Research ArticleAlzheimer’s Disease

Activation of CaMKIV by soluble amyloid-β1–42 impedes trafficking of axonal vesicles and impairs activity-dependent synaptogenesis

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Sci. Signal.  11 Jul 2017:
Vol. 10, Issue 487, eaam8661
DOI: 10.1126/scisignal.aam8661
  • Fig. 1 sAβ1–42 inhibits cLTP-induced synaptogenesis in cultured rat hippocampal neurons at DIV16.

    (A and B) Experimental protocol (A) and representative fluorescent images (B) of FM 1-43–loaded presynaptic boutons. Solid arrow heads, newly formed synapses; empty arrow heads, stable synapses. Scale bar, 10 μm. (C) Average destaining time course of FM 1-43 fluorescence intensity represented in (B), normalized to the maximal FM 1-43 signal. Decay time constant (τ) was determined by fitting the decay trace with a double exponential function. n = 4 independent assays. FSK, forskolin; APs, action potentials. (D and E) Experimental protocol (D) and representative images (E) of GFP-synaptophysin (green)–expressing neurons were treated with forskolin for 30 min, fixed, and stained with SHANK antibody (red). Empty arrow heads, newly formed GFP-synaptophysin–expressing presynaptic boutons; solid arrow heads, corresponding SHANK-positive postsynaptic structures; ICC, immunocytochemistry. Scale bar, 10 μm. (F) Relative fluorescence intensity of preexisting presynaptic boutons before and after forskolin treatment. (G) GFP-synaptophysin–transfected neurons were preincubated with sAβ1–42 alone or sAβ1–42 with 6E10 antibody for 2 hours and treated with 50 μM foskolin. Solid arrow heads, newly formed presynaptic boutons. Scale bar, 5 μm. (H)Number of presynaptic boutons and relative change (as %) (right) per 100 μm of axon length. Results from each of three independent experiment are shown (gray) along with the means ± SEM; *P < 0.05, **P < 0.01 by Student’s t test (C, F, and H).

  • Fig. 2 sAβ1–42 suppresses the intersynaptic vesicle trafficking.

    (A to F) Cultured rat hippocampal neurons coexpressing synaptoPHluorin (sPH)–AP and endoplasmic reticulum–retained biotin ligase (BirA-ER) were treated as indicated and labeled with streptavidin-conjugated QD 605 (1 nM) at DIV14 to DIV16. (A) Experimental protocols for QD labeling and (top to bottom) (B) representative images of QD 605–loaded SVs, raw kymographs of QD 605–loaded SVs between stationary boutons (vertical lines), difference kymographs [DIFF(N − 1)], and cumulative QD flux (Σ-flux,; flattened image), with an overlay of QD 605–loaded SVs (green) and Σ-flux (red). Vertical scale bar, 20 s; horizontal scale bar, 5 μm. Imaging data were analyzed for the cumulative QD flux (a.u., arbitrary fluorescence units) (C), MSD versus time (D), average diffusion coefficient of QD 605–loaded SVs (E), and cumulative fraction of QD 605–loaded intersynaptic SVs versus diffusion coefficients (F). Data are means ± SEM; n = 4 independent experiments, each from ~50 boutons per group. (G to I) Cultured mouse hippocampal neurons from 5XFAD TG and LT control mice were transfected with sPH-AP and BirA-ER and labeled with streptavidin-conjugated QD 605. Shown are MSD of QD 605–loaded intersynaptic SVs (G), diffusion coefficients (H), and mean number (±SEM) (I) of mobile SVs in a 200 μm × 200 μm area of a group of cultured mice hippocampal neurons. Data are means ± SEM; n = 4 independent experiments per group. *P < 0.05, **P < 0.01 by Student’s t test (H and I) or analysis of variance (ANOVA) and Tukey’s post hoc test (C and E).

  • Fig. 3 sAβ1–42 inhibits the synapsin and actin binding through synapsin phosphorylation at Ser9.

