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SHANK3 overexpression causes manic-like behaviour with unique pharmacogenetic properties

Abstract

Mutations in SHANK3 and large duplications of the region spanning SHANK3 both cause a spectrum of neuropsychiatric disorders, indicating that proper SHANK3 dosage is critical for normal brain function. However, SHANK3 overexpression per se has not been established as a cause of human disorders because 22q13 duplications involve several genes. Here we report that Shank3 transgenic mice modelling a human SHANK3 duplication exhibit manic-like behaviour and seizures consistent with synaptic excitatory/inhibitory imbalance. We also identified two patients with hyperkinetic disorders carrying the smallest SHANK3-spanning duplications reported so far. These findings indicate that SHANK3 overexpression causes a hyperkinetic neuropsychiatric disorder. To probe the mechanism underlying the phenotype, we generated a Shank3 in vivo interactome and found that Shank3 directly interacts with the Arp2/3 complex to increase F-actin levels in Shank3 transgenic mice. The mood-stabilizing drug valproate, but not lithium, rescues the manic-like behaviour of Shank3 transgenic mice raising the possibility that this hyperkinetic disorder has a unique pharmacogenetic profile.

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Figure 1: Characterization of EGFP–Shank3 expression in Shank3 transgenic mice.
Figure 2: Shank3 transgenic mice display manic-like behaviours.
Figure 3: Individuals with SHANK3 duplications have hyperkinetic disorders.
Figure 4: Abnormal EEG and altered synaptic excitatory/inhibitory balance of Shank3 transgenic mice.
Figure 5: Shank3 interacts directly with Arp2/3 complex to increase F-actin levels in Shank3 transgenic mice.
Figure 6: Valproate, but not lithium, rescues manic-like behaviours of Shank3 transgenic mice.

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Acknowledgements

We are indebted to the patients and families who participated in this study; to J. W. Belmont and N. Miller for contributing patients to this study; G. Feng for sharing Shank3B mice; G. Schuster for injection of Shank3 BAC; and C. Spencer for behavioural assays training. This project was supported by The Howard Hughes Medical Institute (H.Y.Z.), National Institutes of Health (NIH) ARRA grant (1R01NS070302) (H.Y.Z.), the Baylor Intellectual and Developmental Disabilities Research Center (P30HD024064) confocal, electrophysiology and mouse neurobehavioral cores, and the Cancer Prevention and Research Institute of Texas (CPRIT) RP110784. J.L.H. was supported by an Early Career Award from the Thrasher Research Fund, NIH 2T32NS043124 and the Ting Tsung and Wei Fong Chao Foundation; C.P.S. was supported by the Joan and Stanford Alexander family, the Ting Tsung and Wei Fong Chao Foundation and the Doris Duke Clinical Scientist Development Award.

Author information

Authors and Affiliations

Authors

Contributions

K.H., J.L.H., H.L., H.C., J.T., H.-C.L. and H.Y.Z. designed the experiments. K.H., J.L.H., H.L., H.C., J.T., Z.W., S.H. and H.S. performed the research. K.H., J.L.H., C.P.S., H.L., H.C., H.K., J.T., Z.W., S.H., S.W.C., P.Y., A.M.B., A.P., H.-C.L. and H.Y.Z. collected, analysed and interpreted the data. K.H., J.L.H., C.P.S., H.L., H.K., J.T., H.-C.L. and H.Y.Z. wrote and edited the paper.

