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.

Late in neural development, the expression of growth/differentiation factor (GDF) 15 increases in the germinal epithelium of the murine ganglionic eminence (GE). However, the function of GDF15 in this region is unknown. We here show that ablation of GDF15 leads to an increase in proliferation of apically and subapically dividing progenitors in the GE. This is associated with faster cell cycle progression in both progenitor groups, and an increase in the total number of cycling progenitors. Enhanced proliferation of apically dividing progenitors leads to a permanent significant increase in the number of ependymal and apical neural stem cells (NSCs). Our data also indicate that the extra proliferation of subapically dividing progenitors causes a transient increase in the number of neuronal progenitors, which is compensated by increased apoptosis. Independent of the genotype, activity of endogenous epidermal growth factor (EGFR) signalling is essential for the proliferation of apically and subapically dividing progenitors. However, lack of GDF15 leads to a reduced cell surface expression of EGFR and altered dynamics of MAPK activation in response to EGF stimulation. Application of exogenous GDF15 rescued the effect of the genotype on the expression of EGFR and decreased proliferation in the mutant GE. Taken together, our results indicate that GDF15 modulates proliferation and growth factor responsiveness of apical progenitors in the developing GE, thereby regulating the number of total ependymal and NSCs.

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