Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model
- Department of Cell and Developmental Biology, Division of Biosciences, University College London, United Kingdom
- Division of Psychological Medicine and Clinical Neurosciences, Cardiff University, United Kingdom
- UK Dementia Research Institute, Cardiff University, United Kingdom
Figures
Figure 1
DKK3 mRNA and protein levels are increased in the human AD brain.
(A) Temporal cortex RNAseq dataset logistic regression shows that DKK3 mRNA levels are increased in AD cases relative to controls. Ordinal regression shows that DKK3 is differentially expressed for …
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Figure 1—source data 1
Uncropped western blot gels.
Raw and annotated WB images. The representative western blot images for Figure 1B and C are indicated within a blue square.
- https://cdn.elifesciences.org/articles/89453/elife-89453-fig1-data1-v1.zip
Figure 2 with 3 supplements
DKK3 localizes to dystrophic neurites around Aβ plaques, and DKK3 extracellular levels are increased in the brain of AD mouse models.
(A) Diagram of the components of an Aβ plaque (blue), astrocytes (orange), microglia (green), and dystrophic neurites (red). (B) Confocal images of DKK3 protein (red) and amyloid plaques stained …
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Figure 2—source data 1
Uncropped western blot gels.
Raw and annotated WB images. The representative western blot images for Figure 2E and F are indicated within a blue square.
- https://cdn.elifesciences.org/articles/89453/elife-89453-fig2-data1-v1.zip
Figure 2—figure supplement 1
DKK3 is present in principal neurons of the mouse hippocampus.
(A) Immunoblotting images show the presence or absence of DKK3 protein in hippocampal homogenates of WT and Dkk3-/-ApoE-/- (KO) mice demonstrate the specificity of the antibody against DKK3. Actin …
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Figure 2—figure supplement 1—source data 1
Uncropped western blot gels.
Raw and annotated WB images. The representative western blot images for Figure 2—figure supplement 1A are indicated within a blue square.
- https://cdn.elifesciences.org/articles/89453/elife-89453-fig2-figsupp1-data1-v1.zip
Figure 2—figure supplement 2
DKK3 accumulates in Aβ plaques in the hippocampus of J20 and NLGF mice and DKK3 levels are increased by Aβ.
(A) Confocal images of Aβ plaques (6E10, blue) and DKK3 (red) in the hippocampus of 12-month-old J20 and 8-month-old NLGF mice. Scale bar = 140 μm. (B) Confocal images show that DKK3 (red) …
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Figure 2—figure supplement 2—source data 1
Uncropped western blot gels.
Raw and annotated WB images. The representative western blot images for Figure 2—figure supplement 2F–G are indicated within a blue square.
- https://cdn.elifesciences.org/articles/89453/elife-89453-fig2-figsupp2-data1-v1.zip
Figure 2—figure supplement 3
cLTD, but not cLTP, increases the levels of extracellular DKK3.
(A, B) Hippocampal neurons were subjected to (A) chemical LTP (cLTP) or (B) chemical LTD (cLTD). DKK3 levels were analyzed in the supernatant (extracell) and cell lysates by western blot. …
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Figure 2—figure supplement 3—source data 1
Uncropped western blot gels.
Raw and annotated WB images. The representative western blot images for Figure 2—figure supplement 3A–D are indicated within a blue square.
- https://cdn.elifesciences.org/articles/89453/elife-89453-fig2-figsupp3-data1-v1.zip
Figure 3 with 1 supplement
Gain-of-function of DKK3 leads to opposing effects on the number of excitatory and inhibitory synapses in the hippocampus.
(A) Diagram depicting the treatment of hippocampal brain slices obtained from 3-month-old adult WT mice with vehicle (Ctrl) or recombinant DKK3 protein. Synapses were evaluated by confocal …
Figure 3—figure supplement 1
DKK3 triggers changes in excitatory and inhibitory synapses in the absence of cell death.
(A) Confocal images of the TUNEL assay (red) in the hippocampus CA3 area (DAPI in blue). Graph shows the number of cells positive for TUNEL. Scale bar = 20 μm (n=2 animals). (B) Confocal images show …
Figure 4 with 1 supplement
DKK3 regulates excitatory and inhibitory synapse number through the Wnt/GSK3β and Wnt/JNK pathways respectively.
