Spine nanostructure profiling of cultured neurons from mouse models reveals a schizophrenia-linked role for Ecrg4
- Department of Cellular Neurobiology, Graduate School of Medicine, The University of Tokyo, Japan
- Graduate School of Information Science, University of Hyogo, Japan
- National Institute for Physiological Sciences (NIPS), National Institutes of Natural Sciences, Japan
- Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, Japan
- Laboratory of Animal Resources, Center for Disease Biology and Integrated Medicine, Graduate School of Medicine, The University of Tokyo, Japan
- Department of Bioscience, Faculty of Life Sciences, Tokyo University of Agriculture, Japan
- Laboratory for Imaging Neural Dynamics, RIKEN Center for Brain Science, Japan
Figures
Figure 1 with 2 supplements
Structured illumination microscopy (SIM) imaging of dendritic spines, automated measurements of spine morphology, and generation of subtracted spine density plots for population-level analysis.
(A) Original SIM image of a dendritic shaft stained with lipophilic membrane dye DiI. (B) Binarized image of the same dendrite (top) and segmented spines numbered from 1 to 24 (middle). The bottom image shows segmented spines (white) and the dendritic shaft (blue). (C) Enlarged images of the individual spines shown in (B). The pseudocolor images indicating the relative positions of the spine segments from the base (blue) to the tip (yellow). (D) 3D views of three spines (Nos. 7, 15, and 24) viewed from different angles. (E) Principal component analysis (PCA)-based dimensional reduction of spine characteristics plotted in the plane of principal components (PC)1 and PC2. (F) Process of generating a subtracted density plot. The scatter plot of spine distribution in the PC1–PC2 plane based on morphological parameters was converted into a density plot for each culture source (genotype or treatment group), and the corresponding plots were subtracted to reveal differences in spine morphology at the population level. Bars, 4 μm for A, B, and E, 1 μm for C.
Figure 1—figure supplement 1
Cumulative frequency plots of spine length, surface area, and volume measured in four independent experiments performed >2 months apart.
The cumulative frequency distributions of spine length (A), spine surface area (B), and spine volume (C) were obtained from wild-type data acquired by structured illumination microscopy (SIM) imaging across four mouse models: Nlgn3R451C/(y or R451C) (S1), Syngap1+/− (S2), PogzQ1038R/+ (S3), and 15q11-13dup/+ (S4). The Kolmogorov–Smirnov test detected significant differences in only three of 18 possible pairwise comparisons: surface area of (S1) vs. (S3) (p = 0.017), volume of (S1) vs. (S3) (p = 0.032), and volume of (S3) vs. (S4) (p = 0.038).
Figure 1—figure supplement 2
Subtracted density plots comparing wild-type cultures from 3q29del/+ and 22q11.2del/+ mice (left) and wild-type vs. mutant cultures from the 22q11.2del/+ line (right).
The differences observed between independent wild-type culture preparations were substantially smaller than those observed between wild-type and mutant cultures, indicating that genotype-dependent effects exceeded inter-preparation variability.
Figure 2 with 1 supplement
Spine population profiles for each model and corresponding control mouse line presented as subtracted density plots.
(A) The subtracted density plots for eight disease mouse models (Nlgn3R451C/(y or R451C), Syngap1+/−, PogzQ1038R/+, 15q11-13dup/+, 3q29del/+, 22q11.2del/+, Setd1a+/−, and Camk2aK42R/K42R) and three different culture conditions (immature culture at 13 DIV, AMPA glutamate receptor blocker CNQX treatment, and GABAA receptor blocker bicuculline treatment). The areas with a higher density of spines from mutant disease model mice are shown in yellow, and the areas with reduced density are shown in blue. The total number of spines (first number, n) analyzed from control and mutant mouse neurons and the corresponding number of dendrites (number in parentheses) are as follows: Nlgn3R451C/(y or R451C); n = 1134 (58) and 1204 (59), Syngap1+/−; n = 991 (65) and 1371 (83), PogzQ1038R/+; n = 1341 (72) and 1271 (72), 15q11-13dup/+; n = 1208 (68) and 1099 (63), 3q29del/+; n = 1429 (66) and 1408 (66), 22q11.2del/+; n = 1143 (66) and 914 (63), Setd1a+/−; n = 1381 (71) and 1405 (71), Camk2aK42R/K42R; n = 880 (58) and 763 (54). All data are from three independent culture preparations. (B) Matrix of the 2D cross-correlations among subtracted density plots. In the lower right area, a spine group showing similar morphological changes can be identified. This group corresponded to the mouse models of schizophrenia. (C) Unbiased clustering of spine samples showing two distinct groups corresponding to schizophrenia (cyan) and ASD (red).
