Abstract
The synaptic protein SHANK3 encodes a multidomain scaffold protein expressed at the postsynaptic density of neuronal excitatory synapses. We previously identified de novo SHANK3 mutations in patients with autism spectrum disorders (ASD) and showed that SHANK3 represents one of the major genes for ASD. Here, we analyzed the pyramidal cortical neurons derived from induced pluripotent stem cells from four patients with ASD carrying SHANK3 de novo truncating mutations. At 40–45 days after the differentiation of neural stem cells, dendritic spines from pyramidal neurons presented variable morphologies: filopodia, thin, stubby and muschroom, as measured in 3D using GFP labeling and immunofluorescence. As compared to three controls, we observed a significant decrease in SHANK3 mRNA levels (less than 50% of controls) in correlation with a significant reduction in dendritic spine densities and whole spine and spine head volumes. These results, obtained through the analysis of de novo SHANK3 mutations in the patients’ genomic background, provide further support for the presence of synaptic abnormalities in a subset of patients with ASD.
Highlights
Autism spectrum disorders (ASD) are characterized by atypical social communications, and the presence of restricted and repetitive patterns of behavior
We used the human induced pluripotent stem cells (iPSC)-based model to analyze the effects of heterozygous truncating de novo SHANK3 mutations found in four patients with autism spectrum disorders (ASD), and presenting moderate to severe intellectual disability (ID)
late cortical progenitors (LCP) were differentiated into cortical neurons of the superficial layers II-IV, which correspond to the cortical upper layers as confirmed previously by Cux[1], Cux[2] and Brn[2] immunolabeling[27]
Summary
Autism spectrum disorders (ASD) are characterized by atypical social communications, and the presence of restricted and repetitive patterns of behavior. Results from four studies in which iPSC-derived neurons from patients were used showed a reduction in dendritic spine or excitatory synaptic transmission as compared with controls[19,20,21,22] These effects can be reversed by SHANK3 overexpression, insulin growth factor 1 (IGF1) treatment[19], Cdc2-like kinase 2 (CLK2) inhibition[20] or lithium treatment[21]. The authors showed that heterozygous and homozygous SHANK3 truncating mutations severely impair hyperpolarization-active cation channels (HCN) that are associated with reduced synaptic connectivity and transmission[24] All these results converge towards a synaptic deficit in patients with ASD carrying a SHANK3 mutation, but none of the previous studies has investigated the dendrite morphology of neurons carrying SHANK3 truncating mutations in the patient genetic background. We generated the corresponding iPSCs for their selective reprogramming into cortical neurons in order to examine the effects of SHANK3 haploinsufficiency on the level of SHANK3 mRNA and on the 3D spine morphogenesis organization
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