Adult restoration of Shank3 expression rescues selective autistic-like phenotypes

Abstract

Because autism spectrum disorders are neurodevelopmental disorders and patients typically display symptoms before the age of three, one of the key questions in autism research is whether the pathology is reversible in adults. Here we investigate the developmental requirement of Shank3 in mice, a prominent monogenic autism gene that is estimated to contribute to approximately 1% of all autism spectrum disorder cases. SHANK3 is a postsynaptic scaffold protein that regulates synaptic development, function and plasticity by orchestrating the assembly of postsynaptic density macromolecular signalling complex. Disruptions of the Shank3 gene in mouse models have resulted in synaptic defects and autistic-like behaviours including anxiety, social interaction deficits, and repetitive behaviour. We generated a novel Shank3 conditional knock-in mouse model, and show that re-expression of the Shank3 gene in adult mice led to improvements in synaptic protein composition, spine density and neural function in the striatum. We also provide behavioural evidence that certain behavioural abnormalities including social interaction deficit and repetitive grooming behaviour could be rescued, while anxiety and motor coordination deficit could not be recovered in adulthood. Together, these results reveal the profound effect of post-developmental activation of Shank3 expression on neural function, and demonstrate a certain degree of continued plasticity in the adult diseased brain.

Extended Data Figure 1. Generation of Shank3 conditional knock-in mice

ai–ii, Schematic of the Shank3 gene and the target locus. aiii, Targeted exons 13–16. aiv, Neo-cassette excision via breeding with germline Flp mice. av, Exons 13–16 re-inversion via Cre-expressing mice. b, PCR genotyping showing the bands for Fx, Rescue, and WT. c, Western blot showing rescue of SHANK3 expression upon germline Cre recombination, with the exception of putative Shank3g isoform. This is likely due to the disruption of a putative intronic promoter by the introduction of the LoxP sites. d, Tamoxifen-inducible Cre strategy leads to broad reporter expression. Sagittal sections from pCAGGS-CreER^+/−;Rosa-floxstop-tdTomato^+/− mice after feeding with tamoxifen (left panel) or corn oil (right panel). Results show widespread induction of tdTomato reporter expression upon tamoxifen-induced Cre activation but not in the absence of Cre activity (corn oil feeding); pCAGGS promoter consists of the CMV early enhancer with chicken b-actin promoter. See Supplementary Figure 1 for gel source data.

Extended Data Figure 2. Additional measurements of dorsal striatum synaptic function in tamoxifen rescue (TM)

a, Representative traces (left) and bar graph (right) for AMPAR/NMDAR ratio in WT, KO and TM groups (WT=30, KO=33, TM=33 MSNs). AMPAR/NMDAR ratio calculated as the ratio of the EPSC peak amplitude at 70 mV (AMPAR EPSC) to the amplitude of the EPSC recorded at +40 mV, 50 ms after afferent stimulation. b, Representative traces (left) and bar graph (right) for pharmacologically isolated AMPAR/NMDAR ratio (WT=20, KO=20, TM=23 MSNs). Dual-component evoked EPSC at +40 mV recorded before and after APV bath application. c, Representative traces (left) and bar graph (right) for NR2B/NMDAR ratio in WT, KO and TM groups (WT=16, KO=16, TM=22 MSNs). Dual-component evoked EPSC at +40 mV recorded before and after ifenprodil bath application. Kruskal-Wallis test, with Dunn’s multiple comparison test for a, one-way ANOVA Bonferroni post-hoc test for b and c. All data presented as means ± s.e.m.