Figure 2—figure supplement 1
Cumulative frequency plots of spine length, surface area, and volume for the eight mouse mutants: Nlgn3R451C/(y or R451C), Syngap1+/−, PogzQ1038R/+, 15q11-13dup/+, 3q29del/+, 22q11.2del/+, Setd1a+/−, and Camk2aK42R/K42R.
Figure 3 with 3 supplements
Distinct morphological properties of spines in cultured neurons derived from schizophrenia model mice compared with autism spectrum disorder (ASD) model mice.
(A, C, E) Relative enrichment of four different subgroups of spines in Nlgn3R451C/(y or R451C) neurons compared with wild-type neurons. In the projection plane of PC1 and PC2, four areas with distinct structural properties, namely (1) small and short, (2) small and long, (3) large and short, and (4) large and long, were defined (A). The map of areas with large differences (>3 × SD) between control and mutant mice in spine number per area in the feature space (C) indicates that the area enriched with wild-type spines (area B, yellow region) overlaps with the area of large spines. The area enriched with the mutant spines overlaps with the area of small spines (area A, blue region). The relative enrichment of mutant spines was summarized in (E). (B, D, F) Similar analysis of mutant spine enrichment in 22q11.2del/+ neurons. The map of spine distribution with four different properties (B), the map of areas enriched with wild-type or mutant spines (D), and the graph of relative enrichment of mutant spines (F) show that the mutant neurons were enriched with small and long spines. (G–J) Relative abundance of the four subgroups of spines in 8 mutant mouse models (1: Nlgn3R451C/(y or R451C), 2: Syngap1+/−, 3: PogzQ1038R/+, 4: 15q11-13dup/+, 5: 3q29del/+, 6: 22q11.2del/+, 7: Setd1a+/−, 8: Camk2aK42R/K42R). Three independent culture experiments with paired wild-type and mutant samples were performed. Group comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test. (*p < 0.05, **p < 0.01, ***p < 0.005).
Figure 3—figure supplement 1
Areas where control (A: blue) or mutant (B: yellow) spines show a higher density within the feature space of the PC1–PC2 plane.
The plots for all eight mouse models are presented. The differences in population-level spine properties between autism spectrum disorder (ASD)- and schizophrenia-associated mouse models were preserved within two groups. The areas enriched with mutant spines in Nlgn3R451C/(y or R451C), Syngap1+/−, and PogzQ1038R/+ models were on the right side of the plot, suggesting the abundance of large spines in this group. In contrast, the areas enriched with mutant spines in 3q29del/+, 22q11.2del/+, Setd1a+/−, and Camk2aK42R/K42R models were positioned on the left side of the plot, indicating the abundance of small spines.
Figure 3—figure supplement 2
The relative numbers of spines within areas A and B in the feature space from Figure 3—figure supplement 1.
The densities of mutant spines were higher in area B than in area A.
Figure 3—figure supplement 3
Profiles of different spine populations (spines in the control-dominant area A and the mutant-dominant area B for both wild-type and mutant neurons) were visualized by plotting the radius along the long axis.
