KIF2C regulates synaptic plasticity and cognition in mice through dynamic microtubule depolymerization
- Department of Neurobiology and Department of Rehabilitation of the Children’s Hospital, Zhejiang University School of Medicine, China
- NHC and CAMS Key Laboratory of Medical Neurobiology, Ministry of Education Frontier Science Center for Brain Research and Brain Machine Integration, School of Brain Science and Brain Medicine, Zhejiang University, China
- Department of Orthopedic Surgery, the Second Affiliated Hospital, Zhejiang University School of Medicine, China
- Department of Physiology and Department of Neurology of First Affiliated Hospital, Zhejiang University School of Medicine, China
- JNU-HKUST Joint Laboratory for Neuroscience and Innovative Drug Research, Jinan University, China
- Division of Life Science and The Brain and Intelligence Research Institute, The Hong Kong University of Science and Technology, China
- Innovative Institute of Basic Medical Sciences of Zhejiang University (Yuhang), China
Figures
Figure 1
Expression of KIF2C in the central nervous system.
(A) Expression of KIF2C in different mouse brain regions by immunoblotting. (B) Expression of KIF2C and EB3 in mouse hippocampus at different day in vitro. (C) Developmental expression patterns of …
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Figure 1—source data 1
Original files of blots with the relevant bands.
- https://cdn.elifesciences.org/articles/72483/elife-72483-fig1-data1-v1.zip
Figure 2 with 1 supplement
KIF2C displays translocation after cLTP and is required for synaptic structure plasticity.
(A) Electrophoresis of kif2c and gapdh amplicons from individual control and shkif2c hippocampal neurons. Histograms show percentage changes of kif2c mRNA levels (% of control, **p < 0.01, n = 6, …
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Figure 2—source data 1
Values for kif2c mRNA levels; Values for spine density and spine head width before or after cLTP stimulation in control and shkif2c neurons; Values for KIF2C synaptic expression before or after cLTP stimulation.
- https://cdn.elifesciences.org/articles/72483/elife-72483-fig2-data1-v1.xlsx
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Figure 2—source data 2
Original files of gels and blots with the relevant bands.
- https://cdn.elifesciences.org/articles/72483/elife-72483-fig2-data2-v1.zip
Figure 2—figure supplement 1
Miniature excitatory synaptic currents (mEPSCs) recordings of control and shkif2c neurons.
(A) Total fractions from mCherry-scramble shRNA (control) and mCherry- shkif2c (shkif2c) hippocampus neurons were probed with antibodies to KIF2C, mCherry, and GAPDH. GAPDH was internal controls. …
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Figure 2—figure supplement 1—source data 1
Values for KIF2C protein levels.
Values for mEPSCs of control and shkif2c neurons; Values for kif2c mRNA levels; Values for mEPSCs of control and shkif2c neurons before and after cLTP.
- https://cdn.elifesciences.org/articles/72483/elife-72483-fig2-figsupp1-data1-v1.xlsx
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Figure 2—figure supplement 1—source data 2
Original files of blots with the relevant bands.
- https://cdn.elifesciences.org/articles/72483/elife-72483-fig2-figsupp1-data2-v1.zip
Figure 3
Knockdown KIF2C impairs synaptic transmission and plasticity.
(A) mEPSCs recorded from control and shkif2c CA1 pyramidal neurons. n = 10 per group, n.s., p > 0.05, **p < 0.01, Student’s t-test; For the cumulative probability curves, *p < 0.05, ****p < 0.0001, …
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Figure 3—source data 1
Values for mEPSCs of control and shkif2c neurons.
Values for PPR ratio; Values for input-output curve of AMPAR and NMDAR mediated EPSCs between control and shkif2c neurons; Values for percentage changes of EPSCs amplitudes in control and shkif2c CA1 pyramidal neurons.
- https://cdn.elifesciences.org/articles/72483/elife-72483-fig3-data1-v1.xlsx
Figure 4 with 1 supplement
Abnormal spine formation in KIF2C conditional knockout mice.
(A) Targeting strategy to generate kif2cflox/flox mice. Two LoxP sites were inserted into intron 1–2 and 8–9. (B) Nissl staining of adult mice brain coronal sections and magnified images of …
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Figure 4—source data 1
Values for CA1 and CA3 thickness in WT and cKO mice; Values for Sholl analysis and spine density; Values for PSD thickness and length.
