Transcriptional and functional complexity of Shank3 provides a molecular framework to understand the phenotypic heterogeneity of SHANK3 causing autism and Shank3 mutant mice
© Wang et al.; licensee BioMed Central Ltd. 2014
Received: 24 January 2014
Accepted: 13 March 2014
Published: 25 April 2014
Considerable clinical heterogeneity has been well documented amongst individuals with autism spectrum disorders (ASD). However, little is known about the biological mechanisms underlying phenotypic diversity. Genetic studies have established a strong causal relationship between ASD and molecular defects in the SHANK3 gene. Individuals with various defects of SHANK3 display considerable clinical heterogeneity. Different lines of Shank3 mutant mice with deletions of different portions of coding exons have been reported recently. Variable synaptic and behavioral phenotypes have been reported in these mice, which makes the interpretations for these data complicated without the full knowledge of the complexity of the Shank3 transcript structure.
We systematically examined alternative splicing and isoform-specific expression of Shank3 across different brain regions and developmental stages by regular RT-PCR, quantitative real time RT-PCR (q-PCR), and western blot. With these techniques, we also investigated the effects of neuronal activity and epigenetic modulation on alternative splicing and isoform-specific expression of Shank3. We explored the localization and influence on dendritic spine development of different Shank3 isoforms in cultured hippocampal neurons by cellular imaging.
The Shank3 gene displayed an extensive array of mRNA and protein isoforms resulting from the combination of multiple intragenic promoters and extensive alternative splicing of coding exons in the mouse brain. The isoform-specific expression and alternative splicing of Shank3 were brain-region/cell-type specific, developmentally regulated, activity-dependent, and involved epigenetic regulation. Different subcellular distribution and differential effects on dendritic spine morphology were observed for different Shank3 isoforms.
Our results indicate a complex transcriptional regulation of Shank3 in mouse brains. Our analysis of select Shank3 isoforms in cultured neurons suggests that different Shank3 isoforms have distinct functions. Therefore, the different types of SHANK3 mutations found in patients with ASD and different exonic deletions of Shank3 in mutant mice are predicted to disrupt selective isoforms and result in distinct dysfunctions at the synapse with possible differential effects on behavior. Our comprehensive data on Shank3 transcriptional regulation thus provides an essential molecular framework to understand the phenotypic diversity in SHANK3 causing ASD and Shank3 mutant mice.
KeywordsActivity-dependent gene regulation Alternative splicing Autism spectrum disorder Phenotypic heterogeneity Shank3 isoform
Shank3/ProSAP2 is one of three members of the Shank/ProSAP family of proteins which contain five conserved protein domains – an ankyrin repeat (ANK), a Src homology 3 (SH3), a PSD-95/Discs large/ZO-1 (PDZ), a proline-rich region containing homer- and cortactin-binding sites (Pro), and a sterile alpha motif (SAM)[1–3]. Shank proteins localize in the postsynaptic density (PSD) of excitatory synapses where they function as master scaffolding proteins by interacting directly or indirectly with various proteins, including major types of glutamate receptors – NMDARs, AMPARs, and mGluRs – via different domains[1, 2, 4–8].
Human genetic studies strongly support the notion that molecular defects of SHANK3 contribute to autism spectrum disorders (ASD). In humans, SHANK3 maps to the critical region of the 22q13.3 deletion syndrome (Phelan-McDermid syndrome; PMS), in which autistic behaviors are an important feature. In addition, de novo sequence variants including missense, frame-shift, and splice site mutations across all coding exons of SHANK3 have been identified in ~0.5% of ASD patients with variable clinical presentations[10–14]. Interestingly, SHANK3 mutations were also reported in patients with childhood-onset schizophrenia and intellectual disability. In the cases with point mutations or small deletions of SHANK3, it was noted that clinical features are also quite variable. Shank3 mutant mice with deletions of exons encoding ANK, SH3, and PDZ domains and proline-rich region have been reported[16–20]. These mutant mice shared some similarities but also have significant differences in synaptic defects and behavioral abnormalities. The interpretations for the data from different lines of mutant mice were complicated at the time by the lack of clear understanding of the complexity of Shank3 transcript structure. It was believed that different lines of mutant mice only disrupted a select set of Shank3 isoforms. These observations then demand more knowledge of transcriptional regulation of Shank3 in the brain, and pose an interesting question about the molecular basis underlying the clinical heterogeneity in human patients with SHANK3 defects and the variability in different Shank3 mutant mice.
SHANK3 undergoes complex transcriptional regulation[8, 21–24]. We and others have determined that Shank3 displays multiple intragenic promoters and alternative splicing of coding exons in both mice and humans[12, 18, 23, 25, 26]. The combination of multiple promoters and alternative splicing is predicted to produce an extensive array of mRNA and protein isoforms, but this has not been fully characterized. With the information of presumptive SHANK3 isoforms, point mutations or small exonic deletions of SHANK3 found in ASD patients are predicted to affect selective isoforms of SHANK3. Since each SHANK3 isoform contains a distinct combination of the five different protein-protein interaction domains, each isoform may have a different function at the synapse. One interesting hypothesis is that isoform-specific disruptions by point mutations and small intragenic deletions within the SHANK3 gene contribute to the clinical heterogeneity in humans and variable phenotypes seen in mice.