The numbers of control and mutant spines included in the analysis are as follows: Nlgn3R451C/(y or R451C), n = 775 in area A and n = 513 in area B; Syngap1+/−, n = 668 in area A and n = 500 in area B; PogzQ1038R/+, n = 580 in area A and n = 815 in area B; 15q11-13dup/+, n = 378 in area A and n = 797 in area B; 3q29del/+, n = 627 in area A and n = 824 in area B; 22q11.2del/+, n = 308 in area A and n = 575 in area B; Setd1a+/−, n = 472 in area A and n = 808 in area B; Camk2aK42R/K42R, n = 237 in area A and n = 634 in area B.
Figure 4
Time-lapse imaging of neurons derived from 22q11.2del/+ mice and corresponding control mice.
Images of dendritic segments from control neurons (A) and 22q11.2del/+ neurons (B) at two different time points. Montages of time-lapse images from control neurons (C) and 22q11.2del/+ neurons (D). The curved dendrites were straightened, revealing newly formed spines (arrowheads) as fluorescent objects appearing at the edge of the dendritic shafts. Bar, 4 μm.
Figure 5
Turnover rate, lifetime, and growth/shrinkage profiles of dendritic spines in cultured neurons derived from 22q11.2del/+, Setd1a+/−, and Nlgn3R451C/(y or R451C) mice, as well as the corresponding controls.
(A–C) Spine turnover rates in the three mutant mouse models compared to the controls. Spine turnover rates were analyzed using a linear mixed-effects model with genotype as a fixed effect and plate, cell, and dendrite as nested random effects, showing a significant effect of genotype only in 22q11.2del/+ and Setd1a+/− mice (n = 16 dendrites/9 cells/5 plates in control and 11 dendrites/6 cells/4 plates in 22q11.2del/+, n = 10 dendrites/7 cells/4 plates in control and n = 14 dendrites/9 cells/6 plates in Setd1a+/−, n = 8 dendrites/8 cells/3 plates in control and n = 8 dendrites/8 cells/3 plates in Nlgn3R451C/(y or R451C)). (D–F) Lifetimes of transient spines for the three mutant mouse models compared with corresponding controls. Spine lifetime was analyzed using a linear mixed-effects model accounting for the hierarchical structure of the data (spines nested within dendrites, cells, and culture plates). The analysis revealed a significant effect of genotype only in 22q11.2del/+ and Setd1a+/− neurons (n = 186 spines/11 dendrites/7 cells/5 plates in control and n = 166 spines/7 dendrites/4 cells/4 plates in 22q11.2del/+, n = 82 spines/5 dendrites/5 cells/4 plates in control and n = 202 spines/8 dendrites/8 cells/5 plates in Setd1a+/−, n = 98 spines/8 dendrites/8 cells/3 plates in control and n = 125 spines/8 dendrites/8 cells/3 plates in Nlgn3R451C/(y or R451C)). (G–I) Temporal patterns of spine growth (n = 60 spines/11 neurons/7 cells/5 plates in control and n = 50 spines/7 dendrites/4 cells/4 plates in 22q11.2del/+, n = 25 spines/5 dendrites/5 cells/4 plates in control and n = 65 spines/8 dendrites/8 cells/5 plates in Setd1a+/−, n = 43 spines/8 dendrites/8 cells/3 plates in control and n = 41 spines/7 dendrites/7 cells/2 plates in Nlgn3R451C/(y or R451C)). Spine volume trajectories were analyzed using linear mixed-effects models incorporating nested random effects (spine within dendrite within cell within culture plate) to account for the hierarchical structure of the data. Newly formed spines in both 22q11.2del/+ and Setd1a+/− neurons were significantly smaller than those in wild-type neurons. In contrast, newly formed spines in Nlgn3R451C/(y or R451C) neurons were significantly larger than those in wild-type neurons. (J–L) Temporal patterns of spine shrinkage (n = 39 spines/10 dendrites/6 cells/5 plates in control and n = 37 spines/7 dendrites/4 cells/4 plates in 22q11.2del/+, n = 15 spines/5 dendrites/5 cells/4 plates in control and n = 55 spines/8 dendrites/8 cells/5 plates in Setd1a+/−, n = 28 spines/8 dendrites/8 cells/3 plates in control and n = 35 spines/8 dendrites/8 cells/3 plates in Nlgn3R451C/(y or R451C) neurons). Spine volume trajectories were analyzed using linear mixed-effects models as in (G–I). In the 22q11.2del/+ neurons, spines undergoing elimination were significantly smaller than those in wild-type neurons. This effect was not detected in Setd1a+/− and Nlgn3R451C/(y or R451C) neurons.