- https://cdn.elifesciences.org/articles/72483/elife-72483-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
kif2cflox/flox;NestinCre mice.
(A) kif2cflox/flox;NestinCre mouse was crossed with Ai9 reporter mouse lines and the expression of Cre-recombinase was characterized by observing tdTomato reporter in Ai9;NestinCre mice (1 month). …
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Figure 4—figure supplement 1—source data 1
Values for kif2c mRNA levels; Values for brain weight and body weight.
- https://cdn.elifesciences.org/articles/72483/elife-72483-fig4-figsupp1-data1-v1.xlsx
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Figure 4—figure supplement 1—source data 2
Original files of gels with the relevant bands.
- https://cdn.elifesciences.org/articles/72483/elife-72483-fig4-figsupp1-data2-v1.zip
Figure 5
KIF2C deficiency impairs synaptic transmission and plasticity.
(A) mEPSCs recorded from WT and cKO CA1 pyramidal neurons. n = 21 neurons per group; ***p < 0.001, Student’s t-test; For the cumulative probability curves, n.s. p > 0.05, ****p < 0.0001, …
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Figure 5—source data 1
Values for mEPSCs of WT and cKO neurons.
Values for PPR ratio; Values for input-output curve of AMPAR and NMDAR mediated EPSCs between WT and cKO neurons; Values for NMDAR and AMPAR subunit surface expression; Values for percentage changes of fEPSCs amplitudes in WT and cKO neurons.
- https://cdn.elifesciences.org/articles/72483/elife-72483-fig5-data1-v1.xlsx
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Figure 5—source data 2
Original files of blots with the relevant bands.
- https://cdn.elifesciences.org/articles/72483/elife-72483-fig5-data2-v1.zip
Figure 6 with 1 supplement
Abnormal MT dynamics causes deficit in synaptic transmission and plasticity.
(A) Time-lapse recordings of EB3-tdtomato-infected neurons showing EB3-tdtomato comets in dendrite of WT and cKO hippocampus neuron. Subsequent images show low-pass-filtered time series in row. …
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Figure 6—source data 1
Values for EB3 speed velocity, MT growth speed, MT growth lifetime, and MT growth length.
Values for MT invasion frequency; Values for number of spines containing MTs before and after BDNF treatment; Values for mEPSCs of cKO+ control, cKO+ KIF2 C(WT), and cKO+ KIF2 C(G491A); Values for percentage changes of fEPSCs amplitudes of cKO+ control, cKO+ KIF2 C(WT), and cKO+ KIF2 C(G491A).
- https://cdn.elifesciences.org/articles/72483/elife-72483-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
KIF2C(WT) and KIF2C(G491A) expression.
(A) GFP vector, GFP-KIF2C(WT), and GFP-KIF2C(G491A) mutant were transfected into HEK 293T cells and α-tubulin was stained. White arrow points to transfected cells, yellow arrows point to intact …
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Figure 6—figure supplement 1—source data 1
Original files of blots with the relevant bands.
- https://cdn.elifesciences.org/articles/72483/elife-72483-fig6-figsupp1-data1-v1.zip
Figure 7 with 1 supplement
KIF2C cKO mice exhibit abnormal cognitive behaviors.
(A) Y maze test from WT and cKO. n = 13 per genotype. n.s., p > 0.05, *p < 0.05, **p < 0.01, Student’s t-test. (B–C) Cued fear conditioning test. (B), WT and cKO mice showed no significant …
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Figure 7—source data 1
Values for Y-maze test of WT and cKO mice.
Values for freezing levels during fear conditioning test of WT and cKO mice; Values for close interaction time during three-chamber test of WT and cKO mice; Values for Y-maze test of cKO+ mCherry, cKO+ KIF2 C(WT), cKO+ mNeonGreen, and cKO+ KIF2 C(G491A) mice; Values for close interaction time during three-chamber test of cKO+ mCherry, cKO+ KIF2 C(WT), cKO+ mNeonGreen, and cKO+ KIF2 C(G491A) mice.
- https://cdn.elifesciences.org/articles/72483/elife-72483-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
WT and KIF2C cKO mice behavioral tests.
(A) Open field test from WT and cKO mice. Quantification of velocity, distance of activities and exploration time. n = 13 per genotype. Student’s t-test. (B) Elevated-zero maze test. Time in open …
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Figure 7—figure supplement 1—source data 1
Values for moving distance, velocity and center zone exploration time in the open-field test.