Corresponding author

Correspondence to Huda Y. Zoghbi.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Characterization of expression patterns of EGFP–Shank3 and other synaptic proteins in Shank3 transgenic mice.

a, Description of the Shank3 BAC used for transgenic mice generation (image was modified from UCSC genome browser). b, Western blot images show expression of EGFP–Shank3 in the synaptic fraction of Shank3 transgenic brain lysates. EGFP antibody recognized EGFP–Shank3 (200 kDa) specifically in synaptic fractions of Shank3 transgenic mice. Asterisk indicates non-specific bands detected by the EGFP antibody. Shank3 antibody detected endogenous Shank3 plus EGFP–Shank3 (arrow) in the transgenic samples. S2, soluble fraction; P2, crude synaptosomal fraction; PSD I, synaptic fraction after one time Triton X-100 washout. c, The brain regional expression pattern of EGFP–Shank3 is similar to that of endogenous Shank3. Cb, cerebellum; Ct, cortex; Hp, hippocampus; St, striatum; Th, thalamus. d, The brain developmental expression pattern of EGFP–Shank3 is similar to that of endogenous Shank3. e, The fold changes of Shank3 proteins in 3-week-old transgenic mice (n = 4) are similar to those of 6-week-old mice (Fig. 1d). f, Female transgenic mice (n = 4) show similar fold changes of Shank3 proteins to male transgenic mice (Fig. 1d). g, h, Expression levels of excitatory (g) and inhibitory (h) synaptic proteins are not significantly altered, except Shank3, in the synaptosomal fraction of 8-week-old transgenic hippocampus and striatum (n = 6). All data are presented as mean ± s.e.m. P values (*P < 0.05) were derived from unpaired, two-tailed Student’s t-test.

Extended Data Figure 2 Behavioural characterization of Shank3 transgenic mice.

a, Transgenic mice showed increased locomotor speed during the 30 min open-field assay. b, Female transgenic mice showed increased home-cage activity. c, Transgenic mice have increased body weight compared to wild-type mice measured at 9-weeks-old (male wild type: 28.1 ± 0.9, male transgenic: 30.6 ± 0.7, female wild type: 20.1 ± 0.3, female transgenic: 21.4 ± 0.4, n = 10–13; *P < 0.05; unpaired two-tailed Student’s t-test) and 15-weeks-old (male wild type: 32.4 ± 1.2, male transgenic: 37.0 ± 1.3, female wild type: 23.6 ± 0.4, female transgenic: 26.6 ± 0.9, n = 10–13; *P < 0.05). d, Increased food intake by transgenic mice. Food intake was measured from single-caged female transgenic or wild-type mice from 6- to 10-weeks-old. At 7-weeks-old (wild type: 3.51 ± 0.05, transgenic: 3.69 ± 0.06, n = 9–11; *P < 0.05; unpaired two-tailed Student’s t-test) and 9-weeks-old (wild type: 3.61 ± 0.07, transgenic: 3.86 ± 0.05, n = 9–11; *P < 0.05), food intake by transgenic mice was significantly higher than wild-type mice. e, f, In the 3-chamber assay, male (e) and female (f) transgenic mice did not show significant preference for novel mice to novel objects. g, Transgenic mice spent significantly less time in close interaction with novel social partners. h, i, Stereotypy time (h) and activity count (i) during the 30 min open-field test were normal in transgenic mice. j, Time spent in grooming during 10 min monitoring was normal in transgenic mice. k, Separation-induced ultrasonic vocalization was measured from wild-type and transgenic mice of postnatal day 6 to 13. Transgenic mice made less calls than wild type at postnatal day 13. l, At postnatal day 13, transgenic mice express more Shank3 proteins than wild-type mice. All data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. The summary of statistical analyses for behavioural assays is provided in Supplementary Table 1.

Extended Data Figure 3 Behavioural phenotypes of Shank3 transgenic mice were rescued by crossing with Shank3B+/− mice.

a, Schematic diagram shows possible genotypic combinations and their Shank3 expression levels (arrow) from the crossing between Shank3 transgenic mice and Shank3B+/− mice. b, Quantification of the levels of Shank3 proteins in each genotype (n = 4). The fold changes were compared to the wild-type (Shank3B+/+) controls. Crossing of Shank3 transgenic mice with Shank3B+/− mice significantly decreased the levels of α isoform and total Shank3 protein. c, d, Locomotor activities of male (c) and female (d) Shank3 transgenic mice were rescued by crossing with Shank3B+/− mice. e, f, Immobile time in tail-suspension test of male (e) and female (f) Shank3 transgenic mice were rescued by crossing with Shank3B+/− mice. g, Prepulse inhibition (by 78 dB) of female Shank3 transgenic mice was rescued by crossing with Shank3B+/− mice. All data are presented as mean ± s.e.m. *P < 0.05; ***P < 0.001.