Extended Data Figure 3. Additional measurements of synaptic function in dorsal striatum by cortical and corpus callosum (cc) evoked stimulation (parasagittal and coronal slices respectively)

a, IR-DIC images showing representative placement of the stimulation electrode in the cortex (left) or corpus callosum (right) to evoke EPSCs in dorsal striatum using parasagittal (left) and coronal slices (right). b, Representative traces (right) and summary bar graphs for paired-pulse ratios (PPR) of evoked EPSCs in dorsal striatum MSNs, showing similar magnitude in parasagittal (left bar graph; WT=23, KO=20, TM=20 MSNs) and coronal slices (right bar graph; WT=9, KO=11, TM=10 MSNs). One-way ANOVA Bonferroni post-hoc test. c, Representative traces (right) and summary bar graphs of AMPAR/NMDAR ratios evoked in parasagittal (left bar graph; WT=17, KO=15, TM=16 MSNs) and coronal slices (right bar graph; WT=20, KO=16, TM=15 MSNs) from all 3 genotypes. AMPAR/NMDAR ratio calculated as the ratio of the EPSC peak amplitude at 70 mV (AMPAR EPSC) to the amplitude of the EPSC recorded at +40 mV, 50 ms after afferent stimulation (panel c, right side). Kruskal-Wallis test, with Dunn’s multiple comparison test. All data presented as means ± s.e.m.

Extended Data Figure 4. Whole-cell measurements of excitatory synaptic function in nucleus accumbens (NAc)

a, Representative traces (top) and summary bar graphs (bottom) of mEPSCs in NAc-MSNs (WT=22, KO=16, GR=18 MSNs). b, Representative traces (top) and summary bar graph (bottom) of AMPAR/NMDAR ratio in NAc-MSNs. AMPAR/NMDAR ratio calculated as the ratio of the EPSC peak amplitude at 70 mV (AMPAR EPSC) to the amplitude of the EPSC recorded at +40 mV, 50 ms aft er afferent stimulation (WT=19, KO=16, GR=15 MSNs). c, Representative traces (top) and summary bar graph (bottom) of paired-pulse ratios in NAc-MSNs (WT=25, KO=22, GR=19 MSNs). One-way ANOVA Bonferroni post-hoc test for a and c, Kruskal-Wallis test, with Dunn’s multiple comparison test for b. All data presented as means ± s.e.m.

Extended Data Figure 5. Western blots of synaptosomal preparations from the cortex of the adult treated mice show minimal difference across genotypes

a, Representative Western blot on SHANK3 in the cortex of adult WT, KO and TM mice, showing that most major Shank3 isoforms are restored in the TM mice. b, Representative Western blots of synaptic proteins including scaffolding proteins and neurotransmitter receptors in the adult cortex across genotypes. c, Quantification of multiple synaptic proteins in the cortex of the adult treated mice. All data WT N=3, TM N=3, KO N=3; each sample is from tissue taken from two animals. Student’s two-tailed unpaired t-test. All data presented as means ± s.e.m. See Supplementary Figure 1 for gel source data.

Extended Data Figure 6. Western blots of synaptosomal preparations from the cerebellum of the adult treated mice show minimal difference across genotypes

a, Representative Western blot of SHANK3 in the cerebellum of the adult mice treated with tamoxifen, showing that SHANK3 isoforms are restored in the TM mice. b, Representative Western blots of synaptic proteins in the adult cerebellum across genotypes. c, Quantification of multiple synaptic proteins in the cerebellum of the adult treated mice. All data WT N=3, TM N=3, KO, N=3; each sample is from tissue taken from a single animal. Student’s two-tailed unpaired t-test. All data presented as means ± s.e.m. See Supplementary Figure 1 for gel source data.

Extended Data Figure 7. Whole-cell measurements of excitatory synaptic function in the cortex

a, Summary bar graph of mEPSC frequency in the prefrontal cortex of the adult WT and KO. b, Summary bar graph of mEPSC amplitude in the prefrontal cortex of the adult WT and KO. a,b, WT N=13, KO N=10 cells; Student’s two-tailed unpaired t-test. All data presented as means ± s.e.m. c, Representative traces from mEPSC recordings in the adult prefrontal cortex of the WT and KO.

