A cell culture model for investigation of synapse influenceability: epigenetics, expression and function of gene targets important for synapse formation and preservation in SH-SY5Y neuroblastoma cells differentiated by retinoic acid

摘要

SH-SY5Y neuroblastoma cells are frequently used for different neuronal cell culture models. As there is no "gold-standard", miscellaneous protocols exist to differentiate these cells into a neuronal cell type. Here, the aim was to find a differentiation condition making cells suitable for investigation of influenceability of synapses by environmental conditions in pharmacologic experiments. For this purpose, effects on synapse molecules should be somehow rateable and cells should be usable for functional analysis like calcium imaging. A system like this is desirable for example in basic research concerning schizophrenia, depression, autism or neurodegeneration as synaptic plasticity and neuronal maturation are known to have a significant impact in these diseases. Cells grown on laminin-coated glass cover slips and treated with 50 mu M retinoic acid (RA) turned out to show most convincing morphological signs of neuronal differentiation and attached strongly to the ground, thereby also fulfilling preconditions for functional analysis. Systematic characterisation of this differentiation condition in comparison to non-treated controls revealed lower methylation rates and higher expression of most candidate molecules relevant for formation, preservation and function of synapses as well as differential function. In conclusion, this combination of differentiation strategy and markers seems to be a suitable system to estimate synapse modifications in basic research as it could help to identify possible dedifferentiating effects. To our knowledge, differentiation of SH-SY5Y has not been described as systematic before regarding comprehensiveness of the set of investigated synapse molecules and coverage of applied methods spanning from epigenetics to protein function. Furthermore, this is the first time that SH-SY5Y cells were differentiated on glass cover slips to an extent making them suitable for investigation of synapse molecules as part of stable intercellular connections in downstream functional analyses.