As a first step to test this hypothesis, we conducted a series of experiments to systematically characterize the extent and regulation of isoform-specific expression of Shank3 in mice because of the ready availability of brain tissues and amenability of this model species to experimental manipulation. We discovered that Shank3 undergoes extensive alternative splicing in the exons encoding for conserved protein domains. We report, for the first time, that the expression and alternative splicing of Shank3 isoforms are brain-region and developmentally specific, activity dependent, and involve epigenetic regulation. We also found that different Shank3 isoforms displayed different subcellular distribution and differential effects on dendritic spine morphology, suggesting a different function for each isoform. We propose that isoform diversity of Shank3 is one of the explanations for the phenotypic diversity in humans and mice carrying various Shank3 defects.
All experiments in animals were conducted with approved protocols by the Institutional Animal Care and Use Committee at Duke University.
Primary neuron culture and drug treatments
The methods for primary hippocampal and cortical neuron cultures were described previously. Briefly, hippocampal and cortical tissues were dissected from newborn C57BL/6J pups between postnatal day 0 and day 1, and digested with trypsin. Tissues were pelleted by brief centrifugation and then dissociated in Neurobasal/B27 medium. Cells were plated into 60 mm dishes coated with 0.1 mg/mL poly-D-lysine at a density of 4 × 106 cells/dish. Cells were treated on 8–10 days in vitro with either 30 mM KCl or 5 μM trichostatin A (TSA) for 16 hours. Astrocyte monolayers were derived from the hippocampus of postnatal day 7 C57BL/6J mice as described. Total RNA and protein were prepared for quantitative PCR or western blot analysis.
DNA constructs and transfection
Mouse brain cDNAs were prepared from cerebral cortex of 8-week-old mice. Specific primers for Shank3 isoforms (3a, 3b, 3c, and 3e) were employed to amplify PCR products containing the complete open reading frame of each isoform, and the PCR products were cloned into EGFP-C1 vectors using In-Fusion Cloning Kits (Clontech, CA, USA). COS-7 cells growing on coverslips in 6-well plates were transfected with 2 μg of different Shank3 constructs using FuGENE HD transfection reagent (Promega, WI, USA) according to the manufacturer’s technical manual. The cells were fixed by 4% paraformaldehyde 36 hours post-transfection. Dissociated hippocampal neurons were grown on poly-D-lysine-coated coverslips. After 7 days in vitro, neurons were transfected with Shank3 constructs using Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA). Briefly, on the day of transfection, half of the medium was removed from each well and kept at 4°C. DNA and Lipofectamine 2000 were mixed in serum-free neurobasal medium with a ratio 1:3 (μg:μL). The mixture was added into each well and incubated for 6 hours before replacement with the previously saved conditioned medium. The cells were fixed by 4% paraformaldehyde after 14 days in vitro. To analyze the spine morphology, neurons were co-transfected with Shank3 constructs and a tdTomato plasmid.
Immunochemistry and morphology analysis of dendritic spines
Fixed neurons were permeabilized with 0.2% triton-X 100 in 1× phosphate buffered saline (PBS), blocked with 2% bovine serum albumin, and co-stained with rabbit anti-GFP (Invitrogen) and mouse anti-PSD-95 (UC Davis/NIH NeuroMab Facility) antibodies and corresponding secondary antibodies conjugated with Alexa 488 or Alexa 568. Confocal images were obtained using a 63× objective (Zeiss LSM 510 inverted) with sequential acquisition settings of 1024 × 1024 pixels. Each image was a z-series projection of 3–4 images at 0.5-μm depth intervals and averaged four times. Morphometric analysis and quantification of PSD-95 and dendritic spines were performed using Image J software (NIH, Bethesda, MD, USA) by an experimenter who was blinded to experiment conditions.
RNA isolation and RT-PCR expression analysis
Tissues from different brain regions were dissected from coronal sections of brain slices cut by a Leica VT 1000p microtome (Leica, IL, USA). Total RNA was isolated using the TRIzol method (Life Technologies, CA, USA). Reverse transcription was performed with SuperScript® III first-strand synthesis system (Invitrogen). Real-time quantitative RT-PCR (q-PCR) was carried out using a LightCycler 480 Instrument (Roche Diagnostics, Mannheim, Germany) and QuantiFast SYBR green PCR kit (Qiagen, CA, USA), following the manufacturer’s recommendations. The primers used in this study are listed in Additional file1: Table S1. AccuPrime GC-Rich DNA Polymerase (Invitrogen) was employed to amplify the full-length GC-rich sequence of exons 10–12 of Shank3. The sequences of newly identified splice variants were annotated and deposited in Genbank. Quantification of the bands of splicing variants were carried out using Image J.
Western blot was performed as previously described. Briefly, brain tissues were homogenized and sonicated in modified RIPA buffer (1× PBS, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, and protease inhibitors); 25 μg of proteins were resolved by PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked with 5% milk and incubated with Shank3 (1:5,000 in 5% non-fat milk) antibody (sc-30193, Santa Cruz, Dallas, TX, USA) at 4°C overnight. Following incubation with horseradish peroxidase-conjugated secondary antibody, the membranes were incubated with a Pierce chemiluminescent substrate (Rockford, IL, USA) and exposed to X-ray film.