Extended Data Figure 8. Electrophysiological measurements in the dorsal striatum of germline rescue mice (GR)

a,b, Normal pop spike amplitude (a) and NP1 (b) in germline rescued (GR) mice (insets show representative field traces). c,d, Reduced mEPSC frequency in KO mice compared to WT and GR. A reduction in mEPSC peak current amplitude is observed between KO and WT group only (WT=30, KO=24, GR=26 MSNs). e, Representative traces (left) and bar graph (right) for pharmacologically isolated AMPAR/NMDAR ratio (WT=9, KO=9, GR=8 MSNs). Dual-component evoked EPSC at +40 mV recorded before and after APV bath application. f, Representative traces (left) and bar graph (right) for NR2B/NMDAR ratio in WT, KO and GR groups (WT=6, KO=5, GR=7 MSNs). Dual-component evoked EPSC at +40 mV recorded before and after ifenprodil bath application.). Two-way ANOVA Bonferroni post-hoc test for a and b; one-way ANOVA Bonferroni post-hoc test for c–f. *p<0.05; **p<0.01; ***p<0.001 (ANOVA). Data are means ± s.e.m.

Extended Data Figure 9. Expression of Shank3 at P20–21 rescues some behavioral measurements

a, Representative Western blots showing efficient SHANK3 re-expression in the cortex, striatum, and cerebellum in mice that were treated with tamoxifen at P20–21. b, The total distance traveled as measured by the open field test was not improved in the tamoxifen condition compared to the KO condition. One-way ANOVA, Bonferroni post-hoc test. c, The open field total distance plotted across 5-minute time bins, showing that there is no difference between KO and TM conditions across time. d. Rearing activity measured by open field plotted across time, showing that TM mice perform in between WT and KO for most of the 30-minute test. e. Rearing time measured by open field plotted across time, also showing that the intermediate performance of TM between that of WT and KO. (b to e) WT N=21, KO N=36, TM N=30. Two-way ANOVA, Bonferroni post-hoc test. f. Activity on the zero maze indicates that the TM condition shows significantly reduced anxiety compared to that of the KO condition. WT N=18, KO N=25, TM N=30; outliers were removed using Iglewicz and Hoaglin’s test (two-sided); Kruskal-Wallis test, Dunn’s Multiple Comparison test. All data presented as means ± s.e.m. See Supplementary Figure 1 for gel source data.

Extended Data Figure 10. Expression of Shank3 at P20–21 improves motor coordination

a, Summary data from trial 1 of the accelerating rotarod test on mice treated with TM at P20–21. WT N=19, KO N=35, TM N=33. b, Summary data from trial 2 of the same rotarod test on mice treated at P20–21. WT N=18, KO N=33, TM N=33. c, Summary data from trial 3 of the rotarod on mice treated at P20–21. WT N=20, KO N=36, TM N=33. a–c, Outliers were removed using Iglewicz and Hoaglin’s robust outlier test. One-way ANOVA, Bonferroni post-hoc test. d, Body weight from WT and KO that were unfed (UF) and mice that were fed with either corn oil or TM. WT(UF) N=28, WT(TM) N=14, KO(UF) N=27, KO(CO) N=25, KO (TM) N=26; One-way ANOVA, Bonferroni post-hoc test. All data presented as means ± s.e.m.

Figure 1. Shank3^fx/fx mice have deficits in neurotransmission and behavior

a, Domain structure of SHANK3 protein, with FLExed PDZ domain inverted, which can be re-oriented in the presence of Cre. b, Western blot showing Shank3 expression in striatal PSD from wildtype (WT) and Shank3^fx/fx (KO) mice. c, KO mice show decreased total distance traveled in the open field test compared to WT. d, KO mice show impaired motor coordination in rotarod test. e, KO mice spend less time in the open arm in elevated zero maze test. f, KO mice show decreased pop spike amplitude in extracellular field recordings in the striatum. g, Normal relationship of stimulation intensity to the negative peak 1 amplitude (NP1; action potential component) suggesting unaltered presynaptic function; insets show representative traces. *P< 0.05, **P<0.01, ***P< 0.001; all data presented as means ± s.e.m. (all behavior data from n=6 WT and n=7 KO mice); two-tailed t-test for c (left panel) and e; two-way repeated measures ANOVA with Bonferroni post hoc test for c (right panel), d, f and g (electrophysiology data from n=9 slices; 3 mice per genotype). See Supplementary Figure 1 for gel source data.

