CoChat
Sign Up
Cited by 162Genetics and Neurodevelopmental DisordersAutism Spectrum Disorder ResearchNeuroscience and Neuropharmacology Research

The DOI matches its Crossref record; 33 of the 34 references it cites were found in a registry or an index.

Inherited and de novo SHANK2 variants associated with autism spectrum disorder impair neuronal morphogenesis and physiology

Open access
Berkel, Simone, Tang, Wannan, Treviño, Mario, Vogt, Miriam, Obenhaus, Horst Andreas, Gass, Peter, Scherer, Stephen Wayne, Sprengel, Rolf, Schratt, Gerhard, Rappold, Gudrun Anna·2012·Human Molecular Genetics
DOI 10.1093/hmg/ddr470PMC PMC3276277
Open this paper in CoChat — free accountOpen PDF

Abstract

Mutations in the postsynaptic scaffolding gene SHANK2 have recently been identified in individuals with autism spectrum disorder (ASD) and intellectual disability. However, the cellular and physiological consequences of these mutations in neurons remain unknown. We have analyzed the functional impact caused by two inherited and one de novo SHANK2 mutations from ASD individuals (L1008_P1009dup, T1127M, R462X). Although all three variants affect spine volume and have smaller SHANK2 cluster sizes, T1127M additionally fails to rescue spine volume in Shank2 knock-down neurons. R462X is not able to rescue spine volume and dendritic branching and lacks postsynaptic clustering, indicating the most severe dysfunction. To demonstrate that R462X when expressed in mouse can be linked to physiological effects, we analyzed synaptic transmission and behavior. Principal neurons of mice expressing rAAV-transduced SHANK2-R462X present a specific, long-lasting reduction in miniature postsynaptic AMPA receptor currents. This dominant negative effect translates into dose-dependent altered cognitive behavior of SHANK2-R462X-expressing mice, with an impact on the penetrance of ASD.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1093/hmg/ddr470