Values for time in open quadrants, open entrances, number of 4 paws in open quadrants, number of head dips and number of stretch attendance postures (SAP) during elevated-zero maze test; Values for grooming time; Values for latency to platform and time in quadrants during Morris water maze; Values for freezing time change during fear conditioning test; Values for PPI; Values for time in each chamber during three-chamber test; Values for arm entries during Y-maze test.
- https://cdn.elifesciences.org/articles/72483/elife-72483-fig7-figsupp1-data1-v1.xlsx
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus, male/female) | C57BL/6 | Shanghai SLAC Laboratory Animal C.,Ltd | ||
| Strain, strain background (Mus musculus, male/female) | kif2cflox/flox mouse | Model Animal Research Center of Nanjing University | ||
| Strain, strain background (Mus musculus, male/female) | NestinCre mouse | Zhejiang University | Wang et al., 2019 | |
| Cell line (Homo-sapiens) | HEK 293T | ATCC | CRL-3216 | |
| Genetic reagent (virus) | pAKD-CMV-bGlobin-mcherry-H1-shRNA | ObioTechnology | ||
| Genetic reagent (virus) | pAAV-CMV-KIF2C-3FLAG-P2A-mNeonGreen-CW3SL | ObioTechnology | ||
| Genetic reagent (virus) | pAAV-CMV-KIF2C(G491A)–3FLAG-P2A-mNeonGreen-CW3SL | ObioTechnology | ||
| Genetic reagent (virus) | rAAV-TRE3G- KIF2C-3*Flag –2A-mCherry-WPRE-pA | BrainVTA | ||
| Antibody | Anti-KIF2C, (rabbit polyclonal) | Proteintech | Cat# 12139–1-AP, RRID:AB_2877829 | IF (1:500), WB (1:500) |
| Antibody | Anti-PSD-95, (mouse monoclonal) | Abcam | Cat# ab2723, RRID:AB_303248 | IF (1:1000) WB (1:1000) |
| Antibody | Anti-MAP2, (chicken polyclonal) | Abcam | Cat# ab5392, RRID:AB_2138153 | IF (1:2000) |
| Antibody | anti-GFP (rabbit polyclonal) | Abcam | Cat# ab6556, RRID:AB_305564 | IF (1:2000) |
| Antibody | Anti-α-tubulin, (rabbit monoclonal) | Abcam | Cat# ab52866, RRID:AB_869989 | IF (1:1000) |
| Antibody | Anti-mCherry, (rabbit polyclonal) | Abcam | Cat# ab167453, RRID:AB_2571870 | WB (1:2000) |
| Antibody | Anti-EB3, (rabbit monoclonal) | Abcam | Cat# ab157217, RRID:AB_2890656 | WB (1:10,000) |
| Antibody | Anti-α-tubulin, tyrosinated, (rat monoclonal) | Millipore | Cat# MAB1864-I, RRID:AB_2890657 | IF (1:1000) |
| Antibody | Anti-synaptophysin, (mouse monoclonal) | Millipore | Cat# MAB368, RRID:AB_94947 | IF (1:1000) WB (1:2000) |
| Antibody | Anti-GAPDH, (mouse monoclonal) | Millipore | Cat# MAB374, RRID:AB_2107445 | WB (1:10,000) |
| Antibody | Anti-GluA1, (rabbit monoclonal) | Millipore | Cat# 04–855, RRID:AB_1977216 | WB (1:1000) |
| Antibody | Anti-GluA2, (mouse monoclonal) | Millipore | Cat# MAB397, RRID:AB_2113875 | WB (1:2000) |
| Antibody | Anti-GluN2A, (rabbit polyclonal) | Cell Signaling Technology | Cat# 4205, RRID:AB_2112295 | WB (1:2000) |
| Antibody | Anti- GluN2B, (rabbit polyclonal) | Cell Signaling Technology | Cat# 4207, RRID:AB_1264223 | WB (1:2000) |
| Antibody | Anti-GluN1, (rabbit polyclonal) | Cell Signaling Technology | Cat# 5704, RRID:AB_1904067 | WB (1:2000) |
| Antibody | Anti-flotillin, (mouse monoclonal) | BD Bioscience | Cat# 610820, RRID:AB_398139 | WB (1:1000) |
| Antibody | Anti-β-tubulin, a (mouse monoclonal) | Santa Cruz | Cat# sc-5274, RRID:AB_2288090 | WB (1:2000) |
