Medicine at Dartmouth, and Norris Cotton Cancer Center, Lebanon, New Hampshire, USA

    Edvin Klosi, Erik B Faber, Heidi M Chapman & Mark R Spaller

  11. Tumor Suppression Group, Spanish National Cancer Research Center (CNIO), Madrid, Spain

    Ana Ortega-Molina & Manuel Serrano

  12. Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED),

    Lara Ordóñez-Gutiérrez & Francisco Wandosell

  13. Department of Physiology, Faculty of Medicine, University of Valencia, Valencia, Spain

    Jose Viña

  14. School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong

    Andrew M Chan

Authors
  1. Shira Knafo
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  2. Cristina Sánchez-Puelles
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  3. Ernest Palomer
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  4. Igotz Delgado
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  5. Jonathan E Draffin
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  6. Janire Mingo
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  7. Tina Wahle
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  8. Kanwardeep Kaleka
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  9. Liping Mou
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  11. Edvin Klosi
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  12. Erik B Faber
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  13. Heidi M Chapman
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  14. Laura Lozano-Montes
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  15. Ana Ortega-Molina
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  18. Jose Viña
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  19. Carlos G Dotti
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  20. Randy A Hall
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  21. Rafael Pulido
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  22. Nashaat Z Gerges
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  26. César Venero
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  27. José A Esteban
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Contributions

S.K. performed and analyzed most of the in vitro experiments. C.S.-P. performed most biochemical and imaging experiments. J.E.D. performed the EGFP-GSK3β imaging experiments. T.W., E.P., I.D. and L.L.-M. performed some of the biochemical and imaging experiments under the supervision of C.G.D. I.P.-P. performed some of the behavioral experiments. E.K., E.B.F. and H.M.C. designed and synthesized the PTEN peptides under the supervision of M.R.S. The specificity of PTEN-PDZ peptide using PDZ domain overlays was carried out by L.M. under the supervision of R.A.H. GST pull-down experiments were carried out by J.M. under the supervision of R.P. N.Z.G. and K.K. performed some of the patch-clamp experiments. A.M.C., A.O.-M. and M.S. developed transgenic PTEN mice. L.O.-G., F.W. and J.V. analyzed, characterized and maintained the App/Psen1 mouse colonies. C.V. designed and supervised all in vivo manipulations. J.A.E., S.K. and C.V. designed the study and wrote the paper.

Corresponding authors

Correspondence to Shira Knafo, César Venero or José A Esteban.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 PCR-genotyping of the three mouse models used in this study and controls for behavioral experiments after semi-chronic Pten inhibition.

a-c. DNA from App/Psen1 (a), Ptentg (b) and PtenΔPDZ (c) was analyzed by PCR with the primers described in Supplemental Information. Representative PCR reactions from transgenic animals and wild-type littermates are shown for each case. d. Total time spent exploring in the novel object location task, by App/Psen1 animals and WT littermate controls, with or without VO-OHpic treatment. e. Percentage freezing displayed by App/Psen1 animals and WT littermate controls, with or without VO-OHpic treatment, before fear conditioning (white columns) or after contextual fear conditioning, before tone presentation in the memory test (see Methods). f. Representative western blot of hippocampal homogenates prepared from App/Psen1 mice and their WT littermates after semi-chronic (3-4 weeks) treatment with VO-OHpic. Uncropped version of the western blot is shown in Supplementary Fig. 10.

Supplementary Figure 2 VO-OHpic specificity, characterization of Aβ assemblies and effect on electrophysiological responses

a. Representative western blots of phospho-Akt (pAkt, Thr308), total-Akt (tAkt), phospho-Tyrosine (pTyr), and actin as loading control, from acute hippocampal slices incubated with 50 nM VO-OHpic for 30, 60 or 120 min. Uncropped versions of the western blots are shown in Supplementary Fig. 10. b. Quantification of the experiment described in (a). Relative levels (normalized to vehicle) of pAkt, tAkt, pTyr and actin after treatment with VO-OHpic for different periods of time, as indicated. c. Kinetics of Aβ aggregation resulting from incubation of synthetic Aβ42 alone or with either of the PTEN inhibitors used in this study (VO-OHpic and bPV(HOpic)) as measured with the Thioflavin T assay. The mean fluorescence (at 490nm) ± SEM (in arbitrary units) is plotted against the incubation period (at 37 °C). Each point represents the average of 3 independent experiments d. Aβ42 peptide (final concentration 258 μM) was prepared as described in Methods and incubated for different periods of time at room temperature. The lane of 5 min (5’) of incubation represents the Aβ42 assemblies used for our electrophysiological and imaging experiments. Bands were visualized by western blot analysis, probed with the N-terminal anti-Aβ antibody, 6E10, to identify SDS-stable Aβ42 species. e. Representative electron micrographs of negatively stained Aβ42 showing time-dependent assembly formation (see Methods, protocol I). Note the heterogeneous mixtures of aggregates of different sizes and morphologies and the predominant fibril formation at long incubation times (52 h). f. Representative electron micrographs of negatively stained Aβ42 assemblies, 5 min after preparation (see Methods, protocol II). g. Top: Input-output curves of field excitatory postsynaptic potentials (fEPSPs) plotted against the amplitude of the fiber volley, evoked by stimulation of Schaffer Collaterals, from slices treated with vehicle or Aβ42 assemblies (2 h incubation), as indicated. Bottom: The same data showing only low values of fiber volley. h. Paired-pulse facilitation (PPF) in Aβ- and vehicle-treated hippocampal slices. The values denote the ratio of the second fEPSP amplitude to the first fEPSP amplitude. PPF was tested for 10-, 20-, 50-, 100-, and 400-ms interstimulus intervals. Insets. Sample trace of fEPSP with an interstimulus interval of 50 ms. N, number of slices/mice. Scale bars: 0.1 mV, 50 ms. i. Evaluation of TrkB signaling (monitored by TrkB phosphorylation) from slices overexpressing GFP or Appswe/lnd, with or without PTEN inhibition by VO-OHpic incubation, as indicated.