Figure 6 with 1 supplement
Simulations of long-term spine turnover.
(A) The model of spine dynamics. Five successive phases of spine state transitions were defined. Phase 1: newly formed spines grow at speed V1. Phase 2: nascent spines are eliminated with probability P1. Phase 3: nascent spines are stabilized when volume reaches an upper threshold. Phase 4: stable spines are destabilized with probability P2. Phase 5: spines shrinking at a rate V2 are lost after reaching a lower threshold. (B) Pseudocolor maps of 625 different combinations of these parameters to identify those best fitting the experimental data. The color-coded values indicate the fitness of the simulation results to both the spine lifetime distribution profile and the turnover rate. (C) Frequency histograms of spine lifetimes for the three mouse models and controls (experimental data), and the results of simulations. The bin with a lifetime of 24 hr corresponds to the spines that persisted throughout the imaging period. (D) Differences in V1, V2, P1, and P2 between mutant mice and controls, expressed as differences in the four parameters normalized by the control condition values. (E) Plots of individual spine turnover simulated using parameters that best fit the experimental data from control and schizophrenia-associated mouse models. The upper plots show the progression of spine formation along 200 dendritic segments (50 μm) over 10 days. The lower plots show the enlargement of the last 24 hr of spine turnover. Color indicates spine size.
Figure 6—figure supplement 1
Spine turnover (A), density (B), and size (C) from simulation data.
Simulation parameters were tuned to fit experimental results from control and mutant models. Means and standard deviations are shown.
Figure 7 with 3 supplements
Manipulation of candidate gene expression in wild-type hippocampal neurons and its effects on spine turnover.
(A) Turnover rates of dendritic spines in neurons transfected with an overexpression plasmid encoding Cip4, Npas4, or Ecrg4, or a plasmid encoding an shRNA targeting Met or Arhgap15, together with a green fluorescent protein (GFP) expression plasmid. Spine turnover rates were calculated from the images of GFP-expressing dendrites taken at an interval of 24 hr. Among the five differentially expressed genes (DEGs), only the upregulation of Ecrg4 selectively increased the spine turnover rate. Linear mixed-effects modeling with experiment as a random factor revealed a significant effect of overexpression treatment (F(3,8) = 4.59, p = 0.038), driven by Ecrg4 (p = 0.013), whereas shRNA manipulations showed no significant effect (F(2,6) = 0.29, p = 0.76). (results from n = 22 dendrites/11 neurons/3 culture plates derived from independent primary culture for all conditions except the Met shRNA groups, where n = 21 dendrites from 11 neurons were included in the analysis). (B) Fluorescence images of dendritic segments expressing GFP or GFP plus Ecrg4 on days 1 and 2. Newly formed spines are marked by asterisks. Bar, 2 μm. (C) Images of dendrites and axons expressing HA-tagged Ecrg4 together with GFP. Anti-HA immunocytochemistry revealed the presence of immunopositive puncta both in the dendrites (arrows) and axons (arrowheads). Some clusters could be detected in the extracellular space (asterisks). The upper image is the overlay of the anti-HA signal (magenta) and GFP (green). The lower image shows the distribution of the anti-HA signals. Bar, 5 μm.
Figure 7—figure supplement 1
Pseudocolor presentation of differentially expressed genes (DEGs) between mutant and corresponding control mice.
The number of shared DEGs was higher in the schizophrenia-related mouse models than in the autism spectrum disorder (ASD)-related mouse models. Mouse gene identifiers (ENSMUSG) and gene names for DEGs shared by three or four schizophrenia-associated mouse models and not differentially expressed in ASD-associated mouse models are presented. DEGs analyzed for their effects on spines are shown in red. Color indicates the number of DEGs. The lower table shows all DEGs shared by three or four mouse models, irrespective of their grouping as ASD- or schizophrenia-related.
