Abstract
SHANK2 mutations have been identified in individuals with neurodevelopmental disorders, including intellectual disability and autism spectrum disorders (ASD). Using CRISPR/Cas9 genome editing, we obtained SH-SY5Y cell lines with frameshift mutations on one or both SHANK2 alleles. We investigated the effects of the different SHANK2 mutations on cell morphology, cell proliferation and differentiation potential during early neuronal differentiation. All mutant cell lines showed impaired neuronal differentiation marker expression. Cells with bi-allelic SHANK2 mutations revealed diminished apoptosis and increased proliferation, as well as decreased neurite outgrowth during early neuronal differentiation. Bi-allelic SHANK2 mutations resulted in an increase in p-AKT levels, suggesting that SHANK2 mutations impair downstream signaling of tyrosine kinase receptors. Additionally, cells with bi-allelic SHANK2 mutations had lower amyloid precursor protein (APP) expression compared to controls, suggesting a molecular link between SHANK2 and APP. Together, we can show that frameshift mutations on one or both SHANK2 alleles lead to an alteration of neuronal differentiation in SH-SY5Y cells, characterized by changes in cell growth and pre- and postsynaptic protein expression. We also provide first evidence that downstream signaling of tyrosine kinase receptors and amyloid precursor protein expression are affected.
Highlights
SHANK2 mutations have been identified in individuals with neurodevelopmental disorders, including intellectual disability and autism spectrum disorders (ASD)
To investigate the functional consequences of SHANK2 mutations that have been identified in patients with ASD and intellectual disability, we introduced SHANK2 frameshift mutations in SH-SY5Y cells using CRISPR/Cas9-based genome editing
The non-homologous end-joining repair mechanism of the cells led to the introduction of a heterozygous adenosine insertion, which caused a frameshift and premature stop codon (6H6, p.L837Sfs*49)
Summary
SHANK2 mutations have been identified in individuals with neurodevelopmental disorders, including intellectual disability and autism spectrum disorders (ASD). Mutations in the gene coding for amyloid precursor protein (APP) have been identified in several individuals with AD22,23 Both SHANK2 and APP are expressed during neuronal differentiation and are involved in synapse formation and neural p lasticity[23,24,25], and could potentially interact in the course of ASD and neurodegenerative disorders. SHANK2 gene variants have first been investigated in primary hippocampal cultures of rodents by overexpressing the respective mutated protein[5,26] These analyses point to an impaired signal transduction at glutamatergic synapses, with a reduced synaptic density at d endrites[5,7] and reduced AMPA receptors at the cell s urface[26], which are impairments that potentially underlie different neurodevelopmental disorders. Findings in this cellular model should be reproduced in a more physiological model, in order to strengthen the respective findings
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