Research ArticleNeurodegeneration

Pin1 mediates Aβ42-induced dendritic spine loss

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Science Signaling  20 Mar 2018:
Vol. 11, Issue 522, eaap8734
DOI: 10.1126/scisignal.aap8734
  • Fig. 1 Pin1 KO causes dendritic spine loss in vitro and vivo.

    (A) Total spine counts in Pin1fl/fl neurons after transfection with Td-Tomato and either NLS-GFP [wild-type (WT), black] or NLS-GFP-Cre (KO, gray). Neurons were fixed at DIV21. (B) As described in (A), in DIV21 neurons transduced with TAT-GFP (“G”) or TAT-Pin1 (“P”). (C) Representative images of GFP immunofluorescence in hippocampi of AAV-GFP– or AAV-GFP-Cre–injected 3-month-old Pin1fl/fl mice at 3, 6, 9 and 12 DPI. Sections were counterstained with DAPI. Scale bar, 1000 μm. (D) Total synapse counts at 3, 6, 9, and 12 DPI from 24 mouse hippocampi injected at 3 months old with either AAV-GFP (WT, black) or AAV-GFP-Cre (KO, gray). (E) Imaging of GFP (green) and antibody to synaptophysin (Syn; red) in dendritic spines from hippocampal neurons described in (C). A representative composite Z-stack image of three 0.33-μm-thick step sizes is shown. Scale bar, 10 μm. Data are means ± SEM; n > 100 spines from ≥15 images and ≥3 coverslips per condition were counted. *P < 0.05 by an unpaired t test (A) or Fisher’s least significant difference (LSD) test following two-way analysis of variance (ANOVA) (B and C). NS, not significant.

  • Fig. 2 42 inhibits Pin1 isomerase activity.

    (A) Western blotting for Pin1, SNAP25, βIII-tubulin, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) in soluble fractions of temporal cortex lysates from three Alzheimer’s disease (AD) patients and three age-matched control individuals (not diagnosed with dementia; Con). (B) Pin1 activity assay in equal amounts of soluble protein extract from control and AD patient brain tissues described in (A). (C) Pin1 activity assay with murine SNs that were untreated (red) or treated for 10 min with control brain extract (+HC; black), AD brain extract (+AD; purple), or Aβ42 (green). (D) Pin1 activity assay in SNs that were either untreated (red) or treated with juglone, PiB, TAT-WW, TAT-W34A, or Aβ42. Data are means of n ≥ 8 replicates per treatment. *P < 0.05 by Fisher’s LSD test following two-way ANOVA (B to D).

  • Fig. 3 Calcineurin interacts with Pin1 and mediates Aβ42 signaling.

    (A and B) Total spine counts for DIV21 WT (A) or DIV21 KO (B) neurons transduced with TAT-GFP (“G”) or TAT-Pin1 (“P”) ± Aβ42 (Aβ). Data are means ± SEM; n > 100 spines from ≥15 images and ≥3 coverslips per condition were counted. *P < 0.05 by Fisher’s LSD test following two-way ANOVA. (C) Western blot of catalytic and regulatory subunits of calcineurin in Pin1 immunoprecipitations from SNs prepared from P28 mice. (D) Calcineurin (CN) activity in SNs that were either untreated (−) or treated with Aβ42 (Aβ) for 10 min before lysis. Data are means ± SEM; n = 3 biological replicates. *P < 0.05 by an unpaired t test. (E) Pin1 activity assay in SNs that were either untreated (SN) or treated with Aβ42, FK506, FK506 + Aβ42, CsA, or CsA + Aβ42 before lysis. Data are means from n ≥ 8 replicates per treatment. *P < 0.05 by Fisher’s LSD test following two-way ANOVA.

  • Fig. 4 Dephosphorylation of Pin1 at Ser111 inhibits Pin1 activity.

    (A) Total spine counts for DIV21 WT (black) or KO (gray) neurons ± FK506 (FK) ± Aβ42 (Aβ). (B) Pin1 activity assay in SNs treated with TAT-GFP (GFP), TAT-Pin1-S111D (S111D), or TAT-Pin1-S111A (S111A) ± Aβ42 before lysis. Data are means from n ≥ 8 replicates per treatment. *P < 0.05 by Fisher’s LSD test following two-way ANOVA. (C and D) Total spine counts in DIV21 WT (C) and KO (D) neurons transduced with TAT-GFP (“G”), TAT-Pin1 (“P”), TAT-Pin1-S111A (“A”), or TAT-Pin1-S111D (“D”) ± Aβ42. Data are means ± SEM; n > 100 spines from ≥15 images and ≥3 coverslips per condition were counted. *P < 0.05 by Fisher’s LSD test following two-way ANOVA (A, C, and D).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/522/eaap8734/DC1

    Fig. S1. Pin1 loss causes spine loss.

    Fig. S2. Pin1 successfully recombined in mouse hippocampus after AAV-GFP-Cre.

    Fig. S3. Pin1 reconstitution restores mature spine counts in the presence of Aβ42.

    Fig. S4. FK506 restoration of spines requires Pin1.

    Fig. S5. Pin1-S111D restores spine counts in wild-type or knockout cells treated with Aβ42.

  • Supplementary Materials for:

    Pin1 mediates Aβ42-induced dendritic spine loss

    Nancy R. Stallings, Melissa A. O'Neal, Jie Hu, Ege T. Kavalali, Ilya Bezprozvanny, James S. Malter*

    *Corresponding author. Email: james.malter{at}utsouthwestern.edu

    This PDF file includes:

    • Fig. S1. Pin1 loss causes spine loss.
    • Fig. S2. Pin1 successfully recombined in mouse hippocampus after AAV-GFP-Cre.
    • Fig. S3. Pin1 reconstitution restores mature spine counts in the presence of Aβ42.
    • Fig. S4. FK506 restoration of spines requires Pin1.
    • Fig. S5. Pin1-S111D restores spine counts in wild-type or knockout cells treated with Aβ42.

    [Download PDF]


    © 2018 American Association for the Advancement of Science

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