(A) Diagram of the canonical Wnt pathway through inhibition of GSK3β (Wnt/GSK3β pathway), resulting in elevation of β-catenin and transcriptional activation via TCF/LEF. (B) Confocal images show …
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Figure 4—source data 1
Uncropped western blot gels.
Raw and annotated WB images. The representative western blot images for Figure 4E are indicated within a blue square.
- https://cdn.elifesciences.org/articles/89453/elife-89453-fig4-data1-v1.zip
Figure 4—figure supplement 1
Activation of the Wnt/GSK3β pathway blocks DKK3-induced excitatory synapse loss whereas inhibition of the Wnt/JNK pathway blocks the effect of DKK3 on inhibitory synapses.
(A) Confocal images show excitatory synapses (co-localized vGLUT1 in green and PSD-95 in red) in the CA3 SR after treatment with vehicle (Ctrl) or DKK3 in the absence or presence of CHIR99021 (CHIR) …
Figure 5
In vivo loss-of-function of DKK3 decreases inhibitory synapses but does not affect excitatory synapses in the wild-type hippocampus.
(A) Diagram showing the experimental design. Three-month-old WT mice were injected with AAV9 scrambled (Scr) or Dkk3 shRNA in the CA3 region. Confocal images showing GFP (green) and DKK3 (red) in …
Figure 6 with 1 supplement
In vivo loss-of-function of DKK3 ameliorates synaptic changes in the hippocampus of J20 mice before and after Aβ plaque formation.
(A) Diagram depicting the experimental design. In green, 3-month-old WT and J20 mice were injected bilaterally with AAV9-Scr shRNA or AAV9-Dkk3 shRNA in the CA3 region. The density of synapses was …
Figure 6—figure supplement 1
In vivo DKK3 loss-of-function affects pre- and postsynaptic markers but does not affect Aβ plaque pathology or Dkk1 expression in the mouse hippocampus.
(A) Confocal images of the CA3 area of the hippocampus show that endogenous DKK3 is downregulated in the hippocampus of wildtype and J20 injected with Dkk3 shRNA AAV9 virus. Scale bar = 38 μm. (B, C)…
Figure 7 with 1 supplement
In vivo loss-of-function of DKK3 improves spatial memory in J20 mice.
(A) Diagram depicting that 3-month-old WT and J20 mice were injected bilaterally with AAV9-Scr shRNA or AAV9-Dkk3 shRNA in the CA3 area of the hippocampus. One month later, the behavior of animals …
Figure 7—figure supplement 1
In vivo DKK3 loss-of-function does not affect locomotion or anxiety.
(A) Open-field task. Representative traces of the open-field. Graphs show the total distance traveled in the open-filed (cm, left, Kruskal-Wallis test), mean speed (cm/s, middle, Two-way ANOVA) and …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mouse) | C57BL/6 J | The Jackson Laboratory | JAX: 000664 RRID: MGI:2159769 | WT mice |
| Strain, strain background (Mouse) | B6.Cg-Zbtb20Tg(PDGFB-APPSwInd)20Lms/2Mmjax (hAPP-J20) | The Jackson Laboratory | J20 mice | |
| Strain, strain background (Mouse) | Apptm3.1Tcs/Apptm3.1Tcs (APPNL-G-F) | Professor Takaomi Saido | Saito et al., 2014 | NLGF mice |
| Biological sample (Rat) | Primary neurons | Charles River | RRID: MGI:5651135 | Freshly isolated from Sprague-Dawley E18 embryos |
| Biological sample (Mouse) | Dkk3 knock-out brain tissue | Professor Qingbo Xu | Yu et al., 2017 | |
| Biological sample (Human) | Control and patient brain tissue | Cambridge Brain Bank (CBB), Division of the Human Research Tissue Bank, Addenbrooke’s Hospital, Cambridge, UK | ||
| Antibody | Mouse monoclonal Anti- Aβ (6E10 clone) | Biolegend | Cat# 803001, RRID: AB_2564653 | 1:1,000 for IF and WB |