Figure 2. Rescue of PSD proteins and striatal neurotransmission

a, Experimental groups and TM feeding scheme. b, Western blot from striatal synaptosome preparation after tamoxifen feeding (TM) shows restoration of most SHANK3 isoforms. c, Western blots of striatal synaptosomal fractions; PSD protein levels in KO mice are restored to WT levels in TM group. d, Striatal field response in KO mice was rescued in TM mice. e, Representative traces for WT, KO and TM mice. Indistinguishable relationship of stimulation intensity to the NP1 amplitude among groups suggests unaltered presynaptic function. f, Rescued mESPC frequency in the striatum in the TM compared to the KO. g, Increased spine density in the TM compared to the KO, while spine density in the KO is lower than that of the WT. *P< 0.05, **P<0.01, ***P< 0.001; all data presented as means ± s.e.m. Student’s two-tailed t-test for c (n=3 WT, n=4 TM, n=3 KO; each sample represents combined striatal tissue from 2 mice); Two-way repeated measures ANOVA, with Bonferroni post hoc test for d and e (n=12 slices from 4 WT, n=12 slices from 4 TM and n=12 slices from 4 KO mice). One-way ANOVA with Bonferroni post hoc test for f (n=23 cells for WT, n=26 cells for KO, and n=27 cells for TM). One-Way ANOVA, Newman-Keuls Multiple Comparison test for g (n=32 dendritic segments for WT, n=30 for KO, and n=40 for TM). See Supplementary Figure 1 for gel source data.

Figure 3. Adult Shank3 expression rescued repetitive grooming and social interaction

a,b, Significantly reduced repetitive grooming behavior in TM mice compared to KO mice. c, Representative heat maps from the social interaction test for all groups. d, Unlike WT mice, KO mice showed no preference for interaction with a stranger mouse; this behavior is rescued in TM group. *P<0.05, **P<0.01, ***P<0.001; all data presented as means ± s.e.m; one-way repeated measures ANOVA with Bonferroni post hoc test for a,b (n=9 WT, n=9 TM and n=12 KO mice). Social interaction duration data (d, left panel) was analyzed using one-way repeated measures ANOVA with Bonferroni post hoc test. Social interaction frequency data (d, right panel) was analyzed using Kruskal-Wallis test with Dunn’s multiple comparison test due to the distribution not being normally distributed (n=22 WT, n=30 TM and n=30 KO).

Figure 4. Restoring Shank3 expression in adulthood did not rescue anxiety and rotarod deficits

a,b, Open field tests indicated that Shank3 re-expression in adults (TM) does not rescue reduced locomotion and reduced rearing (axiogenic behavior) in KO mice. c, KO mice spend less time exploring the open arm in elevated zero maze test; this behavior is also not rescued in TM group. d, Motor coordination measurement from rotarod is not rescued in TM group. e–h, Germline rescued Shank3^fx/fx mice (GR) show that all above parameters for open field, elevated zero maze and rotarod tests can be rescued if Shank3 expression is restored at germ cell stage. *P < 0.05, **P<0.01, ***P < 0.001; One way ANOVA for a (left panel), c; Kruskal-Wallis test with Dunn’s multiple comparisons for b (left panel). Two-tailed t-test for e (left panel), f and g (left panel); Two-way repeated measures ANOVA with Bonferroni post hoc test for a (right panel), b (right panel), d, e (right panel), g (right panel) and h; All data presented as means ± s.e.m. (a–c: n=18 WT, n=25 TM and n=27 KO; d: n=13 WT, n=19 TM and n=21 KO; e–h: n=10 WT and n=8 GR mice).