Nature Neuroscience: doi: /nn Supplementary Figure 1
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1 Supplementary Figure 1 PCR-genotyping of the three mouse models used in this study and controls for behavioral experiments after semi-chronic Pten inhibition.
2 a-c. DNA from App/Psen1 (a), Pten tg (b) and Pten PDZ (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.
3 Supplementary Figure 2 VO-OHpic specificity, characterization of A assemblies and effect on electrophysiological responses
4 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 A aggregation resulting from incubation of synthetic A 42 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. A 42 peptide (final concentration 258 M) 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 A 42 assemblies used for our electrophysiological and imaging experiments. Bands were visualized by western blot analysis, probed with the N-terminal anti-a antibody, 6E10, to identify SDS-stable A 42 species. e. Representative electron micrographs of negatively stained A 42 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 A 42 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 A 42 assemblies (2 h incubation), as indicated. Bottom: The same data showing only low values of fiber volley. h. Paired-pulse facilitation (PPF) in A - 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 App swe/lnd, with or without PTEN inhibition by VO-OHpic incubation, as indicated.
5 Supplementary Figure 3 Scatter plot analyses for the effects of App expression on synaptic transmission in organotypic hippocampal slices a. Percentage of neurons overexpressing App swe/lnd with visible intracellular A. b. Scatter plots of AMPAR- and NMDAR-mediated EPSCs recorded from pairs of neurons infected with App swe/lnd EGFP and neighboring uninfected neurons. Each pair of infecteduninfected 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 App MV and EGFP. d. Example traces from uninfected and App MV -EGFP expressing neurons recorded at -60 mv (AMPAR EPSCs), and +40 mv (NMDAR EPSCs). e. Scatter plots of AMPAR- and NMDAR-mediated
6 EPSCs recorded from pairs of neurons infected with App MV EGFP and neighboring uninfected neurons. Each pair of infecteduninfected neuron is represented with a single point. f. Scatter plots of AMPAR- and NMDAR-mediated EPSCs recorded from pairs of neurons infected with App swe/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.
7 Supplementary Figure 4 Electrophysiology controls and A assemblies
8 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, App swe/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 A 42 assembly formation by western blot analysis (left panel) and electron microscopy (right panel). A 42 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 App swe/lnd -IRES-EGFP (blue channel, fluorescence in the nucleus). To visualize A production, slices were fixed, permeabilized and immunostained for A (NU-1 antibody, green channel). Note that neurons expressing App swe/lnd (blue channel, fluorescence in the nucleus) are positive for A staining (green signal), while neurons expressing PTEN-C124S (blue channel, fluorescence excluded from the nucleus, white arrowheads) are negative for A 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 A secreted from neighboring App swe/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 A 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.
9 Supplementary Figure 5 PTEN recruitment to synaptic compartments
10 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 M synthetic A 42 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 M A 42 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 A 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.
11 Supplementary Figure 6 A overproduction does not globally alter the PIP 3 pathway
12 a. Representative blot of PTEN (left) and quantification of PTEN levels (right) from extracts of hippocampal slice cultures injected with App swe/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 GSK3 (c: pgsk3 (S9), tgsk3 ), and tubulin from extracts of hippocampal slice cultures injected with App swe/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-GSK3. Bottom: Quantification of time lapse imaging of the spine/dendrite ratio of WT EGFP-GSK3 up-to 60 min after A 42 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 A 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.
13 Supplementary Figure 7 NMDAR-dependent LTD in WT and Pten PDZ 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 M) of AP5 (b). Some slices were incubated in 1 M A assemblies, as indicated. c. Similar to A, with acute slices from Pten PDZ mice. d. Quantification of the average fepsp maximal slope, at minutes after LTD induction, from the experiments shown in a-c. P value was determined with Wilcoxon test relative to baseline.
14 Supplementary Figure 8 Chemical structure and biochemical analysis of PTEN synthetic peptides and their permeability in neurons
15 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 M) 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 M 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 A 42 assemblies (or vehicle control) with or without preincubation (0.5 h) with 10 M scrambled peptide at 10, 50, 100, 150 and 200 A 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.
16 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.
17 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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