Multiple protein isoforms produced by intragenic promoters in mouse brain
Extensive alternative splicing of Shank3 mRNAs confers further complexity to Shank3 isoforms
Alternative splicing of Shank3 has been suggested[12, 18] but has not been fully characterized. We conducted RT-PCR with primer combinations that cover all exons of Shank3 (Additional file2: Figure S1A) using total RNA from the cerebral cortex of 8-week-old mice. We discovered that the coding exons 10–12, exon 18, exon 21, and exon 22 of Shank3 displayed extensive alternative splicing (Additional file3: Figure S2). Interestingly, the alternatively spliced exons were concentrated in the conserved SH3, proline-rich, and SAM domains of Shank3 and resulted in protein species with different combinations of the five functional domains. The alterative splicing of exons 10–12 resulted in five different splice variants (E10–12S I to V, Figure 1B). E10–12S I represented the full length of this portion of mRNA without alternative splicing. In E10–12S II, a cryptic splicing of 57 nucleotides occurred in exon 11 without a shift of the open reading frame (ORF) of Shank3 mRNA. However, splice variants of E10–12S III and IV resulted in truncated isoforms only containing the ANK domain due to a frame shift of the Shank3 ORF (Figure 1E). Interestingly, skipping of exons 10 to 12 in E10–12S V was predicted to produce a Shank3 isoform without the SH3 domain but presumably retaining the other four protein domains (Figure 1E). Alternative splicing of exons 18–22 was examined by primers specific to promoter 5 for Shank3e (Additional file2: Figure S1A). Four splice variants (E18S I to IV, Figure 1C) were identified. Exon 18 was spliced out in E18S II to IV. The splicing of exon 18 appeared to be in concert with splicing of exons 21/22 or exons 19–22 in Shank3 E18S III and IV, respectively. The frame shift of the Shank3 ORF in E18S III and IV variants is predicted to result in premature stop codons in exon 22 and produced short Shank3e isoforms (Shank3e-1) lacking the proline-rich region and SAM domain (Figure 1E). With a different set of primers, alternative splicing of exons 21 and 22 was examined, and four different variants were observed (E21–22S I to IV, Figure 1D). While the E21–22S I and II variants did not change the ORF of Shank3, the E21–22S III and IV variants were predicted to produce C-terminal truncated Shank3 isoforms lacking the SAM domain due to premature stop codons in exon 22. In summary, these results indicate that Shank3 undergoes extensive alternative splicing, which results in extreme diversity of Shank3 isoforms at mRNA and possibly at protein levels.
Region- and development-specific expression of Shank3 isoforms in mouse brain
Region-, development-, and cell-type-specific alternative splicing of Shank3 isoforms in mouse brain
Activity dependent expression and alterative splicing of Shank3
Expression and alternative splicing of Shank3 are regulated by a histone deacetylase (HDAC) inhibitor
Differential subcellular localization of Shank3 isoforms and their effects on dendritic spines
Using various molecular genetics approaches, we have described an unusually complex transcriptional profile for Shank3, a strong human autism causative gene, in the mouse brain. We have delineated transcript structures for major Shank3 isoforms resulting from the combination of multiple intragenic promoters and alternative splicing. Furthermore, we showed, for the first time, that the isoform-specific expression of Shank3 is temporally and spatially specific and regulated by neural activity. We showed that the isoform-specific expression of Shank3 in cortical neurons involves an epigenetic regulation suggested in previous reports[23, 35]. Further, this is the first report indicating that a selective Shank3 isoform is strictly localized in neuronal nuclei which implies a novel function other than as a scaffolding protein at the PSD. Our results support the hypothesis that different Shank3 isoforms have distinct functions at synapses. These findings will promote our knowledge of the molecular diversity in the brain and help us to understand the phenotypic heterogeneity caused by various SHANK3 defects in humans and mice.