Cites

33 of 33 references verified0 citation errors · 0 not checkable

Statistics

Figures

Figure 1. Overexpression of SHANK2 mutants R462X, L1008_P1009dup and T1127M in primary hippocampal neurons. (A) SHANK2 WT and mutant constructs fused N-terminal with mCherry. SH3 (Src homology 3), PDZ (postsynaptic density 95/discs large/zona occludens-1), proline-rich region and SAM (sterile alpha motif) indicate the functional domains of SHANK2. Binding motifs for Homer (H), Dynamin 2 (D) and Cortactin (C) are indicated. (B) Western blot analysis displaying the sizes of the different SHANK2 constructs. Endogenous SHANK2 is not present in HEK293 cells. (C) Representative hippocampal neurons transfected with GFP in addition to overexpression with mCherry-SHANK2 WT, mCherry-SHANK2-R462X, mCherry-SHANK2-L1008_P1009dup or the mCherry-SHANK2-T1127M mutant. Scale bars: 10 µm (main panels); 2.5 µm (insets). (D) SHANK2 WT, the T1127M and the L1008_P1009dup mutant are located at functional synapses opposed to the presynaptic marker synapsin 1 (indicated by arrows). In contrast, the truncated SHANK2 mutant R462X did not cluster at functional synapses. Scale bars: 2 µm. (E) Quantitative analysis of the clustering behavior of the SHANK2 mutants shown in (A). The average pixel intensity in dendrites versus spine heads was measured to determine the SCR for WT and mutants. SCR = 1 indicates no clustering or even distribution, SCR = 0 indicates complete clustering. (F) Cumulative distribution of SHANK2 WT, L1008_P1009dup and T1127M cluster sizes. The R462X mutant did not cluster at all. SHANK2 WT clusters are larger compared with the two mutants. Kolmogorov–Smirnov test: WT/L1008_P1009dup P< 10−9 D = 0.121; WT/T1127M P< 10−7 D = 0.077 (n = 3, 18 neurons for each condition).
Figure 1. Overexpression of SHANK2 mutants R462X, L1008_P1009dup and T1127M in primary hippocampal neurons. (A) SHANK2 WT and mutant constructs fused N-terminal with mCherry. SH3 (Src homology 3), PDZ (postsynaptic density 95/discs large/zona occludens-1), proline-rich region and SAM (sterile alpha motif) indicate the functional domains of SHANK2. Binding motifs for Homer (H), Dynamin 2 (D) and Cortactin (C) are indicated. (B) Western blot analysis displaying the sizes of the different SHANK2 constructs. Endogenous SHANK2 is not present in HEK293 cells. (C) Representative hippocampal neurons transfected with GFP in addition to overexpression with mCherry-SHANK2 WT, mCherry-SHANK2-R462X, mCherry-SHANK2-L1008_P1009dup or the mCherry-SHANK2-T1127M mutant. Scale bars: 10 µm (main panels); 2.5 µm (insets). (D) SHANK2 WT, the T1127M and the L1008_P1009dup mutant are located at functional synapses opposed to the presynaptic marker synapsin 1 (indicated by arrows). In contrast, the truncated SHANK2 mutant R462X did not cluster at functional synapses. Scale bars: 2 µm. (E) Quantitative analysis of the clustering behavior of the SHANK2 mutants shown in (A). The average pixel intensity in dendrites versus spine heads was measured to determine the SCR for WT and mutants. SCR = 1 indicates no clustering or even distribution, SCR = 0 indicates complete clustering. (F) Cumulative distribution of SHANK2 WT, L1008_P1009dup and T1127M cluster sizes. The R462X mutant did not cluster at all. SHANK2 WT clusters are larger compared with the two mutants. Kolmogorov–Smirnov test: WT/L1008_P1009dup P< 10−9 D = 0.121; WT/T1127M P< 10−7 D = 0.077 (n = 3, 18 neurons for each condition).CC BY-NC · PubMed Central
Figure 2. shRNA-mediated knock-down of endogenous Shank2 reduces spine volume and increases dendritic arborization. (A) Representative figures of rat hippocampal neurons co-transfected with GFP and control shRNA (co-shRNA), shRNA#1 or shRNA#2, directed against Shank2. Knock-down of Shank2 results in many thin filopodia-like dendritic spines, most of them do not contain Shank2, whereas most spines under control conditions are mushroom-shaped and express Shank2 in their spine heads (indicated by arrows). Scale bar, 4 µm. (B) Representative neurons demonstrating that loss of Shank2 increases the complexity of the dendritic arbor. Scale bar, 23 µm. (C) Co-staining of primary hippocampal neurons with the presynaptic marker synapsin 1 (blue) and the postsynaptic