Supplementary Figure 3 Scatter plot analyses for the effects of App expression on synaptic transmission in organotypic hippocampal slices

a. Percentage of neurons overexpressing Appswe/lnd with visible intracellular Aβ. b. Scatter plots of AMPAR- and NMDAR-mediated EPSCs recorded from pairs of neurons infected with Appswe/lnd–EGFP and neighboring uninfected neurons. Each pair of infected-uninfected neuron is represented with a single point. Black circles represent the mean values of EPSCs amplitudes. c. Representative confocal projection image of neurons co-expressing AppMV and EGFP. d. Example traces from uninfected and AppMV-EGFP expressing neurons recorded at -60 mV (AMPAR EPSCs), and +40 mV (NMDAR EPSCs). e. Scatter plots of AMPAR- and NMDAR-mediated EPSCs recorded from pairs of neurons infected with AppMV–EGFP and neighboring uninfected neurons. Each pair of infected-uninfected neuron is represented with a single point. f. Scatter plots of AMPAR- and NMDAR-mediated EPSCs recorded from pairs of neurons infected with Appswe/lnd–EGFP and neighboring uninfected neurons all treated with PTEN inhibitors (15 nM bPV(HOpic) –dark grey circles- or 50 nM VO-OHpic –light grey circles-). Each pair of infected-uninfected neuron is represented with a single point. Black circles represent the mean values of EPSCs amplitudes.

Supplementary Figure 4 Electrophysiology controls and Aβ assemblies

a, c. Time course of AMPAR-mediated synaptic responses of the control (non-induced) pathway from LTP experiments shown in Fig. 4g-j, without (a) and with (c) PTEN inhibition (50 nM VO-OHpic, 5 h after virus injection and during recordings), from neurons in control (uninjected) slices, Appswe/lnd-infected neurons or neighboring uninfected neurons, as indicated. b. Time course of AMPAR-mediated synaptic responses before and after LTP induction from GFP-infected neurons or neighboring uninfected neurons. d. Biochemical characterization of Aβ42 assembly formation by western blot analysis (left panel) and electron microscopy (right panel). Aβ42 was prepared from lyophilized peptide (Invitrogen, #03-112), dissolved in water at 6 mg/ml, and then diluted to 1 mg/ml with PBS and incubated at 37ºC for 36 h (see Methods, protocol II). e. Representative confocal projection image of neurons in organotypic hippocampal slices infected with PTEN-C124S–EGFP (blue channel, fluorescence excluded from the nucleus) or Appswe/lnd-IRES-EGFP (blue channel, fluorescence in the nucleus). To visualize Aβ production, slices were fixed, permeabilized and immunostained for Aβ (NU-1 antibody, green channel). Note that neurons expressing Appswe/lnd (blue channel, fluorescence in the nucleus) are positive for Aβ staining (green signal), while neurons expressing PTEN-C124S (blue channel, fluorescence excluded from the nucleus, white arrowheads) are negative for Aβ staining. See Fig. 5d for a schematic representation of the experimental configuration. f. Scatter plots of AMPAR-mediated EPSCs recorded from pairs of neurons infected with PTEN-C124S–EGFP and neighboring uninfected neurons, all exposed to extracellular Aβ secreted from neighboring Appswe/lnd-expressing neurons (gray circles) or added exogenously (white circles). Each pair of infected-uninfected neuron is represented with a single point. g. Time course of AMPAR-mediated synaptic responses of the control pathway from LTP experiments shown in Fig. 5f, g, with or without Aβ exposure. h. Absolute current flow (holding current) during LTP experiments carried out in whole-cell under voltage-clamp configuration. These data correspond to experiments shown in Fig. 4g-j and Fig. 5f, g of the main text.

Supplementary Figure 5 PTEN recruitment to synaptic compartments

a. Confocal projection image of a primary hippocampal neuron (21 days in vitro) expressing EGFP-tagged WT (full-length) PTEN before (top) and 1 h after treatment with 4 μM synthetic Aβ42 assemblies (bottom). See Methods for preparation of primary neuronal cultures and imaging techniques. b. Quantification of time lapse imaging of the spine/dendrite ratio of WT EGFP-PTEN up-to 60 min after 1 μM Aβ42 application. N represents number of spines from 3 cells analyzed on 3 independent experiments. c. Top: Representative blot of PTEN amount on synaptosomal preparation prepared from hippocampal slices treated with Aβ for different periods of time. Uncropped versions of the western blots are shown in Supplementary Fig. 10. Bottom: Quantification of the blots represented at the top panel. N=5 rats.

