Analysis of copper transport in B104 neuroblastoma cell model

The arising of a number of neurodegenerative disorders in mammals has been correlated to the impairment of copper homeostasis in the CNS that is far from being completely understood. In the attempt to give a contribution to clarify some molecular aspects of this relationship, we used a rat neuroblastoma cell line (B104) as a cell model aiming to examine how extracellular copper availability can affect the protein expression pattern and Cu fluxes. Briefly, B104 cells were cultured under Cu depletion for rising time periods (48, 96 hours). To reveal possible changes in the protein expression pattern (CTR1, DCT1, PRPc, ATP7A) we conducted Real Time PCR and Immunoblotting analysis, that enlightened an expected complexity of cuproprotein genes expression and regulation. Surprisingly the activation of a transcriptional adaptive response involving the cellular prion protein and the high affinity copper transporter CTR1 was evidenced, that hasn’t been reported before. We tried to estimate possible alterations of transmembrane copper fluxes in ion deprived cells by the employment of a fluorimetric method based on the use of the Cu-sensitive probe Phen Green SK. To verify the suitability of such a method with respect to our intent a preliminary study was carried out to verify the dependence of the rate of copper influx on external potassium and the presence of metal ions (Ag, Cd, Mn, Zn), typically associated to CTR1-mediated Cu entry. A confocal microscopy approach was additionally developed to confirm the specificity of the response of the intracellular Phen Green SK probe to copper intake. In conclusion, our results support the hypothesis that the adapting strategies developed by B104 neuroblastoma cell model in response to reduced copper availability pass through the activation of a wide transcriptional adaptive response with suspected implications in the pathophysiology of neurodegenerative disorders.