The implication of molecular complexity of Shank3 to the functional diversity of synapses
Our results reveal that Shank3 displays more isoforms in the brain than it does in peripheral tissues and suggest that each Shank3 isoform has a unique function, which allows us to propose that the complexity of Shank3 contributes to the functional diversity of synapses. First, the major isoforms of Shank3 generated from different promoters contain distinct combinations of the five conserved domains, which provide the functional diversity for Shank3 isoforms. The full-length Shank3a contains all five domains which would have the capacity to interact with all possible interacting proteins. Other isoforms with different combinations of functional domains would then interact with a different subset of synaptic proteins. For instance, the N-terminal truncated Shank3e only contains the proline-rich region and SAM domain and would be able to couple with homer-mGluRs complexes but not GKAP-PSD95-NMDARs complexes[4, 5]. In contrast, the C-terminal truncated Shank3b would be able to interact with NMDARs but not mGluRs. By competing with Shank3a, one of the functions of Shank3b and Shank3e might be the regulation of the cross-talk between NMDARs and mGluRs, which results in the fine tuning of synaptic transmission. Second, alternative splicing contributes to further complexity of Shank3 isoforms and functional diversity. Alterative splicing of Shank3 is predicted to have an impact on the function of Shank3 since it is heavily concentrated in the exons encoding functional protein domains. The splice variants of E10–12S III and IV generate truncated Shank3 isoforms which only contain the ANK domain known to interact with cytoskeleton proteins such as alpha-fodrin and sharpin[30, 36]. Conceptually, these isoforms will compete with the binding of full-length Shank3 to alpha-fodrin and sharpin, thereby modulating the rearrangement of the PSD structure. It is interesting that these isoforms are highly expressed at early stages of brain development and concentrated in the olfactory bulb of adult brain. The splicing variant E18S II showed similar region- and development-specific splicing patterns to that of E10–12S, implying that they may coordinate with each other for some functions. Exon 18 inclusion may be considered a neuronal marker at the mRNA level, as it occurs specifically in neurons, but not in astrocytes or peripheral tissues. Exon 18 contains 24 nucleotides that encode 8 amino acids containing an arginine stretch (RRRK) residues that is similar to the "RXR" motif. It has been shown that the RXR motif serves as an endoplasmic reticulum (ER) retention signal in the NMDA receptor subunit NR1. Whether or not this motif regulates the sorting and trafficking of Shank3 in vivo is an interesting question for further investigation. Third, the functional diversity of Shank3 isoforms is supported by the finding that Shank3 isoforms display different subcellular localization and differential effects on spine morphology. For example, the full length Shank3a increased the density of dendritic spines and PDS-95 puncta, but Shank3b and Shank3e have an opposite impact on dendritic spine development. The nuclear targeting of Shank3b in heterologous cells and neurons implicates that this isoform probably possesses a function other than as a scaffolding protein at the PSD as described to date. In addition, the temporally- and regionally-specific expression of Shank3 isoforms also supports the concept that different isoforms have different functions. Altogether, our data strongly support a notion that different Shank3 isoforms contribute differentially to synaptic function. Follow-up studies are warranted to determine the distinct role for each isoform in synaptic development and function.
The complexity of Shank3 and the specification of synapses
Although the phenomena of alternative splicing and multiple promoters are commonly described in both neuronal and non-neuronal genes, the degree of the complexity described for Shank3 is somewhat unusual. A similar level or even more complexity has been described for genes such as Neurexin family proteins and Brain Derived Neurotrophic Factor[38, 39]. The exact number of Shank3 mRNAs and protein isoforms is not known. Whether all mRNA isoforms are translated into proteins cannot be easily determined. Given the tissue-/cell-type and developmental-stage specific promoter usage and alternative splicing, the number of Shank3 isoforms could be very substantial and possibly reach to more than a hundred. The interesting question is whether there is a biological purpose underlying such complexity as has been observed for Shank3. Humans have billions of neurons and a trillion synapses but a relatively small number of genes in a genome (approximately 20,000). We have a limited understanding of what contributes to the diversity of synapses, and the current methods to classify different types of synapses are extremely simplified. From revealing the complexity of Shank3 and other synaptic proteins, one plausible hypothesis is that the myriad isoforms of Shank3 and other synaptic proteins are destined to contribute to the diversity or specification of types of synapses. One could speculate that the different isoforms of Shank3 may localize differentially in the nano-structure of the PSD, such as central versus peripheral sites in the PSD of the same synapse, or different isoforms of Shank3 are at different excitatory synapses in a given pyramidal neuron. In addition, some protein isoforms may be present only when the neurons are stimulated with certain types of neural activity that lead to local protein translation for specific Shank3 mRNA isoforms. To examine these possibilities, high resolution cellular imaging at single synapses is required and isoform-specific antibodies will be helpful to assist these analyses.
The complex Shank3 transcriptional regulation and phenotypic heterogeneity of Shank3 mutant mice and SHANK3 causing ASD
Considerable clinical heterogeneity in ASD has been well documented[40–42], although the cause underlying this heterogeneity remains largely unknown. Molecular heterogeneity due to the number of genes has been hypothesized to be one of the logical explanations[43, 44], and has been supported by the findings of whole exome or whole genome sequencing of ASD cases[45–48]. However, in the case of SHANK3 causing ASD, considerable heterogeneity is also observed among the cases with different mutations within the SHANK3 gene. We have previously shown that human SHANK3 also displayed a similar pattern of promoter usage and alternative splicing. With the knowledge of the complexity of Shank3 transcriptional regulation reported here, the clinical heterogeneity of SHANK3 causing ASD could be predicted to result from the disruption of different sets of SHANK3 isoforms due to the location of the mutation within the coding exons of SHANK3. This prediction has been supported by Shank3-isoform mutant mouse models, in which phenotypic diversity was demonstrated among mutant mice with disruption of different isoforms[16–20]. A striking example is that disruption of Shank3a to Shank3c results in skin lesion/increased self-grooming phenotype, but this phenotype is absent in Shank3a to Shank3b deficient mice, implicating the unique role of Shank3c in the development of self-induced skin lesions. Although genetic background and environmental factors may also contribute to the phenotypic heterogeneity in SHANK3 causing ASD and Shank3 mutant mice, our analysis of Shank3 isoforms provides a framework at the molecular level to understand the question of phenotypic heterogeneity. A head-to-head comparison between different Shank3 mutant mice in the same genetic background and in vivo analysis of the function of each Shank3 isoform will yield insights into the mechanism underlying phenotypic heterogeneity in SHANK3 causing ASD.
