Abstract
BackgroundSHANK proteins are crucial for the formation and plasticity of excitatory synapses. Although mutations in all three SHANK genes are associated with autism spectrum disorder (ASD), SHANK3 appears to be the major ASD gene with a prevalence of approximately 0.5% for SHANK3 mutations in ASD, with higher rates in individuals with ASD and intellectual disability (ID). Interestingly, the most relevant mutations are typically de novo and often are frameshift or nonsense mutations resulting in a premature stop and a truncation of SHANK3 protein.MethodsWe analyzed three different SHANK3 stop mutations that we identified in individuals with ASD and/or ID, one novel (c.5008A > T) and two that we recently described (c.1527G > A, c.2497delG). The mutations were inserted into the human SHANK3a sequence and analyzed for effects on subcellular localization and neuronal morphology when overexpressed in rat primary hippocampal neurons.ResultsClinically, all three individuals harboring these mutations had global developmental delays and ID. In our in vitro assay, c.1527G > A and c.2497delG both result in proteins that lack most of the SHANK3a C-terminus and accumulate in the nucleus of transfected cells. Cells expressing these mutants exhibit converging morphological phenotypes including reduced complexity of the dendritic tree, less spines, and less excitatory, but not inhibitory synapses. In contrast, the truncated protein based on c.5008A > T, which lacks only a short part of the sterile alpha motif (SAM) domain in the very SHANK3a C-terminus, does not accumulate in the nucleus and has minor effects on neuronal morphology.ConclusionsIn spite of the prevalence of SHANK3 disruptions in ASD and ID, only a few human mutations have been functionally characterized; here we characterize three additional mutations. Considering the transcriptional and functional complexity of SHANK3 in healthy neurons, we propose that any heterozygous stop mutation in SHANK3 will lead to a dysequilibrium of SHANK3 isoform expression and alterations in the stoichiometry of SHANK3 protein complexes, resulting in a distinct perturbation of neuronal morphology. This could explain why the clinical phenotype in all three individuals included in this study remains quite severe - regardless of whether there are disruptions in one or more SHANK3 interaction domains.Electronic supplementary materialThe online version of this article (doi:10.1186/s13229-015-0020-5) contains supplementary material, which is available to authorized users.
Highlights
SHANK proteins are crucial for the formation and plasticity of excitatory synapses
A crucial role of SHANK3 mutations in this context is supported by the following three facts: 1) SHANK3 haploinsufficiency is the critical factor for the development of neuropsychiatric symptoms in 22q13 deletion syndrome, known as Phelan-McDermid syndrome, 2) the current prevalence for SHANK3 mutations in individuals with autism spectrum disorder (ASD) in general is between 0.5% and 0.7%, and 3) data indicate that a SHANK3 mutation is present in approximately 2% of individuals with both ASD and intellectual disability (ID) [16,17,18]
[17] This study overexpressed Shank3b localizes to the nucleus accompanied by a reduced number of mature spines and excitatory synapses [37] - just as we report in this study for overexpression of SHANK3a harboring either c.1527G > A or c.2497delG mutations (Figures 2, 4) and as others have reported in previous studies for the rodent Shank3a homologue harboring either c.3679_3680insG or c.3349C > T [23,25]
Summary
SHANK proteins are crucial for the formation and plasticity of excitatory synapses. mutations in all three SHANK genes are associated with autism spectrum disorder (ASD), SHANK3 appears to be the major ASD gene with a prevalence of approximately 0.5% for SHANK3 mutations in ASD, with higher rates in individuals with ASD and intellectual disability (ID). In vitro examination of a de novo exon 21 nonsense mutation c.2997C > G identified in a boy with ID demonstrated a reduction in neurite nodes, tips, and length, at early stages of neuronal differentiation [22]. Taken together, these in vitro studies show that truncations of the distal C-terminus of Shank3a, caused by the c.3679_3680insG, c.3349C > T or c.2997C > G mutations are sufficient to disrupt neuronal morphology when the truncated variant is overexpressed in primary neuronal cultures
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