The role of Nogo-A in neuronal differentiation in SH-SY5Y cells.

Parkinson's disease is one of the most common neurodegenerative disorders, but to this day the pathophysiology of the loss of dopaminergic neurons in the Substantia nigra is not fully understood. Nogo-A, one of the most potent neurite outgrowth inhibitors of the central nervous system is thought to have a neuroprotective role as well as interfering with therapeutic research, limiting the sprouting of grafted dopaminergic neurons. This study aims at further understanding the role of Nogo-A in neuronal differentiation using the neuroblastoma SH-SY5Y cells. Differentiation of SH-SY5Y cells with staurosporine into cells exhibiting a dopaminergic phenotype was not affected by antogonization of the Nogo-A cascade. SH-SY5Y cells were successfully transduced witha lentiviral vector-based system for delivery of a CRISPR/Cas9 plasmid targeting the Nogo-A gene, thus creating a Nogo-A knock-out cell line. The creation of a Nogo-A knock-out SH-SY5Y cell population enables and facilitates further research on the involvement of Nogo-A in neuronal differentiation and degeneration

Quantitative characterization of phenotypical markers after differentiation of SH-SY5Y cells.

BACKGROUND The human neuroblastoma cell line, SH-SY5Y has been widely used in neuroscience research, especially in studies related to Parkinson's disease. However, differences between clones have been demonstrated, highlighting the importance to characterize the properties of this cell line carefully. OBJECTIVE The aim of this study was to characterize the phenotype of undifferentiated and differentiated SH-SY5Y cells using various differentiation protocols. METHODS A morphological and a quantitative analysis of markers related to dopaminergic and cholinergic neurons, but also other phenotypes, was performed. RESULTS Differentiated cells showed the typical neuronal morphology. Undifferentiated cells expressed low levels of tyrosine hydroxylase (TH) and higher levels of the high-affinity choline transporter (CHT1). Staurosporine (ST)-differentiation resulted in the highest number of TH-immunoreactive cells, followed by phorbol ester phorbol-12-myristate-13-acetat (PMA), whereas differentiation with brain-derived neurotropic factor (BDNF) did not increase TH-immunoreactive cells. TH, dopamine -hydroxylase and vesicular monoamine transporter-2 were also significantly upregulated in ST-differentiated cells compared to both undifferentiated and retinoic acid (RA)-differentiated cells. RA induced the highest number of CHT1-immunoreactive cells while ST- and BDNF-differentiation reduced CHT1-immunoreactive cells, indicating a decrease in the cholinergic phenotype. The presynaptic neuronal protein, αsynuclein, was significantly upregulated in RA- and ST-treated cells compared to undifferentiated cells. Ascorbic acid increased the number of CHT1-immunoreactive cells in all differentiation procedures and ST-differentiated TH-positive cells significantly. CONCLUSIONS Our findings indicate that a quantitative characterization of the phenotype is crucial when using SH-SY5Y cells to study the pathogenesis or evaluate compounds for treatment of neurodegenerative diseases