In summary, we showed a complex transcriptional regulation of Shank3 in mouse brain that resulted in diverse Shank3 isoforms. The regional, developmental, activity-dependent, and epigenetic modulation of isoform-specific expression and alternative splicing of Shank3 suggest a different function for each Shank3 isoform. It is predicted that the SHANK3 defects in reported ASD patients and Shank3 mutant mice are isoform-specific. Our study then provides a molecular framework to dissect clinical heterogeneity of SHANK3 causing human disorders and provide insight to better understand the molecular basis underlying the clinical heterogeneity of ASD in general. In addition, these findings are critically important to interpret the difference between different lines of Shank3 mutant mice and formulate the plan to further analyze these mutant mice to elucidate the contribution of Shank3 to the pathophysiology of ASD.
Autism spectrum disorder
Metabotropic glutamate receptor
Open reading frame
Quantitative real-time polymerase chain reaction
Sterile alpha motif
Src homology 3
We thank Shengli Zhao and Tingting Wang for generous gifts of EGFP plasmids. We thank Xinyu Cao for technical assistance. XW is supported by a postdoctoral fellowship from Phelan-McDermid Syndrome Foundation. QX is supported by a grant from Natural Science Foundation of China, NSFC (No. 81371270) ALB is a pre-doctoral fellow supported by Ruth K. Broad Foundation. YHJ is supported by an Autism Speaks grant, Ruth K. Broad Foundation, and National Institutes of Health grant R01MH098114-01.
- Sheng M, Kim E: The Shank family of scaffold proteins. J Cell Sci. 2000, 113 (Pt 11): 1851-1856.PubMedGoogle Scholar
- Ehlers MD: Synapse structure: glutamate receptors connected by the shanks. Curr Biol. 1999, 9: R848-R850. 10.1016/S0960-9822(00)80043-3.View ArticlePubMedGoogle Scholar
- Grabrucker AM, Schmeisser MJ, Schoen M, Boeckers TM: Postsynaptic ProSAP/Shank scaffolds in the cross-hair of synaptopathies. Trends Cell Biol. 2011, 21: 594-603. 10.1016/j.tcb.2011.07.003.View ArticlePubMedGoogle Scholar
- Tu JC, Xiao B, Naisbitt S, Yuan JP, Petralia RS, Brakeman P, Doan A, Aakalu VK, Lanahan AA, Sheng M, Worley PF: Coupling of mGluR/homer and PSD-95 complexes by the Shank family of postsynaptic density proteins. Neuron. 1999, 23: 583-592. 10.1016/S0896-6273(00)80810-7.View ArticlePubMedGoogle Scholar
- Naisbitt S, Kim E, Tu JC, Xiao B, Sala C, Valtschanoff J, Weinberg RJ, Worley PF, Sheng M: Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin. Neuron. 1999, 23: 569-582. 10.1016/S0896-6273(00)80809-0.View ArticlePubMedGoogle Scholar
- Uchino S, Wada H, Honda S, Nakamura Y, Ondo Y, Uchiyama T, Tsutsumi M, Suzuki E, Hirasawa T, Kohsaka S: Direct interaction of post-synaptic density-95/Dlg/ZO-1 domain-containing synaptic molecule Shank3 with GluR1 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor. J Neurochem. 2006, 97: 1203-1214. 10.1111/j.1471-4159.2006.03831.x.View ArticlePubMedGoogle Scholar
- Verpelli C, Dvoretskova E, Vicidomini C, Rossi F, Chiappalone M, Schoen M, Di Stefano B, Mantegazza R, Broccoli V, Bockers TM, Dityatev A, Sala C: Importance of Shank3 protein in regulating metabotropic glutamate receptor 5 (mGluR5) expression and signaling at synapses. J Biol Chem. 2011, 286: 34839-34850. 10.1074/jbc.M111.258384.PubMed CentralView ArticlePubMedGoogle Scholar
- Jiang YH, Ehlers MD: Modeling autism by SHANK gene mutations in mice. Neuron. 2013, 78: 8-27. 10.1016/j.neuron.2013.03.016.PubMed CentralView ArticlePubMedGoogle Scholar
- Wilson HL, Wong AC, Shaw SR, Tse WY, Stapleton GA, Phelan MC, Hu S, Marshall J, McDermid HE: Molecular characterisation of the 22q13 deletion syndrome supports the role of haploinsufficiency of SHANK3/PROSAP2 in the major neurological symptoms. J Med Genet. 2003, 40: 575-584. 10.1136/jmg.40.8.575.PubMed CentralView ArticlePubMedGoogle Scholar