marker PSD 95 (red). Shank2 knock-down neurons show a decrease in synaptic density. Scale bar, 2.5 µm. (D) Quantification of the Shank2 knock-down in primary hippocampal neurons. Bars represent the overall density of Shank2 puncta within a defined dendritic area. Both shRNAs significantly reduce the Shank2 puncta density (shRNA#1 P = 2.2 × 10−7; shRNA#2 P = 8.9 × 10−9). (E) Quantitative analysis of the relative dendritic spine volume. The shRNAs significantly reduce spine volume (shRNA#1 P = 2 × 10−18; shRNA#2 P = 2 × 10−19) (n = 3, 4200 spines from 21 neurons for each condition). (F) Shank2 knock-down reduces the synaptic density in primary hippocampal neurons. Synapse number was determined by counting the number of dendritic spines positive for PSD-95/synapsin that co-cluster along the dendritic area (shRNA#1 P = 4.3 × 10−12; shRNA#2 P = 4.4 × 10−11). (G) Knock-down of Shank2 had no effect on dendritic spine density (n = 3 experiments, 18 neurons for each condition). (H and I) Quantification of the dendritic complexity by Sholl analysis. (H) Shank2 knock-down increases the total number of intersections (shRNA#1 P = 2.1 × 10−5; shRNA#2 P = 0.002). (I) Loss of Shank2 increases dendritic material proximal to the soma relative to control conditions. P-values are still significant across the different groups after correction for multiple testing by the Bonferroni method P ≤ 0.005 (D–I). All values are shown as mean ± SD. **P ≤ 0.01, ***P ≤ 0.001; shRNA values are always compared with the GFP control. Control shRNA has no significant effect on the parameters. ANOVA P< 0.001, P-values of post hoc Scheffé tests are given (D, F, H and I) (n = 3, 30 neurons for each condition).
Figure 2. shRNA-mediated knock-down of endogenous Shank2 reduces spine volume and increases dendritic arborization. (A) Representative figures of rat hippocampal neurons co-transfected with GFP and control shRNA (co-shRNA), shRNA#1 or shRNA#2, directed against Shank2. Knock-down of Shank2 results in many thin filopodia-like dendritic spines, most of them do not contain Shank2, whereas most spines under control conditions are mushroom-shaped and express Shank2 in their spine heads (indicated by arrows). Scale bar, 4 µm. (B) Representative neurons demonstrating that loss of Shank2 increases the complexity of the dendritic arbor. Scale bar, 23 µm. (C) Co-staining of primary hippocampal neurons with the presynaptic marker synapsin 1 (blue) and the postsynaptic marker PSD 95 (red). Shank2 knock-down neurons show a decrease in synaptic density. Scale bar, 2.5 µm. (D) Quantification of the Shank2 knock-down in primary hippocampal neurons. Bars represent the overall density of Shank2 puncta within a defined dendritic area. Both shRNAs significantly reduce the Shank2 puncta density (shRNA#1 P = 2.2 × 10−7; shRNA#2 P = 8.9 × 10−9). (E) Quantitative analysis of the relative dendritic spine volume. The shRNAs significantly reduce spine volume (shRNA#1 P = 2 × 10−18; shRNA#2 P = 2 × 10−19) (n = 3, 4200 spines from 21 neurons for each condition). (F) Shank2 knock-down reduces the synaptic density in primary hippocampal neurons. Synapse number was determined by counting the number of dendritic spines positive for PSD-95/synapsin that co-cluster along the dendritic area (shRNA#1 P = 4.3 × 10−12; shRNA#2 P = 4.4 × 10−11). (G) Knock-down of Shank2 had no effect on dendritic spine density (n = 3 experiments, 18 neurons for each condition). (H and I) Quantification of the dendritic complexity by Sholl analysis. (H) Shank2 knock-down increases the total number of intersections (shRNA#1 P = 2.1 × 10−5; shRNA#2 P = 0.002). (I) Loss of Shank2 increases dendritic material proximal to the soma relative to control conditions. P-values are still significant across the different groups after correction for multiple testing by the Bonferroni method P ≤ 0.005 (D–I). All values are shown as mean ± SD. **P ≤ 0.01, ***P ≤ 0.001; shRNA values are always compared with the GFP control. Control shRNA has no significant effect on the parameters. ANOVA P< 0.001, P-values of post hoc Scheffé tests are given (D, F, H and I) (n = 3, 30 neurons for each condition).CC BY-NC · PubMed Central
