Figures and data
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 (No. 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 component (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.
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 CaMKIIαK42R/K42R) and three different culture conditions (immature culture at 13 DIV, AMPA glutamate receptor blocker CNQX treatment, and GABAA receptor blocker bicuculine 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 = 1,134 (58) and 1,204 (59), Syngap1+/−; n = 991 (65) and 1,371 (83), POGZQ1038R/+; n = 1,341 (72) and 1,271 (72), 15q11-13dup/+; n = 1, 208 (68) and 1,099 (63), 3q29del/+; n = 1,429 (66) and 1,408 (66), 22q11.2del/+; n = 1,143 (66) and 914 (63), Setd1a+/-; n = 1,381 (71) and 1,405 (71), CaMKIIαK42R/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).
Distinct morphological properties of spines in cultured neurons derived from schizophrenia model mice compared with ASD model mice.
(A and B) Areas with large differences (> 3 × SD) between control and mutant mice in terms of spine number per area in the feature space. The areas either enriched in the control (area A, blue region) or mutant (area B, yellow region) are shown. The positions of A and B are reversed between Nlgn3R451C/(y or R451C) and 22q11.2del/+. The total numbers of spines (n) in areas A and B were as follows: Nlgn3R451C/(y or R451C), control in A = 409, mutant in A = 366, control in B = 226, and mutant in B = 287; 22q11.2del/+, control in A = 195, mutant in A = 113, control in B = 268, and mutant in B = 307. Subtraction of spine numbers per area was performed after normalization by total spine numbers (Nlgn3R451C/(y or R451C), control = 1134 and mutant = 1204; 22q11.2del/+, control = 1143 and mutant = 914). (C and D) The relative number of spines distributed in areas A and B. (E and F) Distributions of spine lengths within the areas A and B. The Nlgn3R451C/(y or R451C) mouse model of ASD was enriched in longer spines, whereas the 22q11.2del/+ mouse model of schizophrenia was enriched in shorter spines. (G and H) Spine volumes within areas A and B. The Nlgn3R451C/(y or R451C) mouse model was enriched in large spines, while the 22q11.2del/+ mouse model was enriched in small spines. (I and J) The profiles of spines along their long axes. The trend observed for spine length and diameter was reversed between Nlgn3R451C/(y or R451C) and 22q11.2del/+ mouse models. Statistical significance was determined using the Wilcoxon rank sum test (Nlgn3R451C/(y or R451C), n = 775 in area A and n = 513 in area B; 22q11.2del/+, n = 308 in area A and n = 575 in area B).
Time-lapse imaging of neurons derived from 22q11.2del/+ schizophrenia model mice and corresponding control mice.
(A and B) Images of dendritic segments from control neurons (A) and 22q11.2del/+ neurons (B) at two different times points. (C and D) 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.
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 (n = 16 dendrites from control neurons and 11 dendrites from 22q11.2del/+ neurons, n = 10 control and n = 14 Setd1a+/- dendrites, n = 8 control and n = 8 Nlgn3R451C/(y or R451C) dendrites). (D-F) Spine lifetimes for the three mutant mouse models compared with corresponding controls. (n = 186 (11) control and n = 166 (7) 22q11.2del/+ spines (neurons), n = 82 (5) control and n = 202 (8) Setd1a+/- spines (neurons), n = 98 (8) control and n = 125 (8) Nlgn3R451C/(y or R451C) spines (neurons)). (G-I) Temporal patterns of spine growth (n = 11 control and n = 7 22q11.2del/+ neurons, n = 5 control and n = 8 Setd1a+/- neurons, n = 7 control and n = 8 Nlgn3R451C/(y or R451C) neurons). (J-K) Temporal patterns of spine shrinkage (n = 10 control and n = 7 22q11.2del/+ neurons, n = 5 control and n = 8 Setd1a+/- neurons, n = 8 control and n = 8 Nlgn3R451C/(y or R451C) neurons). Statistical significance was determined using the Wilcoxon rank sum test for two independent samples (A-F) or two-way ANOVA for genotype and time (G-L).
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 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. (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 h corresponds to the spines that persisted throughout the imaging period. (D) Differences in V1, V2, P1, and P2 between mutant mice and controls. (E) Plots of individual spine turnover simulated using parameters that best fit the experimental data from control and schizophrenia 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 h of spine turnover.
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 GFP expression plasmid. Spine turnover rates were calculated from the images of GFP-expressing dendrites taken at an interval of 24 h. Among the five DEGs, only the upregulation of Ecrg4 selectively increased the spine turnover rate. Statistical significance was determined by one-way ANOVA with post hoc Dunnett’s tests between groups (results from n = 22 dendrites from 11 neurons for all conditions except the Met siRNA 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.
Altered spine population profiles after suppression of Ecrg4 expression in neurons derived from schizophrenia mouse models.