Figure 7—figure supplement 2
Validation of overexpression and shRNA knockdown constructs for DEGs identified in schizophrenia-associated mouse models.
(A–C) Validation of overexpressed tagged constructs by immunocytochemistry in hippocampal neurons. Bar = 20 μm. The data are presented as mean ± SEM (N = 5 neurons each). Red allows; transfected neurons, white arrows; non-transfected neurons. (A) Immunocytochemistry of hippocampal neurons expressing Ecrg4-HA with green fluorescent protein (GFP) using anti-Ecrg4 antibody. Anti-GM130 antibody staining detected the Golgi apparatus. (B) Immunocytochemistry of hippocampal neurons expressing Cip4-HA with GFP using anti-Cip4 antibody. Anti-MAP2 antibody detected neuronal dendrites. (C) Anti-NPAS4 and anti-HA immunocytochemistry of hippocampal neurons expressing NPAS4-Flag-HA with GFP. (D, E) Immunoblot verification of tagged construct expression in COS-7 cells. (D) Immunoblotting in total cell lysate from COS-7 cells expressing Cip4-HA or NPAS4-Flag-HA. The estimated molecular weights of Cip4-HA and NPAS4-Flag-HA are 64 and 89 kDa, respectively. (E) Immunoblotting using both anti-HA and anti-Ecrg4 in total cell lysate from COS-7 cells expressing Ecrg4-HA or Ecrg4-HA-SEP. The estimated molecular weights of Ecrg4, Ecrg4-HA, and Ecrg4-HA-SEP are 17, 18, and 45 kDa, respectively. (F–H) Verification of knockdown effects of shRNAs by immunoblotting of COS-7 cells. Immunoblotting in total cell lysates from COS-7 cells transfected with the expression vectors of either Met-GFP (F), ArhGAP15-GFP (G), or Ecrg4-HA (H), together with dsRed2 and shRNAs targeting the corresponding transcripts. (I) Measurement of spine turnover rate in neurons transfected with a control shRNA construct together with GFP expression vectors. For the experimental control, we used neurons transfected only with GFP. The data are presented as mean ± SEM (n = 15 dendrites from 9 neurons for GFP control, n = 16 dendrites from 8 neurons for GFP plus control shRNA).
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Figure 7—figure supplement 2—source data 1
PDF file containing original western blots for Figure 7—figure supplement 2, indicating the relevant bands and molecular weight standards.
- https://cdn.elifesciences.org/articles/109083/elife-109083-fig7-figsupp2-data1-v1.zip
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Figure 7—figure supplement 2—source data 2
Original files for western blot analysis displayed in Figure 7—figure supplement 2.
- https://cdn.elifesciences.org/articles/109083/elife-109083-fig7-figsupp2-data2-v1.zip
Figure 7—figure supplement 3
Expression and distribution of Ecrg4 in cultured hippocampal neurons.
(A) Preferential localization of Ecrg4 protein in the membrane fraction. Immunoblotting of Ecrg4, GluA1 AMPA-type glutamate receptor, and α-tubulin in the remaining fraction after removal of nuclei (S1), supernatant after centrifugation (S2), and resulting pellet (P2). (B) Images of an immunostained hippocampal neuron expressing HA-tagged Ecrg4 and NPY-GFP. The fluorescence signals were partially colocalized, suggesting Ecrg4 protein accumulation in dense-core vesicles. Bar = 2 μm. (C) Quantification of the overlap between puncta immunopositive for HA-tagged Ecrg4 and NPY-GFP. N = 4 cells from 1 dish. (D) Surface labeling with the anti-GFP nanobody revealing clusters of SEP-tagged Ecrg4 in dsRed2-positive axons (arrows) and dendrites (arrowheads). A nanobody was applied at time t = 0 min. Bar = 2 μm. (E) Enlarged image of a single Ecrg4 puncta in the axon, marked by a yellow square in (D), with the fluorescence intensity profile along the dashed arrow. Bar = 0.5 μm. (F) The spine density of cultured hippocampal neurons 24 hr after administration of either Ecrg4 peptides or scrambled peptides (n = 26 dendrites and 13 neurons for each condition). (G) Spine turnover rate of neurons treated with Ecrg4 peptides or scrambled peptides (n = 26 dendrites and 13 neurons for each condition).