| Antibody | Rabbit monoclonal Anti-Aβ (6E10 clone, chimeric) | Novus Biologicals | Cat# NBP2-62566, RRID: AB_2917960 | 1:1,000 for IF |
| Antibody | Chicken polyclonal anti-Bassoon | Synaptic Systems | Cat# 141 016, RRID: AB_2661779 | 1:1,000 for IF |
| Antibody | Goat polyclonal anti- Mouse DKK-3 Systems | R&D Systems | Cat# AF948, RRID: AB_355734 | 1:1,000 for IF and WB |
| Antibody | Goat polyclonal anti- Human DKK-3 | R&D Systems | Cat# AF1118, RRID: AB_354610 | 1:1,000 for WB |
| Antibody | Rabbit monoclonal anti-GAPDH | Abcam | Cat# ab181602, RRID: AB_2630358 | 1:5,000 for WB |
| Antibody | Rabbit polyclonal anti-Gephyrin | Synaptic Systems | Cat# 147 002, RRID: AB_2619838 | 1:500 for IF |
| Antibody | Chicken polyclonal anti-Glial Fibrillary Acidic Protein (GFAP) | Millipore | Cat# AB5541, RRID: AB_177521 | 1:500 for IF |
| Antibody | Rabbit monoclonal anti-GluA1 | Cell Signaling Technology | Cat# 13185, RRID: AB_2732897 | 1:1,000 for WB |
| Antibody | Rabbit monoclonal anti-phospho GluA1 Serine 845 | Cell Signaling Technology | Cat# 8084, RRID: AB_10860773 | 1:1,000 for WB |
| Antibody | Chicken polyclonal Anti-GFP | Millipore | Cat# 06–896, RRID: AB_310288 | 1:500 for IF |
| Antibody | Rabbit polyclonal anti-Homer1 | Synaptic Systems | Cat# 160 003, RRID: AB_887730 | 1:1,000 for IF |
| Antibody | Rabbit polyclonal anti-Iba1 | FUJIFILM Wako Shibayagi | Cat# 019–1974,1 RRID: AB_839504 | 1:1,000 for IF |
| Antibody | Rabbit polyclonal anti- SAPK/JNK | Cell Signaling Technology | Cat# 9252, RRID: AB_2250373 | 1:1,000 for WB |
| Antibody | Mouse monoclonal anti-Phospho-SAPK/JNK (Thr183/Tyr185) | Cell Signaling Technology | Cat# 9255, RRID: AB_2307321 | 1:500 for WB |
| Antibody | Rat monoclonal anti-LAMP1 | DSHB | Cat# 1D4B, RRID: AB_2134500 | 1:250 for IF |
| Antibody | Rabbit monoclonal Anti- NeuN (D3S3I) | Cell Signaling Technology | Cat# 12943, RRID: AB_2630395 | 1:1,000 for IF |
| Antibody | Rabbit polyclonal Anti- Neurofilament Heavy | Abcam | Cat# ab8135, RRID: AB_306298 | 1:5,000 for IF |
| Antibody | Mouse monoclonal Anti-PSD-95 (7E3-1B8) | Thermo Fisher Scientific | Cat# MA1-046, RRID: AB_2092361 | 1:500 for IF |
| Antibody | Mouse monoclonal anti-Tubulin | Sigma-Aldrich | Cat# T9026, RRID: AB_477593 | 1:5,000 for WB |
| Antibody | Guinea pig polyclonal anti- vesicular GABA Transporter (vGAT) | Synaptic Systems | Cat# 131 004, RRID: AB_887873 | 1:500 for IF |
| Antibody | Guinea pig polyclonal Anti- Vesicular Glutamate Transporter 1 (vGLUT1) | Millipore | Cat# AB5905, RRID: AB_2301751 | 1:2,000 for IF |
| Antibody | Mouse monoclonal Anti-Vinculin | Sigma Aldrich | Cat# v4505, RRID: AB_477617 | 1:2,000 for WB |
| Antibody | Goat polyclonal anti-Wnt7a/b | R&D Systems | Cat# AF3460, RRID: AB_2304437 | 1:1,000 for IF |
| Antibody | Mouse monoclonal Anti- beta Catenin | BD Biosciences | Cat# 610153, RRID: AB_397554 | 1:1,000 for IF |
| Antibody | HRP mouse monoclonal anti-beta Actin | Abcam | Cat# ab8224, RRID: AB_449644 | 1:10,000 for WB |
| Sequence-based reagent | shRNA Scrambled sequence | This paper | shRNA Scrambled sequence | 5’-CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGGTTTTT-3’ |
| Sequence-based reagent | shRNA Dkk3 sequence | This paper | shRNA Dkk3 sequence | 5’-GAGCCATGAATGTATCATTGACTCGAGTCAATGATACATTCATGGCTCTTTTT-3 |
| Peptide, recombinant protein | Recombinant DKK3 protein | R&D Systems | Cat# 1118-DK | 150 ng/ml or 200 ng/ml |
| Peptide, recombinant protein | HFIP-treated Amyloid β 1–42 peptide | Bachem | Cat# 4090148 | 200 nM |