Extended Data Figure 4 Basal synaptic transmission and NMDA receptor-dependent synaptic plasticity are normal at the hippocampal Schaffer collateral-CA1 synapses of Shank3 transgenic mice.

a, Images of cresylviolet staining show normal cytoarchitecture of transgenic brain. b, Normal paired-pulse facilitation ratio (PPR) of evoked EPSCs (eEPSCs) at transgenic Schaffer collateral-CA1 synapses. c, d, Normal synaptic input-output (I-O) relationship at transgenic Schaffer collateral-CA1 synapses. I-O curve (c) and cumulative curve of I-O slope (d) are shown. Red lines in c represent the fitting curves. e, Left, sample traces of action potentials triggered by 200 pA current injection in hippocampal CA1 region of wild-type and transgenic mice. Middle and right, unaltered number of action potentials trigged by the injection of current at different level (middle) and amplitude of the first action potential triggered by 800-ms-long pulse of 200 pA current (right) show normal intrinsic excitability of hippocampal CA1 pyramidal neurons in transgenic mice. f, Amplitude, frequency and decay of mEPSC are not altered in CA1 pyramidal neurons of transgenic mice. g, h, NMDA receptor-dependent long-term potentiation (g) and long-term depression (h) at Schaffer collateral-CA1 pyramidal synapses are normal in transgenic neurons.

Extended Data Figure 5 Generation and characterization of Shank3 in vivo interactome.

a, Isolation of EGFP–Shank3 protein and its interactors from synaptosomal fraction of transgenic mice. b, Venn diagrams show overlaps among the protein lists from Shank3 in vivo immunoprecipitation, Shank3 yeast two-hybrid screening, and either published mouse PSP (postsynaptic proteome) or human PSD. c, Shank3 interactome network. d, Average path length of Shank3 interactome (3.12, red line) is significantly (P < 0.0001) shorter than that of mouse PSP interactome (mean = 3.91, black line), supporting strong connectivity of Shank3 interactome. e, GO and KEGG pathway analysis of Shank3 interactome reveal enrichment of actin cytoskeleton-related function/pathway. The full result of analysis is in Supplementary Table 5. f, Confirmation of the in vivo interactions between Shank3 and actin-related proteins. g, In cultured hippocampal neurons, ARPC2 proteins are co-localized with F-actin (upper panel) and EGFP–Shank3 (lower panel).

Extended Data Figure 6 Shank3 siRNA reversed increased F-actin levels and ARPC2 cluster size in cultured hippocampal pyramidal neurons from Shank3 transgenic mice.

a, Validation of the two siRNAs against Shank3. HEK293T cells were transfected with HA-Shank3 plus control or Shank3 siRNA. EGFP plasmid was co-transfected as an internal control. After 48 h, expression levels of HA–Shank3 were measured by western blot and quantified (n = 3). b, siRNA targeting Shank3 (si-Shank3) reversed the increased F-actin levels of Shank3 transgenic neurons. Image for si-Shank3 #2 is not shown. c, si-Shank3 reversed the increased ARPC2 cluster size of Shank3 transgenic neurons. All data are presented as mean ± s.e.m. from 20–30 neurons per condition. P values (*P < 0.05, **P < 0.01) were derived from one-way ANOVA with post hoc Tukey’s multiple comparison.