(A) Transfection of the Ecrg4-shRNA plasmid or control shRNA plasmid into neurons derived from 22q11.2del/+ and Setd1a+/- mice altered the subtracted density plots of spine populations. The left plots show the effect of Ecrg4 shRNA on the mutant background. The middle plots show the differences between mutant and wild-type neurons both expressing control shRNA. The right plots show the difference between mutant neurons expressing Ecrg4 shRNA and wild-type neurons expressing control shRNA. (B) Morphological properties of spines enriched in neurons derived from 22q11.2del/+ and Setd1a+/- mice after transfection with the Ecrg4 shRNA plasmid or the control shRNA plasmid. The areas with more spines following Ecrg4 silencing are marked as B, while the areas with fewer spines are marked as A. The spine volume was larger in area B for neurons derived from both 22q11.2del/+ and Setd1a+/- mice. Statistical significance was determined using the Wilcoxon rank sum test (22q11.2del/+, n = 1076 in area A and n = 230 in area B; Setd1a+/-, n = 1142 in area A and n = 389 in area B). The profiles of spine radius along the long axis also confirmed the larger sizes of spines when Ecrg4 expression was downregulated by shRNA transfection. The numbers of spines and dendritic segments analyzed (in brackets) for the three conditions (mutant neurons expressing Ecrg4 shRNA, mutant neurons expressing control shRNA, wild-type neurons expressing control shRNA) are as follows: 22q11.2del/+; n = 968 (65), 954 (66), and 965 (66); Setd1a+/-: n = 1,015 (66), 1,001 (66), and 1,168 (66).
Cumulative frequency plots of spine length, surface area, and volume measured in four independent experiments performed > 2 months apart.
The cumulative frequency profiles shown as A to D were derived from 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).
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 CaMKIIαK42R/K42R.
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 8 mouse models are presented.
The relative numbers of spines within areas A and B in the feature space from Supplementary Figure 3.
The densities of mutant spines were higher in area B than in area A.
Spine lengths within areas A and B from Supplementary Figure 3.
For three ASD mouse models (Nlgn3R451C/(y or R451C), Syngap1+/−, and POGZQ1038R/+) and two schizophrenia mouse models (3q29del/+ and Setd1a+/-), spines in mutant-dominant area B were longer than in area A. For one ASD mouse model (15q11-13dup/+) and two schizophrenia mouse models (22q11.2del/+ and CaMKIIαK42R/K42R), spines in mutant-dominant area B were shorter than in area A. Statistical significance was determined by the Wilcoxon rank sum test. 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; CaMKIIαK42R/K42R, n = 237 in area A and n = 634 in area B.
Spine volumes within areas A and B from Supplementary Figure 3.
For three ASD mouse models (Nlgn3R451C/(y or R451C), Syngap1+/−, and POGZQ1038R/+), spines in mutant-dominant area B were larger than in area A. For one ASD mouse model (15q11-13dup/+) and four schizophrenia mouse models (3q29del/+, 22q11.2del/+, Setd1a+/-; and CaMKIIαK42R/K42R), spines in mutant-dominant area B were smaller than in area A. Statistical significance was determined using the Wilcoxon rank sum test. The numbers of spines included in the analysis are provided in the legend for Supplementary Figure 5.
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.
Spine numbers included in the analysis are the same as in Supplementary Figure 5.
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.
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 ASD-related mouse models. Mouse gene identifiers (ENSMUSG) and gene names for DEGs shared by more than two schizophrenia mouse models and not differentially expressed in ASD mouse models are presented. DEGs analyzed for their effects on spines are in red characters.
Expression and distribution of Ecrg4 in cultured hippocampal neurons.
(A) Preferential localization of Ecrg4 protein in the membrane fraction. Immunoblotting of Ecrg4, GluA1AMPA-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) Immunoblotting of Ecrg4-HA or Ecrg4-HA-SEP in total cell lysates from COS-7 cells. The molecular weights of these exogenously expressed proteins detected using anti-HA and anti-Ecrg4 antibodies were consistent with those of Ecrg4-HA (18 kDa) and Ecrg4-HA-SEP (45 kDa). (G) Reduced Ecrg4-HA protein expression by transfection of Ecrg4 sh1 or sh2 in COS-7 cells expressing Ecrg4-HA and GFP. Bars, 5 μm.
Subtracted density plots between neurons from schizophrenia model mice (22q11.2del/+ and Setd1a+/-) and neurons from wild-type mice both expressing control shRNA.
A comparison of the spine populations within control-dominant area A and mutant-dominant area B revealed smaller spine volumes in area B. The spine profiles along the long axis revealed an elongated morphology in area A for both 22q11.2del/+ and Setd1a+/- mutant mice. Statistical significance was determined using the Wilcoxon rank sum test (22q11.2del/+, n = 237 in area A and n = 789 in area B; Setd1a+/-, n = 630 in area A and n = 937 in area B).
