Characterization of a Human Neuroblastoma Cell Line and Its Differentiation into Dopamine Neurons
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Dopamine (DA) synthesizing neurons are important because of their role in the control of motor movement, cognition and motivation. The development of DA neurons is marked by the presence of transcription factors and proteins that regulate specific stages during development. Given their specificity, these signals can be used as molecular markers for each developmental stage. The transcription factors NURR1 and PITX3 are two markers for post-mitotic DA neurons (neurons with the potential to become dopaminergic) whereas the presence of the dopamine transporter (DAT), a protein important for the reuptake of DA from the synaptic cleft, is used to identify a mature DA neuron (neurons that can synthesize and reuptake dopamine). The molecular mechanisms that govern DA neurons’ development are not fully understood and the lack of ideal cell models that recapitulate the phenotype of DA neurons makes challenging the study of the regulation of DA gene expression. To provide further insights into the role of NURR1 and PITX3 on the transcriptional regulation of DA marker genes such as the DAT, we studied their gene expression in two SK-N-AS cell lines, C line which expresses NURR1 at low basal levels, and E line engineered to over-express NURR1 by two fold. Our results have shown that indeed NURR1 and DAT are expressed in the SK-N-AS cell line, validating previously published research, while PITX3 is not expressed in these cells. Thus we suggest that PITX3 is not required for the expression of DAT gene, contrary to some previous data. Additionally, we have characterized the SK-N-AS morphologically by comparing the C-line with the E- line using the transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The most notable difference between the C and the E-line was the cytoplasmic content detected by TEM. Our results point toward the presence of more lysosomes autophagic vacuoles, residual bodies, and multivesicular bodies in the E-line. All these structures represent various stages in a dynamic process of intracellular digestion in which primary lysosomes fuse with their heterophagic vacuoles resulting in the degradation of their contents. As a result of degradation, residual bodies and multivesicular bodies are formed and they are recognized by the presence of a pigment called lipofucsin. Our findings suggests that SK-N-AS cells are not expressing several of the key signals involved in the DA pathway, so probably it is not an adequate model system to study DA molecular mechanisms. Morphologically, the over-expression of NURR1 in the E line changes the cells phenotypically by incrementing the amount of cytoplasmic content.