Department of Human Molecular Genetics, Institute of Human Genetics, University Hospital Heidelberg, 69120, Heidelberg, Germany.
Department of Internal Medicine V, University Hospital Heidelberg, Heidelberg, Germany.
Sci Rep. 2021 Jan 22;11(1):2128. doi: 10.1038/s41598-021-81241-4.
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 突变已在包括智力障碍和自闭症谱系障碍（ASD）在内的神经发育障碍患者中被发现。使用 CRISPR/Cas9 基因组编辑，我们获得了 SH-SY5Y 细胞系，这些细胞系在一个或两个 SHANK2 等位基因上具有移码突变。我们研究了不同的 SHANK2 突变对早期神经元分化过程中细胞形态、细胞增殖和分化潜能的影响。所有突变细胞系均显示神经元分化标志物表达受损。具有双等位基因 SHANK2 突变的细胞显示凋亡减少和增殖增加，以及早期神经元分化过程中神经突生长减少。双等位基因 SHANK2 突变导致 p-AKT 水平升高，表明 SHANK2 突变会损害酪氨酸激酶受体的下游信号。此外，与对照相比，具有双等位基因 SHANK2 突变的细胞中淀粉样前体蛋白（APP）的表达降低，这表明 SHANK2 和 APP 之间存在分子联系。总之，我们可以证明一个或两个 SHANK2 等位基因上的移码突变导致 SH-SY5Y 细胞中神经元分化的改变，其特征是细胞生长和前突触和后突触蛋白表达的变化。我们还提供了第一个证据，表明酪氨酸激酶受体的下游信号和淀粉样前体蛋白的表达受到影响。
