The online version of this article (doi:10.1186/2040-2392-5-30) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
XW: Conception and design, data collection and analysis, manuscript writing, critical revision, and final approval of the manuscript. QX: Data collection and analysis, manuscript writing, and final approval of the manuscript. ALB: Data collection, manuscript writing and editing, critical revision, and final approval of the manuscript. YL: Data collection, manuscript writing, and final approval of the manuscript. YHJ: Conception and design, financial support, manuscript writing, and final approval of the manuscript. All authors read and approved the final manuscript.
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.
Additional file 1: Table S1: List of Shank3 primers used in this study. (XLSX 10 KB)13229_2014_125_MOESM1_ESM.xlsx
Additional file 2: Figure S1: Brain-specific expression of Shank3 intragenic promoters. (A) Shank3 gene structure. Intragenic promoters are shown as black arrows. Exons in red are alternatively spliced exons. In lower panel, positions of primers for Shank3 isoforms and splice variants used in this study are indicated. Note that splicing forward primer (in red) for E18S is specific for promoter 5. (B) The presence of multiple intragenic promoters supported by in silico data from ENCODE project using mouse mm10 genome assembly (https://genomebrowser.wustl.edu/). The CHIP sequence data using antibodies against RNA polymerase II (Pol II) and trimethylated lysine 4 of histone 3 (H3K4me3), two landmarks for active promoters, displayed several corresponding peaks (blue rectangular, dash line indicates a weak promoter) near Shank3 promoters (arrows below Shank3 gene) in brain tissues but not in heart and kidney, indicating these intragenic promoters are brain specific. (C) Brain specific expression of Shank3 isoforms from intragenic promoters. Left panel: Western blot analysis using a Shank3 antibody against C-terminus of SHANK3 (sc-30193, Santa Cruz, CA, USA) revealed multiple bands across different tissues. The bands with predicted size of Shank3c, 3d, and 3e are observed in cerebral cortex and cerebellum but not in heart, liver, and kidney. Shank3b is not detectable because it does not contain the C-terminal sequence of Shank3 protein. This result supports that the intragenic promoters are brain-specific. Right panel: Ponceau S staining of the PVDF membrane on the left panel to show that an equal amount of protein (30 μg) was loaded to each lane. Note there is variation of endogenous protein composition across different tissues. (TIFF 1 MB)
Additional file 3: Figure S2: Extensive splicing of Shank3 mRNAs. (A–B) Shank3 exons 10–12 spliced (E10–12S) variants. At least five different products of E10–12S are identified by RT-PCR (A) and the sequences of E10–12S variants were illustrated (B). Arrows indicate the position of primers. I: no splicing. II: exon 11 partially spliced out. III: exon 11 spliced out. IV: exons 11 to 12 spliced out. V: exons 10 to 12 spliced out. Note that regular PCR reactions yield mostly spliced variants as the full-length of this sequence without alternative splicing can only be amplified in GC-rich buffer due to the extremely high GC percentage (79%) in exon 11 (see Materials and methods). (C–D) Shank3e spliced variants (E18S). Different sets of primers yielded various PCR products (C): left panel with primers 1 and 2, right panel with primers 1 and 3. The forward primer 1 is specific for Shank3e isoforms from promoter 5. Gene structure of full-length Shank3e and its spliced variants are illustrated in (D), with arrows showing the position for primers. I: no splicing. II: exon 18 spliced out. III: exons 18, 21, and 22 (partial) spliced out. IV: exons 18 to 21 and 22 (partial) spliced out. E–F, Splicing variants of Shank3 exon 21 and exon 22 (E21–22S). The PCR products are shown in (E), with left panel using primers 2 and 3, right panel using primers 1 and 3. Gene structure of exons 19–22 and its splicing variants were illustrated in (F), with primers labeled as arrows. I: no splicing. II: exon 22 partially spliced out. III: exon 21 spliced out. IV: exon 21 and exon 22 (partial) spliced out. Gene bank accession numbers are shown in red for novel splicing variants identified by the current study. Accession numbers in black are splicing variants identified in the previous study by Wang et al.. (TIFF 810 KB)
Additional file 4: Figure S3: Differential expression and alternative splicing of Shank3 isoforms in neurons and astrocytes. mRNAs from cultured hippocampal neurons and astrocytes were analyzed by RT-PCR. All major isoforms of Shank3 mRNAs were expressed abundantly in neurons as expected. Shank3a, 3c, 3d, 3e, and 3e-1 were low but readily detectable in astrocytes. Alternative splicing of exon 18 was mutually exclusive between cell types, with exon18 inclusion (E18S I) in neurons and exon18 exclusion (E18S II) in astrocytes. Similarly, the E18S III variant was exclusively observed in neurons while E18S IV was only found in astrocytes. N, neuron. A, astrocyte. (TIFF 579 KB)13229_2014_125_MOESM4_ESM.tiff
Additional file 5: Figure S4: Differentiated cellular distribution of Shank3 isoforms. Shank3 isoforms tagged with GFP (green) were expressed in COS-7 cells (A) or primary hippocampal neurons (B). Cells were co-stained with 4′,6-diamidino-2-phenylindole (DAPI) to label the nuclei (blue). PSD-95 antibody was employed to distinguish hippocampal neurons. Note that Shank3b localizes in nuclei in both COS-7 cells and hippocampal neurons. Shank3 E22: EGFP-Shank3 exon 22 that only encodes SAM domain. Scale bar: 10 μm. (TIFF 2 MB)13229_2014_125_MOESM5_ESM.tiff
Additional file 6: Figure S5: Shank3 isoforms are not localized in inhibitory synapses. Shank3 isoforms tagged with GFP (green) were expressed in primary hippocampal neurons and co-stained with an inhibitory post-synaptic marker Gephyrin (red). Note that there is no overlapping of Gephyrin with Shank3 isoforms. Scale bar: 10 μm. (TIFF 846 KB)13229_2014_125_MOESM6_ESM.tiff
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Sheng M, Kim E: The Shank family of scaffold proteins. J Cell Sci. 2000, 113 (Pt 11): 1851-1856. PubMed
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. CrossRefPubMed
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. PubMedCentralCrossRefPubMed
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. PubMedCentralCrossRefPubMed
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. PubMedCentralCrossRefPubMed
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. PubMedCentralCrossRefPubMed
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. PubMedCentralCrossRefPubMed
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. PubMedCentralCrossRefPubMed
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. PubMedCentralCrossRefPubMed
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. PubMed
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. PubMedCentralCrossRefPubMed
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. PubMedCentralCrossRefPubMed
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. PubMedCentralCrossRefPubMed
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. CrossRefPubMed
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. PubMed
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. PubMedCentralCrossRefPubMed
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. PubMedCentralCrossRefPubMed
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. PubMedCentralCrossRefPubMed
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. PubMedCentralCrossRefPubMed
- Transcriptional and functional complexity of Shank3 provides a molecular framework to understand the phenotypic heterogeneity of SHANK3 causing autism and Shank3 mutant mice
Alexandra L Bey
- BioMed Central
Neu in den Fachgebieten Neurologie und Psychiatrie
Meistgelesene Bücher in der Neurologie & Psychiatrie