Extended Data Figure 7 Normal excitatory and inhibitory synapse numbers of GAD-6-positive inhibitory neurons of Shank3 transgenic mice.

a, Expression of EGFP–Shank3 in GAD-6-positive inhibitory neurons of Shank3 transgenic mice. The yellow circles of dotted line and solid line indicate neuronal cell bodies of a GAD-6-negative excitatory neuron and a GAD-6-positive inhibitory neuron, respectively. The box 1 shows dendritic segment of the excitatory neuron and the box 2 shows that of the inhibitory neuron. b, Quantification of dendritic EGFP–Shank3 intensity in excitatory and inhibitory transgenic neurons. Inhibitory neurons express less EGFP–Shank3 (excitatory neurons: 1.00 ± 0.06, inhibitory neurons: 0.74 ± 0.09, n = 10; *P < 0.05; unpaired two-tailed Student’s t-test). c, Normal excitatory synapse number of GAD-6-positive inhibitory neurons of Shank3 transgenic mice. d, Normal inhibitory synapse number of GAD-6-positive inhibitory neurons of Shank3 transgenic mice. e, Quantification of (c) (wild type: 1.00 ± 0.10, transgenic: 0.99 ± 0.07, n = 18; P > 0.05; unpaired two-tailed Student’s t-test) and (d) (wild type: 1.00 ± 0.12, transgenic: 0.95 ± 0.09, n = 20; P > 0.05; unpaired two-tailed Student’s t-test).

Extended Data Figure 8 Shank3 interactome is connected with gephyrin-interacting actin-related proteins, Mena, profilin1 and profilin2.

Based on a literature search, we selected candidate actin-related proteins (Mena/VASP, profilin1 and profilin2) that directly interact with inhibitory postsynaptic protein gephyrin. To understand potential interactions among these proteins and the Shank3 interactome, we generated interaction network using the information from our yeast two-hybrid screening and IRefIndex PPI database (http://irefindex.org). The interaction network shows that Mena/VASP, profilin1 and profilin2 are connected with Shank3 interactome mainly through actin-related proteins. Note that one of the gephyrin-interacting proteins, profilin2, was also identified by our Shank3 in vivo immunoprecipitation (Supplementary Table 4).

Extended Data Figure 9 Mania-like behaviours of Shank3 transgenic mice are resistant to lithium treatment.

a, Basal and amphetamine-induced locomotor activities of female transgenic mice were not affected by lithium treatment. b–e, Abnormal acoustic startle response (b, d) and PPI (c, e) of male and female transgenic mice were not rescued by lithium treatment. f, g, Immobile time of male (f) and female (g) transgenic mice in tail-suspension test was not rescued by lithium treatment. Wild-type female mice showed decreased immobile time upon lithium treatment, which is an expected response to high dose lithium treatment in wild-type mice. All data are presented as mean ± s.e.m. *P < 0.05; ***P < 0.001.

Extended Data Figure 10 Valproate treatment does not affect synaptic protein and F-actin levels in neurons of Shank3 transgenic mice.

a, One hour after final valproate injection (200 mg per kg), synaptosomal fraction was prepared from brains, and indicated proteins were detected by western blotting. Neither wild-type nor transgenic mice showed significant change in the levels of synaptic proteins by valproate treatment (n = 7). b, Representative confocal images of Shank3 transgenic cultured hippocampal pyramidal neurons (DIV 14) treated with different concentrations of valproate (0, 0.01, 0.1 and 1 mM) for 30 or 60 min. c, Quantification of (b). There was no significant change in F-actin levels by valproate treatment (n = 16 per condition). All data are presented as mean ± s.e.m.

Supplementary information

Supplementary Data

This file contains Supplementary data and Supplementary Tables 4-5. (PDF 406 kb)

Supplementary Data

This file contains Supplementary Tables 1-3. (XLSX 35 kb)

Spontaneous seizure of Shank3 transgenic mice

This video shows spontaneous seizure from an 8-week old female Shank3 transgenic mouse in the home-cage. (MP4 5938 kb)

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Han, K., Holder Jr, J., Schaaf, C. et al. SHANK3 overexpression causes manic-like behaviour with unique pharmacogenetic properties. Nature 503, 72–77 (2013). https://doi.org/10.1038/nature12630

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