Supplementary Figure 6 Aβ overproduction does not globally alter the PIP3 pathway

a. Representative blot of PTEN (left) and quantification of PTEN levels (right) from extracts of hippocampal slice cultures injected with Appswe/lnd–EGFP Sindbis virus and uninfected slices. b, c. Representative blot (left) and quantification (right) of phosphorylated and total AKT (b: pAKT(T308), tAKT), phosphorylated and total GSK3β (c: pGSK3β(S9), tGSK3β), and tubulin from extracts of hippocampal slice cultures injected with Appswe/lnd-EGFP Sindbis virus or control slices. d-f. Similar to a-c with extracts of hippocampi from App/Psen1 mice and their WT littermates. N represents the number of experiments. Statistical significance was calculated according to the Mann-Whitney test. g. Top: High magnification images of dendritic spines from neurons expressing WT EGFP-GSK3β. Bottom: Quantification of time lapse imaging of the spine/dendrite ratio of WT EGFP-GSK3β up-to 60 min after Aβ42 application. N represents number of spines from 3 cells analyzed on 3 independent experiments. h. Top: Representative blot of pAKT and tAKT amount on synaptosomal preparation prepared from hippocampal slices treated with Aβ for different periods of time. Bottom: Quantification of the blots represented at the top panel. N=6 rats. Uncropped versions of the western blots in panels a-f and h are shown in Supplementary Fig. 10.

Supplementary Figure 7 NMDAR-dependent LTD in WT and PtenΔPDZ mice

a, b. LTD was induced in WT acute hippocampal slices (900 pulses at 1 Hz) in the absence (a) or in the presence of a saturating concentration (50 μM) of AP5 (b). Some slices were incubated in 1 μM Aβ assemblies, as indicated. c. Similar to A, with acute slices from PtenΔPDZ mice. d. Quantification of the average fEPSP maximal slope, at 50-60 minutes after LTD induction, from the experiments shown in a-c. P value was determined with Wilcoxon test relative to baseline.

Supplementary Figure 8 Chemical structure and biochemical analysis of PTEN synthetic peptides and their permeability in neurons

a. Top panels: Chemical structure and Liquid Chromatography-Mass Spectrometry (LC-MS) analysis for each of the peptides used for this study. Bottom panels: Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) analysis of each peptide. b. Primary hippocampal neurons (21 days in vitro) were incubated with Fluorescein-tagged PTEN-PDZ peptide (10 μM) dissolved in DMSO (final concentration 0.1%). Top: Confocal projection image of an individual neuron before (0’) and after (2’and 50’) the addition of PTEN-PDZ peptide, demonstrating initial accumulation of the peptide at the plasma membrane (2’), followed by diffusion into the cytosol (50’). Bottom: brightfield images of the same neuron. c. N-terminus biotinylated PTEN PDZ peptide was overlaid onto an array of PDZ domains. Bound peptide was visualized with the ABC kit from Vectasatin, based on its biotin modification. “Control” represents signal detected without peptide incubation. Uncropped version of the blot is shown in Supplementary Fig. 10. d. GST pull-down of recombinant PTEN bound to a GST fusion of the PDZ2 domain of hDlg, or to GST alone (left-most lane). GST beads were incubated with recombinant PTEN in the presence of 200 μM of the indicated peptide, or in the absence of peptide, as control. Pulled-down PTEN was then visualized by western blot, with an anti-PTEN antibody. Binding is normalized to the conditions without peptide incubation. Uncropped version of the western blot is shown in Supplementary Fig. 10. e. Top: superimposed representative fEPSPs evoked by stimulation of Schaffer Collaterals after 2 h incubation in Aβ42 assemblies (or vehicle control) with or without preincubation (0.5 h) with 10 μM scrambled peptide at 10, 50, 100, 150 and 200 μA of stimulation intensity. Bottom: Input-output curves of fEPSPs. P values were determined with two-way ANOVA with Bonferroni post-tests (results without peptide preincubation are replotted from Fig. 8e as lines). Scale bars: 0.5 mV, 20 ms. Data are represented as mean +/− SEM.

Supplementary Figure 9 Uncropped versions of blots from main figures

Uncropped versions of western blots from Figure panels 1d, 1e, 2d, 3b, 3c, 3e and 7b are shown, as indicated.

Supplementary Figure 10 Uncropped versions of blots from supplementary figures

Uncropped versions of western blots from Supplementary Figure panels 1f, 2a, 5c, 6a-f, 6h, 8d, and overlay blot from Supplementary Figure 8c are shown, as indicated.

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Knafo, S., Sánchez-Puelles, C., Palomer, E. et al. PTEN recruitment controls synaptic and cognitive function in Alzheimer's models. Nat Neurosci 19, 443–453 (2016). https://doi.org/10.1038/nn.4225

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