| Peptide, recombinant protein | HFIP-treated Reverse Amyloid β 42–1 | Bachem | Cat# 4107743 | 200 nM |
| Commercial assay or kit | TUNEL assay | Abcam | Cat# ab66110 | |
| Commercial assay or kit | BCA kit | Thermo Fisher Scientific | Cat# 23225 | |
| Commercial assay or kit | DirectZol Miniprep RNA Kit | Zymo Research | Cat# R2052 | |
| Commercial assay or kit | RevertAid H Minus First Strand cDNA Synthesis kit | Thermo Fisher Scientific | Cat# K1632 | |
| Commercial assay or kit | GoTaq qPCR Master Mix | Promega | Cat# A6002 | |
| Chemical compound, drug | Fluoromount-G | Southern Biotech | Cat# 0100–01 | |
| Chemical compound, drug | FluorSave Reagent | Merck Millipore | Cat# 345789 | |
| Chemical compound, drug | TRIzol Reagent | Thermo Fisher Scientific | Cat# 15596018 | |
| Chemical compound, drug | DAPI | Thermo Fisher Scientific | Cat# 62248 | 1:50,000 dilution |
| Chemical compound, drug | Thioflavin S | Sigma Aldrich | Cat# T1892 | 1% dilution |
| Chemical compound, drug | BIO | Calbiochem | Cat# 361550 | 0.5 μM |
| Chemical compound, drug | CC-930 | Cayman Chemical | Cat# CAY22466 | 60 nM |
| Chemical compound, drug | CHIR99021 | Calbiochem | Cat# 361559 | 1 µM |
| Chemical compound, drug | NMDA | Tocris Bioscience | Cat# 0114 | 20 μM |
| Chemical compound, drug | Glycine | Fisher Chemical | Cat# 10070150 | 200 μM |
| Chemical compound, drug | D-APV | Tocris Bioscience | Cat# 0106 | 20 μM or 50 μM |
| Chemical compound, drug | Brefeldin A | Biolegend | Cat# 420601 | 10 μg/ml |
| Software, algorithm | ImageJ-FIJI | NIH | https://imagej.net/software/fiji/ RRID:SCR_001935 | |
| Software, algorithm | GraphPad Prism version 8 | GraphPad software | https://www.graphpad.com/scientific-software/prism RRID:SCR_002798 | |
| Software, algorithm | Image Lab | Bio-Rad | https://www.bio-rad.com/en-uk/product/image-lab-software?ID=KRE6P5E8Z RRID:SCR_014210 | |
| Software, algorithm | Volocity 3D Image Analysis version 6.5.1 | Quorum Technologies | https://www.volocity4d.com/ RRID:SCR_002668 | |
| Software, algorithm | CFX Manager Software 3.1 | Bio-Rad | https://www.bio-rad.com/en-uk/sku/1845000-cfx-manager-software?ID=1845000 | |
| Software, algorithm | SMART Video tracking | Panlab | https://www.panlab.com/en/products/smart-video-tracking-software-panlab RRID:SCR_002852 | |
| Software, algorithm | WinEDR | University of Strathclyde | http://spider.science.strath.ac.uk/sipbs/software_ses.htm | |
| Software, algorithm | WinWCP | University of Strathclyde | http://spider.science.strath.ac.uk/sipbs/software_ses.htm RRID:SCR_014713 | |
| Software, algorithm | RNASeQC | DeLuca et al., 2012 | https://software.broadinstitute.org/cancer/cga/rna-seqc | |
| Other | Confocal microscope | Leica | SP8 | Materials and Methods, Image acquisition and analyses |
| Other | Vibratome | Leica | VT100S | Materials and Methods, Treatment of acute hippocampal slices and Electrophysiology |
| Other | Cryostat | Leica | CM1850 | Materials and Methods, Tissue processing for immunofluorescence microscopy |
| Other | Patch-clamp amplifier | Molecular Devices | Axopatch 200B | Materials and Methods, Electrophysiology |
Additional files
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Supplementary file 1
Human brain samples information.
M=Male, F=Female, PMI = Post-mortem interval.
- https://cdn.elifesciences.org/articles/89453/elife-89453-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/89453/elife-89453-mdarchecklist1-v1.pdf