Figure 3. Rescue of Shank2 knock-down. (A) Human SHANK2 WT protein normalizes spine volume in Shank2 knock-down neurons, whereas the SHANK2 R462X and the T1127M mutants have lost this ability (shRNA#2 P = 4.4 × 10−7; SHANK2-WT P = 0.787; R462X mutant P = 4.7 × 10−11, T1127M mutant P = 1.4 × 10−5; compared against GFP; n = 3, 21 neurons for each condition). The L1008_P1009dup mutant rescues the spine volume to some extent but is not as efficient as WT protein (GFP/shRNA#2 P = 4.4 × 10−7, GFP/L1008_P1009dup P = 0.066). (B and C) Human WT SHANK2, the T1127M and the duplication variants normalize dendritic arborization in SHANK2 knock-down neurons, as determined by Scholl analysis. In contrast, the R462X mutation is not able to rescue the increased dendritic branching. (B) Analysis of the total amount of intersections. Pair-wise P-values: GFP/shRNA P = 0.001, GFP/SHANK2-WT P = 0.024, SHANK2-WT/R462X P = 2.3 × 10−6; SHANK2-WT/T1127M and SHANK2-WT/L1008_P1009dup P-values are not significant. Only the R462X mutant fails to rescue the excessive dendritic branching, whereas the two missense variants behave like WT. (C) Distribution of intersections across the neuron. The R462X mutant is not able to rescue the increase in branching near the cell body within a distance of 50 µm (n = 3, 30 neurons for each condition). Values for T1127M, L1008_P1009dup and Shank2-WT are similar to the GFP control. *P ≤ 0.05, ***P ≤ 0.001, ANOVA P< 0.001; P-values of post hoc Scheffé test are given.
Figure 3. Rescue of Shank2 knock-down. (A) Human SHANK2 WT protein normalizes spine volume in Shank2 knock-down neurons, whereas the SHANK2 R462X and the T1127M mutants have lost this ability (shRNA#2 P = 4.4 × 10−7; SHANK2-WT P = 0.787; R462X mutant P = 4.7 × 10−11, T1127M mutant P = 1.4 × 10−5; compared against GFP; n = 3, 21 neurons for each condition). The L1008_P1009dup mutant rescues the spine volume to some extent but is not as efficient as WT protein (GFP/shRNA#2 P = 4.4 × 10−7, GFP/L1008_P1009dup P = 0.066). (B and C) Human WT SHANK2, the T1127M and the duplication variants normalize dendritic arborization in SHANK2 knock-down neurons, as determined by Scholl analysis. In contrast, the R462X mutation is not able to rescue the increased dendritic branching. (B) Analysis of the total amount of intersections. Pair-wise P-values: GFP/shRNA P = 0.001, GFP/SHANK2-WT P = 0.024, SHANK2-WT/R462X P = 2.3 × 10−6; SHANK2-WT/T1127M and SHANK2-WT/L1008_P1009dup P-values are not significant. Only the R462X mutant fails to rescue the excessive dendritic branching, whereas the two missense variants behave like WT. (C) Distribution of intersections across the neuron. The R462X mutant is not able to rescue the increase in branching near the cell body within a distance of 50 µm (n = 3, 30 neurons for each condition). Values for T1127M, L1008_P1009dup and Shank2-WT are similar to the GFP control. *P ≤ 0.05, ***P ≤ 0.001, ANOVA P< 0.001; P-values of post hoc Scheffé test are given.CC BY-NC · PubMed Central
Figure 4. Expression of SHANK2-R462X in the hippocampal CA1 cells is correlated with reduced mEPSCs in mice and causes morphological changes in vivo. (A) Top: Diagram of rAAV constructs containing SHANK2-WT and SHANK2-R462X. Bottom (left): Fluorescence confocal images of the hippocampal area CA1 in brain sections of adult mice injected at P42 with rAAV-Venus2A-SHANK2-WT and rAAV-Venus-SHANK2-R462X. Bottom (center): Cumulative amplitude distributions for mEPSCs and mIPSCs recorded at P69 ± 1 from CA1 pyramidal cells in brain slices from SHANK2-WT (green) and SHANK2-R462X (blue) infected mice. SHANK2-R462X mice display intact inhibitory signaling, but ∼26% reduced AMPAR-mediated transmission. This is reflected as a leftward shift of the distribution of mEPSC amplitudes. Group-average mEPSC and mIPSC traces acquired from the same cells (see Materials and Methods). Decay-time constants for the miniature currents are all similar (mIPSC, control: 14.8 ± 0.3, n = 16; SHANK2-WT: 17.3 ± 1.1, n = 18; SHANK2-R462X: 16.8 ± 1.3, n = 21; F(2,51) = 1.6119, P = 0.1339). Right: Summary graphs for averaged mEPSC and mIPSC amplitudes and frequencies. No differences in mEPSC frequency were detected. This indicates that the mutation affects postsynaptic excitatory transmission. The anti-GFP immunostaining reflects a volume