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Figure 7—figure supplement 3—source data 1
PDF file containing original western blots for Figure 7—figure supplement 3, indicating the relevant bands and molecular weight standards.
- https://cdn.elifesciences.org/articles/109083/elife-109083-fig7-figsupp3-data1-v1.zip
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Figure 7—figure supplement 3—source data 2
Original files for western blot analysis displayed in Figure 7—figure supplement 3.
- https://cdn.elifesciences.org/articles/109083/elife-109083-fig7-figsupp3-data2-v1.zip
Figure 8
Altered spine population profiles after suppression of Ecrg4 expression in neurons derived from two schizophrenia-associated mouse models: the 22q11.2del/+ mouse model (A–I) and Setd1a+/− mouse model (J–R).
(A, J) Density plot obtained by subtracting wild-type spines from mutant spines. Both wild-type and mutant neurons expressed control shRNA. (B, K) Area enriched with the mutant spines under the control shRNA condition. (C, L) Area depleted of the mutant spines under the control shRNA condition. (D, M) Density plot obtained by subtraction of the wild-type spines under control shRNA from the mutant spines under Ecrg4 shRNA. This subtraction illustrates the normalizing effect of Ecrg4 shRNA. (E, N) Reduction of mutant spines in area A by Ecrg4 shRNA. The density plot, obtained by subtracting the mutant spines under control shRNA from those under Ecrg4 shRNA, shows the extent of normalization by Ecrg4 shRNA. (F, O) The increase of mutant spines in area B by Ecrg4 shRNA using the same subtraction method. (G, P) The extent of rescue by Ecrg4 shRNA in areas A and B for three independent culture experiments using either 22q11.2del/+ or Setd1a+/− mouse models. The red lines show the values corresponding to statistical significance (p = 0.05), estimated from permutation analyses shown in (H, I, Q, R). (H, Q) Permutation analysis for area A to estimate the 95th percentile of the shuffled data. (I, R) Permutation analysis for area B.
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL/6J | Japan SLC, Inc | N/A | |
| Strain, strain background (M. musculus) | C57BL/6N | Japan SLC, Inc | N/A | |
| Strain, strain background (M. musculus) | Nlgn3 mutant | Tabuchi et al., 2007 | PMID:17823315 | Background: C57BL/6J Provided by Dr. Tabuchi |
| Strain, strain background (M. musculus) | Syngap1 mutant | Komiyama et al., 2002 | PMID:12427827 | Background: C57BL/6J Provided by Dr. Grant |
| Strain, strain background (M. musculus) | Pogz mutant | Matsumura et al., 2020 | PMID:32103003 | Background: C57BL/6N Provided by Dr. Nakazawa |
| Strain, strain background (M. musculus) | 15q11-13dup/+ mutant | Nakatani et al., 2009 | PMID:19563756 | Background: C57BL/6J Provided by Dr. Takumi |
| Strain, strain background (M. musculus) | 3q29 mutant | Baba et al., 2019 | PMID:31216562 | Background: C57BL/6J Provided by Dr. Nakazawa |
| Strain, strain background (M. musculus) | 22q11.2del/+ mutant | Saito et al., 2021 | PMID:33845872 | Background: C57BL/6N Provided by Dr. Aiba |
| Strain, strain background (M. musculus) | Setd1a mutant | Nagahama et al., 2020 | PMID:32937141 | Background: C57BL/6N Provided by Dr. Kano |