- Hamdan FF, Gauthier J, Araki Y, Lin DT, Yoshizawa Y, Higashi K, Park AR, Spiegelman D, Dobrzeniecka S, Piton A, Tomitori H, Daoud H, Massicotte C, Henrion E, Diallo O, S2D Group, Shekarabi M, Marineau C, Shevell M, Maranda B, Mitchell G, Nadeau A, D'Anjou G, Vanasse M, Srour M, Lafrenière RG, Drapeau P, Lacaille JC, Kim E, Lee JR: Excess of de novo deleterious mutations in genes associated with glutamatergic systems in nonsyndromic intellectual disability. Am J Hum Genet. 2011, 88: 306-316. 10.1016/j.ajhg.2011.02.001.PubMed CentralView ArticlePubMedGoogle Scholar
- Moessner R, Marshall CR, Sutcliffe JS, Skaug J, Pinto D, Vincent J, Zwaigenbaum L, Fernandez B, Roberts W, Szatmari P, Scherer SW: Contribution of SHANK3 mutations to autism spectrum disorder. Am J Hum Genet. 2007, 81: 1289-1297. 10.1086/522590.PubMed CentralView ArticlePubMedGoogle Scholar
- Durand CM, Betancur C, Boeckers TM, Bockmann J, Chaste P, Fauchereau F, Nygren G, Rastam M, Gillberg IC, Anckarsater H, Sponheim E, Goubran-Botros H, Delorme R, Chabane N, Mouren-Simeoni MC, de Mas P, Bieth E, Rogé B, Héron D, Burglen L, Gillberg C, Leboyer M, Bourgeron T: Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are associated with autism spectrum disorders. Nat Genet. 2007, 39: 25-27. 10.1038/ng1933.PubMed CentralView ArticlePubMedGoogle Scholar
- Schaaf CP, Sabo A, Sakai Y, Crosby J, Muzny D, Hawes A, Lewis L, Akbar H, Varghese R, Boerwinkle E, Gibbs RA, Zoghbi HY: Oligogenic heterozygosity in individuals with high-functioning autism spectrum disorder. Hum Mol Genet. 2011, 20 (17): 3366-3375. 10.1093/hmg/ddr243.PubMed CentralView ArticlePubMedGoogle Scholar
- Waga C, Okamoto N, Ondo Y, Fukumura-Kato R, Goto Y, Kohsaka S, Uchino S: Novel variants of the SHANK3 gene in Japanese autistic patients with severe delayed speech development. Psychiatr Genet. 2011, 21: 208-211. 10.1097/YPG.0b013e328341e069.View ArticlePubMedGoogle Scholar
- Gauthier J, Champagne N, Lafreniere RG, Xiong L, Spiegelman D, Brustein E, Lapointe M, Peng H, Cote M, Noreau A, Hamdan FF, Addington AM, Rapoport JL, Delisi LE, Krebs MO, Joober R, Fathalli F, Mouaffak F, Haghighi AP, Néri C, Dubé MP, Samuels ME, Marineau C, Stone EA, Awadalla P, Barker PA, Carbonetto S, Drapeau P, Rouleau GA, S2D Team: De novo mutations in the gene encoding the synaptic scaffolding protein SHANK3 in patients ascertained for schizophrenia. Proc Natl Acad Sci U S A. 2010, 107: 7863-7868. 10.1073/pnas.0906232107.PubMed CentralView ArticlePubMedGoogle Scholar
- Bozdagi O, Sakurai T, Papapetrou D, Wang X, Dickstein DL, Takahashi N, Kajiwara Y, Yang M, Katz AM, Scattoni ML, Harris MJ, Saxena R, Silverman JL, Crawley JN, Zhou Q, Hof PR, Buxbaum JD: Haploinsufficiency of the autism-associated Shank3 gene leads to deficits in synaptic function, social interaction, and social communication. Molecular Autism. 2010, 1: 15-10.1186/2040-2392-1-15.PubMed CentralView ArticlePubMedGoogle Scholar
- Peca J, Feliciano C, Ting JT, Wang W, Wells MF, Venkatraman TN, Lascola CD, Fu Z, Feng G: Shank3 mutant mice display autistic-like behaviours and striatal dysfunction. Nature. 2011, 472: 437-442. 10.1038/nature09965.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang X, McCoy PA, Rodriguiz RM, Pan Y, Je HS, Roberts AC, Kim CJ, Berrios J, Colvin JS, Bousquet-Moore D, Lorenzo I, Wu G, Weinberg RJ, Ehlers MD, Philpot BD, Beaudet AL, Wetsel WC, Jiang YH: Synaptic dysfunction and abnormal behaviors in mice lacking major isoforms of Shank3. Hum Mol Genet. 2011, 20: 3093-3108. 10.1093/hmg/ddr212.PubMed CentralView ArticlePubMedGoogle Scholar
- Schmeisser MJ, Ey E, Wegener S, Bockmann J, Stempel AV, Kuebler A, Janssen AL, Udvardi PT, Shiban E, Spilker C, Balschun D, Skryabin BV, Dieck S, Smalla KH, Montag D, Leblond CS, Faure P, Torquet N, Le Sourd AM, Toro R, Grabrucker AM, Shoichet SA, Schmitz D, Kreutz MR, Bourgeron T, Gundelfinger ED, Boeckers TM: Autistic-like behaviours and hyperactivity in mice lacking ProSAP1/Shank2. Nature. 2012, 486: 256-260.PubMedGoogle Scholar
- Kouser M, Speed HE, Dewey CM, Reimers JM, Widman AJ, Gupta N, Liu S, Jaramillo TC, Bangash M, Xiao B, Worley PF, Powell CM: Loss of predominant shank3 isoforms results in hippocampus-dependent impairments in behavior and synaptic transmission. J Neurosci. 2013, 33: 18448-18468. 10.1523/JNEUROSCI.3017-13.2013.PubMed CentralView ArticlePubMedGoogle Scholar