labeling of soluble Venus released in Venus-2A-SHANK2-WT-expressing cells, whereas the Venus-SHANK2-R462X is expressed as Venus fusion protein with an enriched nuclear localization as also noticed in transfected primary neurons. (B) Top: Confocal fluorescence images of Venus-positive pyramidal cells in the hippocampal area CA1 and cortical layer 2/3 (right) injected at P0 with rAAV-Venus2A-SHANK2-WT and rAAV-Venus-SHANK2-R462X. Scale bar, 10 μm. Bottom (left): Cumulative amplitude distributions for mEPSCs and mIPSCs recorded from CA1 pyramidal cells from Shank2-WT (green) and SHANK2-R462X (blue) P0-infected mice at P100 ± 2 (average of 300 mPSCs per cell per mouse). P0-injected mice produced consistent results: SHANK2-R462X displays an ∼33% reduction in AMPAR-mediated transmission without differences in mPSC decay-kinetics [mEPSC (in ms), SHANK2-WT: 4.4 ± 0.6, n = 20; SHANK2-R462X: 3.4 ± 0.3, n = 22; F(1,39) = 1.4303, P = 0.2387; mIPSC decay-time (in ms), SHANK2-WT: 16.1 ± 0.9, n = 20; SHANK2-R462X: 14.0 ± 2.0, n = 22; F(1,39) = 3.0959, P = 0.0861] or in mPSC frequencies [mIPSC frequencies (in Hz), SHANK2-WT: 8.6 ± 0.5, n = 20; SHANK2-R462X: 7.7 ± 0.5, n = 22; F(1,39) = 1.8317, P = 0.1835]. Bottom (right): Cumulative amplitude distributions for mEPSCs and mIPSCs recorded from infected pyramidal neurons in EC layer 2/3 at P69 + 1. SHANK2-R462X mice present an ∼17% reduction of AMPAR but no change in GABAA-R-mediated transmission [mean mIPSC amplitude (in pA), SHANK2-WT: 23.6 ± 1.2, n = 21; SHANK2-R462X: 23.8 ± 1.3, n = 24; one-way ANOVA, F(1,41) = 0.0127, P = 0.9106]. mPSC frequency [mIPSC frequency (in Hz), SHANK2-WT: 5.9 ± 0.6, n = 21; SHANK2-R462X: 5.9 ± 0.4, n = 24; F(1,41) = 0.0090, P = 0.9246] and mPSC decay kinetics [mEPSC-decay time, SHANK2-WT: 6.1 ± 0.3, n = 20; SHANK2-R462X: 5.7 ± 0.6, n = 21; F(1,41) = 0.9733, P = 0.3294; mIPSC-decay time (in ms), SHANK2-WT: 13.3 ± 0.7, n = 21; SHANK2-R462X: 11.2 ± 0.4, n = 24; F(1,41) = 3.9014, P = 0.5119] were similar for both groups. (C) GluA1 cluster staining of SHANK2-R462X mice compared with GFP control overexpressing mice. Left panel shows the overview of GluA1 staining in the CA1 region of a P0 infected hippocampus. Scale bar, 10 μm. Confocal stacks were taken from rAAV-Syn-Venus-SHANK2-R462X and rAAV-Syn-GFP infected stratum radiatum regions of the hippocampus. Center and right panels: Overlaying red dots show the GluA1 stained clusters as they were counted in ImageJ. Scale bar, center, 5 μm; right, 2.5 μm. (D) Statistical analysis shows significant differences of GluA1 cluster number and cluster size between SHANK2-R462X and GFP overexpressing animals (P < 0.0001). (E) Golgi staining of SHANK2-R462X P0 infected hippocampal CA1 neurons and PBS P0 treated hippocampal CA1 neurons. The SHANK2-R462X mice showed densely packed filopodia structures instead of mature spines. Scale bar, 5 μm. *P ≤ 0.05, ***P ≤ 0.001.
Figure 4. Expression of SHANK2-R462X in the hippocampal CA1 cells is correlated with reduced mEPSCs in mice and causes morphological changes in vivo. (A) Top: Diagram of rAAV constructs containing SHANK2-WT and SHANK2-R462X. Bottom (left): Fluorescence confocal images of the hippocampal area CA1 in brain sections of adult mice injected at P42 with rAAV-Venus2A-SHANK2-WT and rAAV-Venus-SHANK2-R462X. Bottom (center): Cumulative amplitude distributions for mEPSCs and mIPSCs recorded at P69 ± 1 from CA1 pyramidal cells in brain slices from SHANK2-WT (green) and SHANK2-R462X (blue) infected mice. SHANK2-R462X mice display intact inhibitory signaling, but ∼26% reduced AMPAR-mediated transmission. This is reflected as a leftward shift of the distribution of mEPSC amplitudes. Group-average mEPSC and mIPSC traces acquired from the same cells (see Materials and Methods). Decay-time constants for the miniature currents are all similar (mIPSC, control: 14.8 ± 0.3, n = 16; SHANK2-WT: 17.3 ± 1.1, n = 18; SHANK2-R462X: 16.8 ± 1.3, n = 21; F(2,51) = 1.6119, P = 0.1339). Right: Summary graphs for averaged mEPSC and mIPSC amplitudes and frequencies. No differences in mEPSC frequency were detected. This indicates that the mutation affects postsynaptic excitatory transmission. The anti-GFP immunostaining reflects a volume labeling of soluble