Unexpected Compensatory Increase in Shank3 Transcripts in Shank3 Knock-Out Mice Having Partial Deletions of Exons

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.

A chimeric mouse model to study human iPSC-derived neurons: the case of a truncating SHANK3 mutation

“…In contrast to our previous in vitro results, we did not observe any significant difference in morphological parameters of dendrites and spines from transplanted CTR and ASD neurons. Since SHANK3 is known to play a crucial role in both synaptic development and function, our results suggest that compensatory changes may occur under in vivo conditions, as was recently observed in Shank3 knock-out mice having partial deletions of exons 32 . Other authors also reported unexpected levels of complexity and modularity of SHANK3 functions in vivo using mouse models 33 .…”

Truncating mutations in SHANK3 associated with global developmental delay interfere with nuclear β‐catenin signaling

“…One might argue that mRNAs coding for truncated mutant forms of Shank3, as observed in our patient, will be degraded by the mechanism of nonsense‐mediated decay (NMD), to the effect that the truncated protein will not be produced. However, analysis of Shank3 transcripts in two mouse lines with truncating deletions in exons 13‐16 (central part of the protein, (Jin et al, 2019)) or exon 21 (C‐terminal part; (Qin et al, 2018)) demonstrated a strongly increased expression of transcripts coding for the remaining N‐terminal part of the protein. Thus, the lack of post‐synaptic Shank3 leads to a compensatory activation of upstream Shank3 promoters.…”

The Neomycin Resistance Cassette in the Targeted Allele of Shank3B Knock-Out Mice Has Potential Off-Target Effects to Produce an Unusual Shank3 Isoform

“…The results were analyzed using the comparative Ct method and were normalized against the levels of housekeeping gene Gapdh ( Han et al, 2013a ). The primer sequences for real-time PCR were described previously ( Jin et al, 2019a ) or are as follows:…”

“…Shank3B KO mice ( Peca et al, 2011 ) in which exons 13–16 of the Shank3 gene are replaced with the Neo cassette are among the most extensively characterized ASD models. We recently identified an unexpected increase in the mRNA expression of Shank3 exons 1–12 (i.e., 5′ of the Neo cassette) in the brains of Shank3B KO mice ( Jin et al, 2019a ; Lee et al, 2019a ). In particular, the mRNA levels of Shank3 exons 1–12 increased significantly in the cortex, striatum, and hippocampus of both juvenile and adult Shank3B KO mice.…”

“…In particular, the mRNA levels of Shank3 exons 1–12 increased significantly in the cortex, striatum, and hippocampus of both juvenile and adult Shank3B KO mice. Furthermore, in multiple brain regions of Shank3B KO mice, the increased mRNA of Shank3 exons 1–12 possibly produced an unusual ∼60 kDa Shank3 protein isoform containing the N-terminal domains of Shank3, which was not expressed in wild-type (WT) brains ( Jin et al, 2019a ). In a previous study, we speculated that the increase in mRNA levels of Shank3 exons 1–12 could be due to the compensatory activation of distal Shank3 promoters in response to reduced Shank3 protein levels in Shank3B KO brains ( Jin et al, 2019a ).…”