Abstract
Variants of the SH3 and multiple ankyrin repeat domains 3 (SHANK3), which encodes postsynaptic scaffolds, are associated with brain disorders. The targeted alleles in a few Shank3 knock-out (KO) lines contain a neomycin resistance (Neo) cassette, which may perturb the normal expression of neighboring genes; however, this has not been investigated in detail. We previously reported an unexpected increase in the mRNA expression of Shank3 exons 1–12 in the brains of Shank3B KO mice generated by replacing Shank3 exons 13–16 with the Neo cassette. In this study, we confirmed that the increased Shank3 mRNA in Shank3B KO brains produced an unusual ∼60 kDa Shank3 isoform (Shank3-N), which did not properly localize to the synaptic compartment. Functionally, Shank3-N overexpression altered the dendritic spine morphology in cultured neurons. Importantly, Shank3-N expression in Shank3B KO mice was not a compensatory response to a reduction of full-length Shank3 because expression was still detected in the brain after normalizing the level of full-length Shank3. Moreover, in another Shank3 KO line (Shank3 gKO) with a similar Shank3 exonal deletion as that in Shank3B KO mice but without a Neo cassette, the mRNA expression levels of Shank3 exons 1–12 were lower than those of wild-type mice and Shank3-N was not detected in the brain. In addition, the expression levels of genes neighboring Shank3 on chromosome 15 were altered in the striatum of Shank3B KO but not Shank3 gKO mice. These results suggest that the Neo cassette has potential off-target effects in Shank3B KO mice.
Highlights
The SRC homology 3 (SH3) and multiple ankyrin repeat domains protein 3 (SHANK3) gene encodes core scaffolding proteins that organize the macromolecular protein complex in the postsynaptic density of glutamatergic excitatory synapses (Sheng and Kim, 2000, 2011)
To better determine the identity of the ∼60 kDa Shank3 protein expressed in Shank3B KO brains, we expressed an untagged Shank3 protein in HEK293T cells (Shank3-N, aa 1–536 of 1,730 aa full-length Shank3), corresponding to the Shank3 protein residues encoded by Shank3 exons 1–12 (Figure 1B)
Because Shank3-N did not contain the C-terminal sterile alpha motif (SAM) domain of full-length Shank3 (Figure 1B), which is critical for synaptic targeting and multimerization (Boeckers et al, 2005; Baron et al, 2006; Hayashi et al, 2009), we investigated its subcellular distribution in the striatum, cortex, and hippocampus of Shank3B KO mice
Summary
The SH3 and multiple ankyrin repeat domains protein 3 (SHANK3) gene encodes core scaffolding proteins that organize the macromolecular protein complex in the postsynaptic density of glutamatergic excitatory synapses (Sheng and Kim, 2000, 2011). To understand the detailed neurobiological mechanisms underlying SHANK3-associated brain disorders, more than 10 different Shank knock-out (KO) mouse lines carrying deletions of different Shank exons (of total 22 exons) have been generated to date, and molecular, cellular, and functional changes in their brains have been characterized (Jiang and Ehlers, 2013; Monteiro and Feng, 2017; Eltokhi et al, 2018). The Neo cassette can affect the expression of neighboring genes in the genome (i.e., off-target effects) (Pham et al, 1996; Scacheri et al, 2001; Meier et al, 2010; West et al, 2016). It is possible that the expression levels of the remaining Shank exons as well as neighboring genes on mouse chromosome 15 are affected by the Neo cassette in these Shank KO lines; this has not been carefully considered or investigated
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