- Lim S, Naisbitt S, Yoon J, Hwang JI, Suh PG, Sheng M, Kim E: Characterization of the Shank family of synaptic proteins. Multiple genes, alternative splicing, and differential expression in brain and development. J Biol Chem. 1999, 274: 29510-29518. 10.1074/jbc.274.41.29510.View ArticlePubMedGoogle Scholar
- Leblond CS, Heinrich J, Delorme R, Proepper C, Betancur C, Huguet G, Konyukh M, Chaste P, Ey E, Rastam M, Anckarsäter H, Nygren G, Gillberg IC, Melke J, Toro R, Regnault B, Fauchereau F, Mercati O, Lemière N, Skuse D, Poot M, Holt R, Monaco AP, Järvelä I, Kantojärvi K, Vanhala R, Curran S, Collier DA, Bolton P, Chiocchetti A: Genetic and functional analyses of SHANK2 mutations suggest a multiple hit model of autism spectrum disorders. PLoS Genet. 2012, 8: e1002521-10.1371/journal.pgen.1002521.PubMed CentralView ArticlePubMedGoogle Scholar
- Maunakea AK, Nagarajan RP, Bilenky M, Ballinger TJ, D’Souza C, Fouse SD, Johnson BE, Hong C, Nielsen C, Zhao Y, Turecki G, Delaney A, Varhol R, Thiessen N, Shchors K, Heine VM, Rowitch DH, Xing X, Fiore C, Schillebeeckx M, Jones SJ, Haussler D, Marra MA, Hirst M, Wang T, Costello JF: Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature. 2010, 466: 253-257. 10.1038/nature09165.PubMed CentralView ArticlePubMedGoogle Scholar
- Waga C, Asano H, Sanagi T, Suzuki E, Nakamura Y, Tsuchiya A, Itoh M, Goto YI, Kohsaka S, Uchino S: Identification of two novel Shank3 transcripts in the developing mouse neocortex. J Neurochem. 2014, 128 (2): 280-293. 10.1111/jnc.12505.View ArticlePubMedGoogle Scholar
- Zhu L, Wang X, Li XL, Towers A, Cao X, Wang P, Bowman R, Yang H, Goldstein J, Li YJ, Jiang YH: Epigenetic dysregulation of SHANK3 in brain tissues from individuals with autism spectrum disorders. Hum Mol Genet. 2014, 23 (6): 1563-1783. 10.1093/hmg/ddt547.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang X, Bey AL, Chung L, Krystal AD, Jiang YH: Therapeutic approaches for shankopathies. Dev Neurobiol. 2014, 74: 123-135. 10.1002/dneu.22084.View ArticlePubMedGoogle Scholar
- Kim HJ, Magrane J: Isolation and culture of neurons and astrocytes from the mouse brain cortex. Methods Mol Biol. 2011, 793: 63-75. 10.1007/978-1-61779-328-8_4.View ArticlePubMedGoogle Scholar
- Schneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012, 9: 671-675. 10.1038/nmeth.2089.View ArticlePubMedGoogle Scholar
- Wang XM, Li J, Feng XC, Wang Q, Guan DY, Shen ZH: Involvement of the role of Chk1 in lithium-induced G2/M phase cell cycle arrest in hepatocellular carcinoma cells. J Cell Biochem. 2008, 104: 1181-1191. 10.1002/jcb.21693.View ArticlePubMedGoogle Scholar
- Bockers TM, Mameza MG, Kreutz MR, Bockmann J, Weise C, Buck F, Richter D, Gundelfinger ED, Kreienkamp HJ: Synaptic scaffolding proteins in rat brain. Ankyrin repeats of the multidomain Shank protein family interact with the cytoskeletal protein alpha-fodrin. J Biol Chem. 2001, 276: 40104-40112. 10.1074/jbc.M102454200.View ArticlePubMedGoogle Scholar
- Ehlers MD: Activity level controls postsynaptic composition and signaling via the ubiquitin-proteasome system. Nat Neurosci. 2003, 6: 231-242. 10.1038/nn1013.View ArticlePubMedGoogle Scholar
- Flavell SW, Greenberg ME: Signaling mechanisms linking neuronal activity to gene expression and plasticity of the nervous system. Annu Rev Neurosci. 2008, 31: 563-590. 10.1146/annurev.neuro.31.060407.125631.PubMed CentralView ArticlePubMedGoogle Scholar
- Fischer A, Sananbenesi F, Mungenast A, Tsai LH: Targeting the correct HDAC(s) to treat cognitive disorders. Trends Pharmacol Sci. 2010, 31: 605-617. 10.1016/j.tips.2010.09.003.View ArticlePubMedGoogle Scholar
- Schor IE, Rascovan N, Pelisch F, Allo M, Kornblihtt AR: Neuronal cell depolarization induces intragenic chromatin modifications affecting NCAM alternative splicing. Proc Natl Acad Sci U S A. 2009, 106: 4325-4330. 10.1073/pnas.0810666106.PubMed CentralView ArticlePubMedGoogle Scholar