| Antibody | Anti- bassoon, (mouse monoclonal) | Enzo Life Sciences | Cat# ADI-VAM-PS003-F, RRID:AB_11181058 | IF (1:1000) |
| Antibody | Anti- N-cadherin, (rabbit polyclonal) | Bioworld Technology | Cat# BS2224, RRID:AB_1664028 | WB (1:1000) |
| Chemical compound, drug | CNQX | Sigma-Aldrich | Cat# C239; CAS: 115066-14-3 | 20 μM |
| Chemical compound, drug | AP-V | Sigma-Aldrich | Cat# A8054; CAS: 79055-68-8 | 50 μM |
| Chemical compound, drug | Doxycycline Hyclate | Selleck | Cat# S4163; CAS:24390-14-5 | 40 μg/ml |
| Chemical compound, drug | Glycine | Sigma-Aldrich | Cat# G8898; CAS:56-40-6 | 200 μM |
| Chemical compound, drug | bicuculline | Abcam | Cat# ab120108; CAS: 40709-69-1 | 20 μM |
| Chemical compound, drug | Picrotoxin | Sigma-Aldrich | Cat# P1675; CAS: 124-87-8 | 100 μM |
| Commercial assay or kit | SYBR Green qPCR Master Mix | QIAGEN | Cat# 204,057 | |
| Commercial assay or kit | Nissl staining Kit | Beyotime | Cat# C0117 | |
| Commercial assay or kit | FD Rapid GolgiStain Kit | FD NeuroTechnologies | Cat# PK401 | |
| Commercial assay or kit | BCA Protein Assay Kit | Thermo Fisher | Cat# 23,227 | |
| Software, algorithm | GraphPad Prism (8.0) | GraphPad Software | https://www.graphpad.com/scientific-software/prism/ | RRID: SCR_002798 |
| Software, algorithm | ImageJ | National Institutes of Health | https://imagej.nih.gov/ij/ | RRID:SCR_003070 |
| Software, algorithm | ANY-maze tracking software | Stoelting Co | https://www.stoeltingco.com/anymaze.html | RRID:SCR_014289 |
| Software, algorithm | PlusTipTracker | UTSouthwestern Medical Center Danuser Lab | https://www.utsouthwestern.edu/labs/danuser/software/ | |
| Software, algorithm | Sigmaplot | systatsoftware | http://www.systatsoftware.cn/ | RRID:SCR_003210 |
| Software, algorithm | MATLAB R2017b | MathWorks | https://se.mathworks.com/products/matlab.html | RRID: SCR_001622 |
| Sequence-based reagent | KIF2C floxP _F | This paper | PCR primers | GGT CCA GCT CTT TAC TGA TGT GTT C |
| Sequence-based reagent | KIF2C floxP _R | This paper | PCR primers | ACA AAG CAA GTC CAG GTC CAA G |
| Sequence-based reagent | Nestin-Cre_F | This paper | PCR primers | TGC AAC GAG TGA TGA GGT TC |
| Sequence-based reagent | Nestin-Cre_R | This paper | PCR primers | GCT TGC ATG ATC TCC GGT AT |
| Sequence-based reagent | kif2c_F | This paper | PCR primers | TGC CGT TGT TGA TGG TCA GTG |
| Sequence-based reagent | kif2c_R | This paper | PCR primers | GGA GAC ACT TGC TGG GAA CAG |
| Sequence-based reagent | shkif2c_F | This paper | PCR primers | TGG ATC GAA GGA GGT ACC AC |
| Sequence-based reagent | shkif2c_R | This paper | PCR primers | CAC TGA CCA TCA ACA ACG GCA |
| Sequence-based reagent | gapdh_F | This paper | PCR primers | AGG TCG GTG TGA ACG GAT TTG |
| Sequence-based reagent | gapdh_R | This paper | PCR primers | TGT AGA CCA TGT AGT TGA GGT CA |
| Peptide, recombinant protein | BDNF | Sigma-Aldrich | Cat# GF301 | 50 ng/mL |
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/72483/elife-72483-transrepform1-v1.docx
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Source data 1
All original files of blots and gels with the relevant bands.
- https://cdn.elifesciences.org/articles/72483/elife-72483-data1-v1.zip