Venus released in Venus-2A-SHANK2-WT-expressing cells, whereas the Venus-SHANK2-R462X is expressed as Venus fusion protein with an enriched nuclear localization as also noticed in transfected primary neurons. (B) Top: Confocal fluorescence images of Venus-positive pyramidal cells in the hippocampal area CA1 and cortical layer 2/3 (right) injected at P0 with rAAV-Venus2A-SHANK2-WT and rAAV-Venus-SHANK2-R462X. Scale bar, 10 μm. Bottom (left): Cumulative amplitude distributions for mEPSCs and mIPSCs recorded from CA1 pyramidal cells from Shank2-WT (green) and SHANK2-R462X (blue) P0-infected mice at P100 ± 2 (average of 300 mPSCs per cell per mouse). P0-injected mice produced consistent results: SHANK2-R462X displays an ∼33% reduction in AMPAR-mediated transmission without differences in mPSC decay-kinetics [mEPSC (in ms), SHANK2-WT: 4.4 ± 0.6, n = 20; SHANK2-R462X: 3.4 ± 0.3, n = 22; F(1,39) = 1.4303, P = 0.2387; mIPSC decay-time (in ms), SHANK2-WT: 16.1 ± 0.9, n = 20; SHANK2-R462X: 14.0 ± 2.0, n = 22; F(1,39) = 3.0959, P = 0.0861] or in mPSC frequencies [mIPSC frequencies (in Hz), SHANK2-WT: 8.6 ± 0.5, n = 20; SHANK2-R462X: 7.7 ± 0.5, n = 22; F(1,39) = 1.8317, P = 0.1835]. Bottom (right): Cumulative amplitude distributions for mEPSCs and mIPSCs recorded from infected pyramidal neurons in EC layer 2/3 at P69 + 1. SHANK2-R462X mice present an ∼17% reduction of AMPAR but no change in GABAA-R-mediated transmission [mean mIPSC amplitude (in pA), SHANK2-WT: 23.6 ± 1.2, n = 21; SHANK2-R462X: 23.8 ± 1.3, n = 24; one-way ANOVA, F(1,41) = 0.0127, P = 0.9106]. mPSC frequency [mIPSC frequency (in Hz), SHANK2-WT: 5.9 ± 0.6, n = 21; SHANK2-R462X: 5.9 ± 0.4, n = 24; F(1,41) = 0.0090, P = 0.9246] and mPSC decay kinetics [mEPSC-decay time, SHANK2-WT: 6.1 ± 0.3, n = 20; SHANK2-R462X: 5.7 ± 0.6, n = 21; F(1,41) = 0.9733, P = 0.3294; mIPSC-decay time (in ms), SHANK2-WT: 13.3 ± 0.7, n = 21; SHANK2-R462X: 11.2 ± 0.4, n = 24; F(1,41) = 3.9014, P = 0.5119] were similar for both groups. (C) GluA1 cluster staining of SHANK2-R462X mice compared with GFP control overexpressing mice. Left panel shows the overview of GluA1 staining in the CA1 region of a P0 infected hippocampus. Scale bar, 10 μm. Confocal stacks were taken from rAAV-Syn-Venus-SHANK2-R462X and rAAV-Syn-GFP infected stratum radiatum regions of the hippocampus. Center and right panels: Overlaying red dots show the GluA1 stained clusters as they were counted in ImageJ. Scale bar, center, 5 μm; right, 2.5 μm. (D) Statistical analysis shows significant differences of GluA1 cluster number and cluster size between SHANK2-R462X and GFP overexpressing animals (P < 0.0001). (E) Golgi staining of SHANK2-R462X P0 infected hippocampal CA1 neurons and PBS P0 treated hippocampal CA1 neurons. The SHANK2-R462X mice showed densely packed filopodia structures instead of mature spines. Scale bar, 5 μm. *P ≤ 0.05, ***P ≤ 0.001.CC BY-NC · PubMed Central
Figure 5. Strong expression of SHANK2-R462X in the forebrain of mice can be monitored by changes in the behavior. (A) Grouping of rAAV-Syn-GFP P0 injected mice. Fluorescence overview images of one brain hemisphere from eight mice at P210 ± 2 (mouse numbers are indicated). Scale bar, 500 µm. On the right, a fluorescence overview image of a coronal brain section from mouse number1 (#1) is depicted. Scale bar, 500 µm. (B) Immunoblot analysis of hippocampal extracts prepared from mice shown in (A). (C) Grouping of rAAV-Syn-Venus-SHANK2-R462X P0 injected mice. Diagram of rAAV-Syn-Venus-SHANK2-R462X vector. Overview image of one brain hemisphere from eight injected mice at P210 ± 2. Exposure conditions were kept constant compared with the middle row in (A). A color-adjusted overview image of the coronal brain section from mouse number 2 (#2) is given on the right. (D) Immunoblot analysis of hippocampal extracts from the mice described in the middle rows. The anti-SHANK2 and anti-GFP antibodies identified relative levels of SHANK2-R462X expression (relative quantification from left to right 2.1, 2.2, 3.4, 3.3, 3.7, 1.9, 3.0, 1.9). Mice were divided into low-expression (L) and high-expression (H) groups. Immunosignals obtained with anti-GluA1, anti-GluA2 were comparable between all animals. (E) Anti-Phospho-Ser831-GluA1 and anti-Phospho-Ser845-GluA1 antibody showed no difference of phosphorylated GluA1 between low-expression and high-expression animals.