    (A) Relative amount of phosphosynapsin (P-syn) to synapsin I (syn I) in cultured rat hippocampal neurons at DIV16. Blots are representative of four experiments. (B) Phosphosynapsin relative to synapsin I in cultured mice hippocampal neurons (DIV16) from LT and 5XFAD. Blots are representative of four experiments. (C) Representative blot from three independent experiments assessing coimmunoprecipitation of actin with synapsin I from cultured rat hippocampal neuron lysates at DIV16. Aβ1–42 ab (1), Aβ1–42-specific antibody (Aβ1–42-1); IgG, immunoglobulin G. (D) Mean amount of actin coimmunoprecipitated with synapsin I, relative to control group; n = 3 blots. (E) Relative amount of phosphosynapsin to synapsin I. Data were normalized to the control group; n = 3 blots. (F) Immunoblots of transfected human embryonic kidney (HEK) 293T cells to confirm expression of wild-type (WT) or mutant (S9A) synapsin Ia. (G and H) Neurons were cotransfected with GFP-synaptophysin and mCherry–synapsin Ia (WT or S9A) and treated with either vehicle or sAβ1–42 (200 nM). MSD curves versus time (G) and diffusion coefficients (H) were then assessed from more than or equal to seven independent experiments. Data are means ± SEM; *P < 0.05, **P < 0.01 by Student’s t test (A, B, D, and E) or ANOVA and Tukey’s post hoc test (H).

  • Fig. 4 The inhibition of CaMKK prevents the sAβ1–42-induced hyperphosphorylation of synapsin and CaMKIV.

    (A) Representative Western blots of phosphosynapsin or phospho-CaMKIV in cultured rat hippocampal neurons at DIV16 treated as indicated for 2 hours. (B) Average relative amount of phosphosynapsin or phospho-CaMKIV relative to the respective total protein, each normalized to the control group. n = 6 blots. (C to E) Cultured rat hippocampal neurons were cotransfected with sPH-AP and BirA-ER, treated as indicated, and labeled with streptavidin-conjugated QD 605. (C) Representative [DIFF(N − 1)] kymographs showing the trafficking of QD 605–loaded SVs (diagonal lines) in each group; scale bars, 20 s (right) and 10 μm (bottom). (D) MSD curves versus time (D) and diffusion coefficients (E) were calculated from 1 min of tracking of QD-loaded intersynaptic vesicles. n = 4 independent experiments per group. (F) Immunoblot assay confirming the expression of Flag-tagged CaMKIV and dominant inhibitory CaMKIV (K75E) in HEK-293T cells. (G and H) Cultured rat hippocampal neurons were transfected with GFP-synaptophysin and treated as indicated. Representative kymographs (G) and diffusion coefficients (H) assessing the trafficking of GFP-synaptophysin–expressing SVs (diagonal lines) in each group. Scale bars, 10 s (right) and 20 μm (bottom). Data are means ± SEM; n = 4 independent experiments per group; *P < 0.05, **P < 0.01 by ANOVA and Tukey’s post hoc test. Asterisks at the bottom of the bars in (B) and (E) represent the significance compared to the control.

  • Fig. 5 sAβ1–42 increases the size of the resting but not that of the recycling pool.

    (A) Representative pseudocolor images of vesicular glutamate transporter conjugated with ecliptic GFP (vGlut-pH)–transfected presynaptic boutons in cultured rat hippocampal neurons before and after 1800 APs [20 Hz, with bafilomycin A1 (Baf)], followed by NH4Cl. Scale bar, 10 μm. (B) Average time course of vGlut-pH fluorescence traces. Each pool size was normalized to the total vesicle pool size. (C and D) Average fraction values of recycling pool (C) and resting pool (D). (E to H) Distribution within a single bouton of recycling versus resting pool size. Slope was fitted by the linear plot. (I to K) Mean values of the raw intensity without normalization showing recycling pool size (I), resting pool size (J), and total pool size (K). Data are means ± SEM; n= 4 to 6 independent experiments per group (n > 300 boutons per independent experiment for each group). **P < 0.01 by ANOVA and Tukey’s post hoc test.

  • Fig. 6 Inhibition of CaMKIV phosphorylation rescues the sAβ1–42-induced presynaptic defects.

