SHANK2 mutations impair apoptosis, proliferation and neurite outgrowth during early neuronal differentiation in SH-SY5Y cells

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.

SHANK2 Mutations Result in Dysregulation of the ERK1/2 Pathway in Human Induced Pluripotent Stem Cells-Derived Neurons and Shank2(−/−) Mice

“…Increased proliferation and reduced apoptosis have already been shown in SH-SY5Y cells with SHANK2 mutations, mimicking early neuronal development (Unsicker et al, 2021). Unsicker et al (2021) linked these findings to increased phospho-AKT expression, which was not observed in our study.…”

“…Increased proliferation and reduced apoptosis have already been shown in SH-SY5Y cells with SHANK2 mutations, mimicking early neuronal development (Unsicker et al, 2021). Unsicker et al (2021) linked these findings to increased phospho-AKT expression, which was not observed in our study. However, Unsicker et al (2021) used a less complex model system and, in addition, only observed increased phospho-AKT levels in the biallelic SHANK2 mutation, possibly triggering certain signaling pathways stronger than our heterozygous deletion.…”

Cellular Models in Schizophrenia Research

“…The CRISPR/Cas9 system has been used to introduce mono-or bi-allelic SHANK2 frameshift mutations into SH-SY5Y cells. These mutations impair early neuronal differentiation in SH-SY5Y cells, change cell growth properties and reduce pre-and postsynaptic protein expression [26]. These data support the constructive validity of the SH-SY5Y cell line.…”

Gene Dosage- and Age-Dependent Differential Transcriptomic Changes in the Prefrontal Cortex of Shank2-Mutant Mice

“…However, our GSEA on Shank2mutant transcriptomes did not detect any significant changes in mTOR signaling-related gene expressions, although altered mTOR signaling should be directly tested at the protein level using protein phosphorylation/activity as readouts. Intriguingly, bi-allelic but not mono-allelic deletion of Shank2 has been shown to increase Akt phosphorylation without changes in total protein levels during early neuronal differentiation in SH-SY5Y cells, which involved decreased apoptosis and increased cell proliferation (Unsicker et al, 2021), suggesting that Shank2 regulates mTOR signaling downstream of receptor tyrosine kinase activation. Intriguingly, mitochondria-related gene expressions are changed largely in parallel with those of ribosome/translationrelated genes in W3-HT, W12-HT, and W12-HM mice.…”