Figure 5. Strong expression of SHANK2-R462X in the forebrain of mice can be monitored by changes in the behavior. (A) Grouping of rAAV-Syn-GFP P0 injected mice. Fluorescence overview images of one brain hemisphere from eight mice at P210 ± 2 (mouse numbers are indicated). Scale bar, 500 µm. On the right, a fluorescence overview image of a coronal brain section from mouse number1 (#1) is depicted. Scale bar, 500 µm. (B) Immunoblot analysis of hippocampal extracts prepared from mice shown in (A). (C) Grouping of rAAV-Syn-Venus-SHANK2-R462X P0 injected mice. Diagram of rAAV-Syn-Venus-SHANK2-R462X vector. Overview image of one brain hemisphere from eight injected mice at P210 ± 2. Exposure conditions were kept constant compared with the middle row in (A). A color-adjusted overview image of the coronal brain section from mouse number 2 (#2) is given on the right. (D) Immunoblot analysis of hippocampal extracts from the mice described in the middle rows. The anti-SHANK2 and anti-GFP antibodies identified relative levels of SHANK2-R462X expression (relative quantification from left to right 2.1, 2.2, 3.4, 3.3, 3.7, 1.9, 3.0, 1.9). Mice were divided into low-expression (L) and high-expression (H) groups. Immunosignals obtained with anti-GluA1, anti-GluA2 were comparable between all animals. (E) Anti-Phospho-Ser831-GluA1 and anti-Phospho-Ser845-GluA1 antibody showed no difference of phosphorylated GluA1 between low-expression and high-expression animals.CC BY-NC · PubMed Central
Figure 6. Behavioral analyses of adult mice expressing SHANK2_R462X (shown in Fig. 5). (A) Both SHANK2-R462X-L and -H cohorts showed unaltered activity in the open field when compared with GFP-expressing controls. (B) In the novel object test, group comparisons by two-way ANOVA exhibited differences in the goal region depending on the presence of the novel object (F(1,26) = 7.951; P< 0.01). Post hoc tests demonstrated that only GFP-expressing controls exhibited significantly higher object approaches after introducing the object into the center of the arena. (C) No difference of anxiety-related behavior (time spent in lit compartment) was found in the light–dark box test. (D) In the puzzle box test, the transition from the light to the dark compartment through a gate (trials 1 and 11) or a tunnel (trials 2–9) requires altering strategies as indicated at the bottom. The tunnel is open in trials 2–4, filled with sawdust in trials 5–7 and covered by a plug in trials 8–10, whereas trial 11 corresponds to trial 1. Repeated-measures ANOVA exhibited a significant genotype effect (F(2,130) = 4,298; P< 0.05), whereas Bonferroni-corrected analyses showed a significant impairment of SHANK2-R462X-high expressing mice compared with both other groups during trial 10. *P ≤ 0.01.
Figure 6. Behavioral analyses of adult mice expressing SHANK2_R462X (shown in Fig. 5). (A) Both SHANK2-R462X-L and -H cohorts showed unaltered activity in the open field when compared with GFP-expressing controls. (B) In the novel object test, group comparisons by two-way ANOVA exhibited differences in the goal region depending on the presence of the novel object (F(1,26) = 7.951; P< 0.01). Post hoc tests demonstrated that only GFP-expressing controls exhibited significantly higher object approaches after introducing the object into the center of the arena. (C) No difference of anxiety-related behavior (time spent in lit compartment) was found in the light–dark box test. (D) In the puzzle box test, the transition from the light to the dark compartment through a gate (trials 1 and 11) or a tunnel (trials 2–9) requires altering strategies as indicated at the bottom. The tunnel is open in trials 2–4, filled with sawdust in trials 5–7 and covered by a plug in trials 8–10, whereas trial 11 corresponds to trial 1. Repeated-measures ANOVA exhibited a significant genotype effect (F(2,130) = 4,298; P< 0.05), whereas Bonferroni-corrected analyses showed a significant impairment of SHANK2-R462X-high expressing mice compared with both other groups during trial 10. *P ≤ 0.01.CC BY-NC · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Cited by