    (A and B) GFP-synaptophysin–transfected rat hippocampal neurons at DIV16 were preincubated for 2 hours with sAβ1–42 alone or sAβ1–42 with STO-609 and then treated with foskolin (50 μM). Shown are time-lapse images (A) and the mean number of (left three graphs) and relative change (far right) in presynaptic boutons per 100-μm axon length (B). All three independent experiments for each group are noted (gray lines) along with the mean (bars; black lines). Solid arrowheads mark newly formed presynaptic boutons. Scale bar, 5 μm. (C) Box-and-whiskers graph of presynaptic boutons stained with synaptophysin antibody. n ≥ 19 independent experiments per group (n = ~70,000 boutons assessed in each group). Data are means ± SEM; *P < 0.05, **P < 0.01 by Student’s t test (B) or ANOVA and Tukey’s post hoc test (C).

  • Fig. 7 Model of the mechanism by which sAβ1–42 impairs the intersynaptic vesicular movements.

    Mobile SVs destined to the intersynaptic pool bind to the actin through the SV-synapsin-actin complex. By forming this complex, SVs can move to intersynaptic spaces because actin is suggested to function to convey mobile SVs through microtubule-based axonal transport machinery. Our data suggest that sAβ1–42 impedes Ca2+ clearance, which activates CaMKK-to-CaMKIV pathway, and subsequently phosphorylates synapsin, resulting in dissociation of the SVs-synapsin-actin complex. Hence, sAβ1–42 prevents SVs from entering into the vesicle pool for intersynaptic trafficking, which could prevent new synapse formation and synaptic plasticity.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/487/eaam8661/DC1

    Fig. S1. Forskolin induces presynaptic potentiation (cLTP).

    Fig. S2. Acute treatment of nanomolar sAβ1–42 does not induce cell death or alter the neuronal excitability.

    Fig. S3. Determination of the QD 605–labeling method.

    Fig. S4. Additional confirmation of sAβ1–42-induced inhibition of the intersynaptic vesicle mobility and chronic sAβ1–42 treatment effect.

    Fig. S5. The effect of γ-secretase inhibitor and naturally secreted Aβ on intersynaptic vesicular trafficking.

    Fig. S6. Nanomolar concentration of sAβ1–42 does not disrupt the mitochondria transport or microtubule structures.

    Fig. S7. Nonradioactive CaMKIV activity assay.

    Fig. S8. Inhibition of CaMKK-to-CaMKIV pathway rescues the sAβ1–42-induced defects.

    Fig. S9. PKA-dependent synapsin Ser9 phosphorylation.

    Fig. S10. sAβ1–42 increases presynaptic Ca2+.

    Table S1. Numerical data underlying each graph in the figures.

  • Supplementary Materials for:

    Activation of CaMKIV by soluble amyloid-β1–42 impedes trafficking of axonal vesicles and impairs activity-dependent synaptogenesis

    Daehun Park, Myeongsu Na, Jung Ah Kim, Unghwi Lee, Eunji Cho, Mirye Jang, Sunghoe Chang*

    *Corresponding author. Email: sunghoe{at}snu.ac.kr

    This PDF file includes:

    • Fig. S1. Forskolin induces presynaptic potentiation (cLTP).
    • Fig. S2. Acute treatment of nanomolar sAβ1–42 does not induce cell death or alter the neuronal excitability.
    • Fig. S3. Determination of the QD 605–labeling method.
    • Fig. S4. Additional confirmation of sAβ1–42-induced inhibition of the intersynaptic vesicle mobility and chronic sAβ1–42treatment effect.
    • Fig. S5. The effect of γ-secretase inhibitor and naturally secreted Aβ on intersynaptic vesicular trafficking.
    • Fig. S6. Nanomolar concentration of sAβ1–42 does not disrupt the mitochondria transport or microtubule structures.
    • Fig. S7. Nonradioactive CaMKIV activity assay.
    • Fig. S8. Inhibition of CaMKK-to-CaMKIV pathway rescues the sAβ1–42-induced defects.
    • Fig. S9. PKA-dependent synapsin Ser9 phosphorylation.
    • Fig. S10. sAβ1–42 increases presynaptic Ca2+.
    • Table S1. Numerical data underlying each graph in the figures.

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    Citation: D. Park, M. Na, J. A. Kim, U. Lee, E. Cho, M. Jang, S. Chang, Activation of CaMKIV by soluble amyloid-β1–42 impedes trafficking of axonal vesicles and impairs activity-dependent synaptogenesis. Sci. Signal. 10, eaam8661 (2017).

    © 2017 American Association for the Advancement of Science