- Beri S, Tonna N, Menozzi G, Bonaglia MC, Sala C, Giorda R: DNA methylation regulates tissue-specific expression of Shank3. J Neurochem. 2007, 101: 1380-1391. 10.1111/j.1471-4159.2007.04539.x.View ArticlePubMedGoogle Scholar
- Lim S, Sala C, Yoon J, Park S, Kuroda S, Sheng M, Kim E: Sharpin, a novel postsynaptic density protein that directly interacts with the shank family of proteins. Mol Cell Neurosci. 2001, 17: 385-397. 10.1006/mcne.2000.0940.View ArticlePubMedGoogle Scholar
- Scott DB, Blanpied TA, Swanson GT, Zhang C, Ehlers MD: An NMDA receptor ER retention signal regulated by phosphorylation and alternative splicing. J Neurosci. 2001, 21: 3063-3072.PubMedGoogle Scholar
- Missler M, Sudhof TC: Neurexins: three genes and 1001 products. Trends Genet. 1998, 14: 20-26. 10.1016/S0168-9525(97)01324-3.View ArticlePubMedGoogle Scholar
- Aid T, Kazantseva A, Piirsoo M, Palm K, Timmusk T: Mouse and rat BDNF gene structure and expression revisited. J Neurosci Res. 2007, 85: 525-535. 10.1002/jnr.21139.PubMed CentralView ArticlePubMedGoogle Scholar
- Lenroot RK, Yeung PK: Heterogeneity within autism spectrum disorders: what have We learned from neuroimaging studies?. Front Hum Neurosci. 2013, 7: 733-PubMed CentralView ArticlePubMedGoogle Scholar
- Rice K, Moriuchi JM, Jones W, Klin A: Parsing heterogeneity in autism spectrum disorders: visual scanning of dynamic social scenes in school-aged children. J Am Acad Child Adolesc Psychiatry. 2012, 51: 238-248. 10.1016/j.jaac.2011.12.017.PubMed CentralView ArticlePubMedGoogle Scholar
- Betancur C: Etiological heterogeneity in autism spectrum disorders: more than 100 genetic and genomic disorders and still counting. Brain Res. 2011, 1380: 42-77.View ArticlePubMedGoogle Scholar
- Abrahams BS, Geschwind DH: Advances in autism genetics: on the threshold of a new neurobiology. Nat Rev Genet. 2008, 9: 341-355. 10.1038/nrg2346.PubMed CentralView ArticlePubMedGoogle Scholar
- Szatmari P: Heterogeneity and the genetics of autism. J Psychiatry Neurosci. 1999, 24: 159-165.PubMed CentralPubMedGoogle Scholar
- Jiang YH, Yuen RK, Jin X, Wang M, Chen N, Wu X, Ju J, Mei J, Shi Y, He M, Wang G, Liang J, Wang Z, Cao D, Carter MT, Chrysler C, Drmic IE, Howe JL, Lau L, Marshall CR, Merico D, Nalpathamkalam T, Thiruvahindrapuram B, Thompson A, Uddin M, Walker S, Luo J, Anagnostou E, Zwaigenbaum L, Ring RH: Detection of clinically relevant genetic variants in autism spectrum disorder by whole-genome sequencing. Am J Hum Genet. 2013, 93: 249-263. 10.1016/j.ajhg.2013.06.012.PubMed CentralView ArticlePubMedGoogle Scholar
- Sanders SJ, Murtha MT, Gupta AR, Murdoch JD, Raubeson MJ, Willsey AJ, Ercan-Sencicek AG, DiLullo NM, Parikshak NN, Stein JL, Walker MF, Ober GT, Teran NA, Song Y, El-Fishawy P, Murtha RC, Choi M, Overton JD, Bjornson RD, Carriero NJ, Meyer KA, Bilguvar K, Mane SM, Sestan N, Lifton RP, Günel M, Roeder K, Geschwind DH, Devlin B, State MW: De novo mutations revealed by whole-exome sequencing are strongly associated with autism. Nature. 2012, 485: 237-241. 10.1038/nature10945.PubMed CentralView ArticlePubMedGoogle Scholar
- Iossifov I, Ronemus M, Levy D, Wang Z, Hakker I, Rosenbaum J, Yamrom B, Lee YH, Narzisi G, Leotta A, Kendall J, Grabowska E, Ma B, Marks S, Rodgers L, Stepansky A, Troge J, Andrews P, Bekritsky M, Pradhan K, Ghiban E, Kramer M, Parla J, Demeter R, Fulton LL, Fulton RS, Magrini VJ, Ye K, Darnell JC, Darnell RB: De novo gene disruptions in children on the autistic spectrum. Neuron. 2012, 74: 285-299. 10.1016/j.neuron.2012.04.009.PubMed CentralView ArticlePubMedGoogle Scholar
- Neale BM, Kou Y, Liu L, Ma’ayan A, Samocha KE, Sabo A, Lin CF, Stevens C, Wang LS, Makarov V, Polak P, Yoon S, Maguire J, Crawford EL, Campbell NG, Geller ET, Valladares O, Schafer C, Liu H, Zhao T, Cai G, Lihm J, Dannenfelser R, Jabado O, Peralta Z, Nagaswamy U, Muzny D, Reid JG, Newsham I, Wu Y: Patterns and rates of exonic de novo mutations in autism spectrum disorders. Nature. 2012, 485: 242-245. 10.1038/nature11011.PubMed CentralView ArticlePubMedGoogle Scholar
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