Sorted by citations

See all 162

Related

See all

Metadata

Authors
Berkel, S., Tang, W., Treviño, M., Vogt, M., Obenhaus, H. A., Gass, P., Scherer, S. W., Sprengel, R., Schratt, G., & Rappold, G. A.
Affiliations
  • Heidelberg University (DE)
  • Max Planck Institute for Medical Research (DE)
  • University of Toronto (CA)
  • Philipps University of Marburg (DE)
Corresponding author
Rappold, Gudrun Anna
ORCID
Venue
Human Molecular Genetics
Publisher
Oxford University Press (OUP)
Published
2011-10-12
Volume
21
Issue
2
Pages
344-357
ISSN
  • 0964-6906 (ISSN-L)
  • 1460-2083
DOI
10.1093/hmg/ddr470
PMC
PMC3276277
OpenAlex
W2120195741
Cited by
162
References
34
Citations by year
2026: 6 · 2025: 4 · 2024: 23 · 2023: 7 · 2022: 10 · 2021: 12 · 2020: 11 · 2019: 8 · 2018: 14 · 2017: 9 · 2016: 9 · 2015: 14 · 2014: 14 · 2013: 9 · 2012: 11
FWCI
6.98
Type
journal article
Language
en
Open access
Licence
cc-by-nc
Locations
  • Human Molecular Genetics (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • Max Planck Digital Library (submittedVersion)
  • MPG.PuRe (Max Planck Society) (submittedVersion)
Funders
  • GlaxoSmithKline
  • Deutsche Forschungsgemeinschaft (EXC 81, SFB636.A4, SFB 636, SFB 488)
  • Bundesministerium für Bildung und Forschung (01GS08168, EXC81)
  • University of Toronto
  • Hospital for Sick Children
  • Canadian Institutes of Health Research
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetics and Neurodevelopmental Disorders
  • Life Sciences › Neuroscience › Cognitive Neuroscience › Autism Spectrum Disorder Research
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
Keywords
Shank2, autism spectrum disorder, de novo mutations, postsynaptic scaffolding, dendritic spine morphology, synaptic transmission, intellectual disability, cognitive behavior, dominant negative effect, penetrance, neuronal morphogenesis
MeSH terms
Animals, Child, Child Development Disorders, Pervasive, Hippocampus, Humans, Morphogenesis, Mutation, Nerve Tissue Proteins, Neurons, Mice, Gene Knockdown Techniques

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review