Abstract
Genetic variants of the SH3 and multiple ankyrin repeat domains 3 (SHANK3) gene, which encodes excitatory postsynaptic core scaffolds cause numerous brain disorders. Several lines of Shank3 knock-out (KO) mice with deletions of different Shank3 exons have previously been generated and characterized. The different Shank3 KO mouse lines have both common and line-specific phenotypes. Shank3 isoform diversity is considered a mechanism underlying phenotypic heterogeneity, and compensatory changes through regulation of Shank3 expression may contribute to this heterogeneity. However, whether such compensatory changes occur in Shank3 KO mouse lines has not been investigated in detail. Using previously reported RNA-sequencing analyses, we identified an unexpected increase in Shank3 transcripts in two different Shank3 mutant mouse lines (Shank3B and Shank3ΔC) having partial deletions of Shank3 exons. We validated an increase in Shank3 transcripts in the hippocampus, cortex, and striatum, but not in the cerebellum, of Shank3B heterozygous (HET) and KO mice, using qRT-PCR analyses. In particular, expression of the N-terminal exons 1–12, but not the more C-terminal exons 19–22, was observed to increase in Shank3B mice with deletion of exons 13–16. This suggests a selective compensatory activation of upstream Shank3 promoters. Furthermore, using domain-specific Shank3 antibodies, we confirmed that the increased Shank3 transcripts in Shank3B KO mice produced a small Shank3 isoform that was not detected in wild-type mice. Taken together, our results illustrate another layer of complexity in the regulation of Shank3 expression in the brain, which may also contribute to the phenotypic heterogeneity of different Shank3 KO mouse lines.
Highlights
Deletions, duplications, and various point mutations of the SH3 and multiple ankyrin repeat domains 3 (SHANK3) gene that encodes neuronal excitatory postsynaptic core scaffolds are causally associated with numerous brain disorders, including autism spectrum disorders, bipolar disorder, intellectual disability, and schizophrenia (Grabrucker et al, 2011; Monteiro and Feng, 2017)
In recent RNA-sequencing analyses of the striatum of adult Shank3B HET and KO mice (Lee et al, 2019), we had unexpectedly observed significantly increased total Shank3 transcripts in the KO striatum when compared to the WT striatum (Figure 1B)
To understand whether this increase in Shank3 transcripts was specific to the Shank3B KO line alone, we consulted another recently reported RNA-sequencing analysis (Qin et al, 2018) of the prefrontal cortex of Shank3∆C HET mice in which exon 21 of the Shank3 gene was targeted (Kouser et al, 2013)
Summary
Duplications, and various point mutations of the SH3 and multiple ankyrin repeat domains 3 (SHANK3) gene that encodes neuronal excitatory postsynaptic core scaffolds are causally associated with numerous brain disorders, including autism spectrum disorders, bipolar disorder, intellectual disability, and schizophrenia (Grabrucker et al, 2011; Monteiro and Feng, 2017). More than ten different lines of Shank KO mice having deletions of different Shank exons were generated and mutant phenotypes both common and specific to certain lines were identified (Monteiro and Feng, 2017). In addition to isoform diversity, any compensatory changes in these Shank regulatory mechanisms may contribute to variable phenotypes in different Shank KO mouse lines. We identified and validated an unexpected increase in Shank transcripts in the brain regions of Shank3B mice, in which exons 13–16 of the Shank gene are targeted. Our results reveal a novel compensatory change with respect to regulating Shank expression in the brain, which may contribute to the phenotypic heterogeneity of different Shank KO mouse lines
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