| Strain, strain background (M. musculus) | Camk2a mutant | Yamagata et al., 2009 | PMID:19515929 | Background: C57BL/6J Provided by Dr. Yamagata |
| Antibody | Anti-HA tag (rabbit polyclonal) | MBL International | Cat#561 RRID:AB_591839 | 1:500 in immunostaining |
| Antibody | Anti-HA tag (mouse, monoclonal) | BioLegend | Cat#901517 RRID:AB_2629622 | 1:100 in immunostaining |
| Antibody | Anti-β-galactosidase (mouse, monoclonal) | Sigma-Aldrich | Cat#G6282 | 1:500 in immunostaining |
| Antibody | Anti-Ecrg4 (rabbit, polyclonal) | BioLegend | Cat#901517 RRID:AB_2629622 | 1:100 in immunostaining; 1:1000 in immunoblot |
| Antibody | Anti-GM130 (mouse, monoclonal) | BD Biosciences | Cat#610823 RRID:AB_398142 | 1:100 in immunostaining |
| Antibody | Anti-TRIP10/Cip4 (rabbit, polyclonal) | Proteintech | Cat#10798-1-AP RRID:AB_2209248 | 1:100 in immunostaining |
| Antibody | Anti-NPAS4 (rabbit, monoclonal) | Activity Signalling | Cat#AS-AB18A RRID:AB_3678717 | 1:100 in immunostaining |
| Antibody | Anti-MAP2 (chicken, polyclonal) | PhosphoSolutions | Cat#1100-MAP2 RRID:AB_2492141 | 1:500 in immunostaining |
| Antibody | Alexa Fluor 546 conjugated anti-Rabbit IgG (goat, polyclonal) | Thermo Fisher Scientific | Cat#A-11035 RRID:AB_2534093 | 1:500 in immunostaining |
| Antibody | Alexa Fluor 647 conjugated anti-Rabbit IgG (goat, polyclonal) | Thermo Fisher Scientific | Cat#A-21235 RRID:AB_2535804 | 1:500 in immunostaining |
| Antibody | Alexa Fluor 647 conjugated anti-Chicken IgY (goat, polyclonal) | Thermo Fisher Scientific | Cat#A-21449 RRID:AB_2535866 | 1:500 in immunostaining |
| Antibody | Alexa Fluor 647 conjugated anti-GFP nanobody | Proteintech | Cat#gb2AF647 RRID:AB_2827575 | 1:4000 in immunostaining |
| Antibody | Anti-GFP (rabbit, polyclonal) | MBL International | Cat#598 RRID:AB_591819 | 1:3000 in immunoblot |
| Antibody | Anti-GluA1 (rabbit, polyclonal) | Nittobo Medical | Cat# MSFR102280 RRID:AB_2571752 | 1:1000 in immunoblot |
| Antibody | Anti-DsRed (rabbit, polyclonal) | Nittobo Medical | Cat#MSFR101400 RRID:AB_2571647 | 1:4000 in immunoblot |
| Antibody | Anti-α-tubulin (mouse, monoclonal) | Sigma-Aldrich | Cat#62204 RRID:AB_1965960 | 1:3000 in immunoblot |
| Antibody | ECL HRP-conjugated anti-mouse IgG (sheep, polyclonal) | Cytiva | Cat#NA931 RRID:AB_772210 | 1:10,000 in immunoblot |
| Antibody | ECL HRP-conjugated anti-rabbit IgG (donkey, polyclonal) | Cytiva | Cat#NA934 RRID:AB_772206 | 1:10,000 in immunoblot |
| Recombinant DNA reagent | Cip4-HA (plasmid) | This paper | N/A | Mouse Trip10 (Cip4; NM_001242391) |
| Recombinant DNA reagent | Npas4-FH (plasmid) | This paper | N/A | Mouse Npas4 (NM_153553) |
| Recombinant DNA reagent | Ecrg4-HA (plasmid) | This paper | N/A | Mouse Ecrg4 (NM_024283) |
| Recombinant DNA reagent | Ecrg4-HA-SEP (plasmid) | This paper | N/A | SEP (superecliptic pHluorin) tagged mutant |
| Recombinant DNA reagent | Met-GFP (plasmid) | This paper | N/A | Mouse Met (NM_008591) |
| Recombinant DNA reagent | Arhgap15-GFP (plasmid) | This paper | N/A | Mouse Arhgap15 (NM_153820) |
| Recombinant DNA reagent | NPY-GFP (plasmid) | Addgene | Cat#74629 RRID:Addgene_74629 | Provided by Dr. Taraska |
