Modeling autism by SHANK gene mutations in mice

Abstract

Shank family proteins (Shank1, Shank2, and Shank3) are synaptic scaffolding proteins that organize an extensive protein complex at the postsynaptic density (PSD) of excitatory glutamatergic synapses. Recent human genetic studies indicate that SHANK family genes (SHANK1, SHANK2, and SHANK3) are causative genes for idiopathic autism spectrum disorders (ASD). Neurobiological studies of Shank mutations in mice support a general hypothesis of synaptic dysfunction in the pathophysiology of ASD. However, the molecular diversity of SHANK family gene products, as well as the heterogeneity in human and mouse phenotypes, pose challenges to modeling human SHANK mutations. Here, we review the molecular genetics of SHANK mutations in human ASD and discuss recent findings where such mutations have been modeled in mice. Conserved features of synaptic dysfunction and corresponding behaviors in Shank mouse mutants may help dissect the pathophysiology of ASD, but also highlight divergent phenotypes that arise from different mutations in the same gene.

Figure 1

SHANK Mutations Causing Autism-Related Phenotypes (A) SHANK3 is part of a large gene cluster associated with deletions in chromosome 22q13.3 deletion syndrome, or Phelan-McDermid syndrome, associated with autistic behaviors and intellectual disability (select genes are shown). The deletion sizes vary from 17 kb within SHANK3 to 10 Mb in 22q13.3. SHANK3 gene structure, mutations, and protein domains are shown. Human SHANK3 has 23 exons as deduced from cDNA AB569469 deposited in GenBank. Exon 11a is a newly identified exon. The positions of six identified promoters are indicated as black arrows. The exons in red are alternatively spliced. The positions of point mutations are indicated as blue arrows and the nature of point mutations are as described above the arrow. c.601-1G>A splicing mutation in intron 5 (Hamdan et al., 2011), p.Q312R in exon 8 (Moessner et al., 2007), p.G440_P446del and p.A447fs in exon 11 (Boccuto et al., 2012; Waga et al., 2011), c.1820-4 G>A splicing mutation in intron 15 (Boccuto et al., 2012), p.R656H in exon 16 (Waga et al., 2011), c.2265+1delG splicing mutation in intron 19 (Gauthier et al., 2009), and p.R1117X, p.A1227fs, p.E1311fs in exon 21 (Boccuto et al., 2012; Durand et al., 2007; Gauthier et al., 2010). Protein domains are shown and aligned to corresponding exons (ANK, ankyrin repeat domain; SH3, Src homology 3 domain; PDZ, PSD-95/Discs large/ZO-1 domain; Pro, a proline-rich region containing homer- and cortactin-binding sites; SAM, sterile alpha motif domain). CpG islands are sites of differential methylation and are indicated by green bars. (B) SHANK2 gene structure, protein domains, isoforms, microdeletions and mutations in ASD. Exons in red are alternatively spliced exons. Microdeletions found in ASD are intragenic and within SHANK2 genomic region. Point mutations found in ASD are indicated as blue arrows and the nature of mutations are described above. Three identifiable promoters corresponding to SHANK2E, 2A, 2B are indicated as black arrow. Two alternative stop codons (TAA) are indicated as yellow arrows. (C) SHANK1 gene structure, protein domains, isoforms, and microdeletions in ASD. Two small deletions including SHANK1 and two other genes are shown. No pathological point mutations in SHANK1 have been reported in ASD. The positions for two promoters are shown (Lim et al., 1999). The alternative splicing and isoforms have not been fully characterized. In (A)-(C), genomic distance and exons are not drawn to scale.

Figure 2

Shank Protein Interactions and Isoform-Specific Domain Structure (A) Schematic of the partial Shank protein interactome at the PSD with Shank3 as a model. A more complete list of Shank family interacting proteins is shown in Table 2. Protein domains in Shank family members are similar. Many interacting proteins interact with all three Shank family proteins (Shank1, Shank2, and Shank3) in in vitro assays. The proteins in red font are altered in Shank3 mutant mice. (B) Diagram of SHANK3 protein isoforms SHANK3a-f. Protein domain structure was deduced from confirmed mRNAs expressed from different promoters in human and mouse brains. Polypeptides have not been validated due to the lack of isoform-specific antibodies. Pro, proline rich region. (C) Diagram of SHANK2 protein isoforms of SHANK2A, 2B, 2C, and 2E [modified from (Leblond et al., 2012)]. SHANK2C has an alternative stop codon due to the alternative splicing of exons 19 and 20 as shown in Figure 1B.

Figure 3

Targeted Mutations in Shank Genes in Mice (A) Schematic of the mouse Shank3 gene structure deduced from cDNA AB230103 deposited in GenBank. The promoters are shown by arrows and alternatively spliced exons are indicated in red. The positions of targeted mutations in five different lines of Shank3 mutant mice are shown. The transcripts from promoters upstream of deleted exons are predicted to be truncated or disrupted (red arrows) and the transcripts from promoter downstream of deleted exons are predicted to be intact in each mutant line of mice (black arrows). Bottom panels depict predicted isoform-specific expression of Shank3 mRNA and proteins in Shank3 mutant mice. The “−” indicates that the isoform is disrupted and “+” indicates the isoform remains intact. The full complement of Shank3 mRNA and protein isoforms that derive from combinations of alternative promoters and mRNA splicing remains unknown. Therefore, the pattern of isoform-specific expression and disruption by specific mutations is likely more complex than indicated. (B) Schematic of mouse Shank2 gene structure and mouse mutations of Shank2. The sequence of Shank2 full length mRNA is not available and the intron-exon structure is deduced from a longest rat Shank2 cDNA deposited in GenBank (NM_201350). Exons in red are alternatively spliced. The human isoforms of SHANK2E, SHANK2A, and SHANK2B are conserved in mice (Shank2e, Shank2a, and Shank2b) but the status of SHANK2C is unknown. The Δex7 and Δex6-7 mutations (exon numbering based on the sequences of Shank2a/ProSAP1A cDNAs NM_001113373 or AB099695) described in the Shank2 mutant mice are shown (Schmeisser et al., 2012; Won et al., 2012). Exons 6 and 7 in Shank2a are deduced to correspond to exons 16 and 17 in the full length Shank2 gene structure diagram. The Shank2 Δex7 or Δex6-7 is predicted to cause truncation of all transcripts of Shank2 from the promoters upstream of exons 6-7 (red arrows). (C). Schematic of mouse Shank1 gene structure and mouse mutation of Shank1. The deletion of exons 14-5 is predicted to disrupt all isoforms of Shank1 (Shank1a and 1b in red arrows) (Hung et al., 2008). In (A)-(C), genomic distance and exons are not drawn to scale.