| Recombinant DNA reagent | pSilencer-mouse-Met shRNA (plasmid) | This paper | N/A | 5′-GCAGTGAATTAGTTCGCTA-3 |
| Recombinant DNA reagent | pSilencer-mouse-Arhgap15 shRNA (plasmid) | This paper | N/A | 5′-AAGACAGATGTGAACATAC-3 |
| Recombinant DNA reagent | pSilencer-mouse-Ecrg4 shRNA (plasmid) | This paper | N/A | 5′-GAGGCTAAATTTGAAGAT-3′ |
| Peptide, recombinant protein | Ecrg4 peptide | BEX Co, Ltd | N/A | Ecrg4 C-terminal 16-mer peptide |
| Peptide, recombinant protein | Scramble peptide | BEX Co, Ltd | N/A | Corresponding scrambled control peptide |
| Commercial assay or kit | QuickGene DNA tissue kit | KURABO Industries Ltd | Cat#DT-S | |
| Commercial assay or kit | RNeasy Mini Kit | Qiagen | Cat#74104 | |
| Commercial assay or kit | RNase-Free DNase Set | Qiagen | Cat#79254 | |
| Commercial assay or kit | NEBNext Ultra Directional RNA Library Prep Kit | New England BioLabs | Cat#E7420 | |
| Commercial assay or kit | KAPA HTP Library Preparation Kit | KAPA Biosystems | Cat#KK8234 | |
| Commercial assay or kit | Trident Membrane Protein Extraction Kit | GeneTex International Corporation | Cat#GTX16373 | For separation into cytosolic and membrane fractions |
| Commercial assay or kit | Syn-PER Synaptic Protein Extraction Reagent | Thermo Fisher Scientific | Cat#87793 | For isolation of synaptosome fractions |
| Chemical compound, drug | CNQX | Tocris Bioscience | Cat#0190 | |
| Chemical compound, drug | Bicuculline | Tocris Bioscience | Cat#0109 | |
| Chemical compound, drug | DiI (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate) | Thermo Fisher Scientific | Cat#D3911 | |
| Software, algorithm | SIM image processing (MATLAB) | This paper | N/A | https://shigeookabe.github.io/download-page-SIM/ |
| Software, algorithm | DESeq2 | Love et al., 2014 | RRID:SCR_015687 |
Additional files
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Supplementary file 1
Structured illumination microscopy (SIM) imaging conditions.
Information about mouse models, genetic background, culture data, genotype, number of cells analyzed, number of dendrites analyzed, the day of fixation after plating, and the presence or absence of manipulation. Data for eight disease models and for both 22q11.2del/+ and Setd1a+/− with expression of shRNA for Ecrg4 were presented.
- https://cdn.elifesciences.org/articles/109083/elife-109083-supp1-v1.xlsx
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Supplementary file 2
Upregulated gene names Ensembl gene ID, gene symbols, log2-fold changes, and adjusted p values for upregulated genes found in culture preparations derived from eight disease models.
- https://cdn.elifesciences.org/articles/109083/elife-109083-supp2-v1.xlsx
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Supplementary file 3
Downregulated gene names Ensembl gene ID, gene symbols, log2-fold changes, and adjusted p values for downregulated genes found in culture preparations derived from eight disease models.
- https://cdn.elifesciences.org/articles/109083/elife-109083-supp3-v1.xlsx
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Spine nanostructure profiling of cultured neurons from mouse models reveals a schizophrenia-linked role for Ecrg4
eLife 14:RP109083.
https://doi.org/10.7554/eLife.109083.4
