Regulation of the noradrenaline transporter in a model neuronal cell line: effects of cell signalllng pathways and the tricyclic antidepressant drug, desipramine

Abstract

The noradrenaline transporter (NAT) is found in presynaptic noradrenergic nerve terminals. It regulates noradrenergic neurotransmission by mediating reuptake of noradrenaline (NA) from the synapse. The NAT is inhibited by the tricyclic antidepressant agent, desipramine (DMI). While a single dose of DMI rapidly inhibits noradrenaline reuptake, repeated administration is required for therapeutic effects. This thesis aimed to investigate whether DMI has cellular effects in addition to the inhibition of the NAT. To research this hypothesis, the effect of DMI on protein expression and regulation of the NAT in cultured neuronal cells was investigated. Short-term DMI treatment was shown to increase the expression of phosphorylated CREB in several mammalian neuronal cell lines, as shown by immunocytochemistry. These results were confirmed by western blotting in human neuroblastoma SH-SY5Y cells, but not for the other cell lines. Short-term DMI treatment also appeared to augment the expression of phosphorylated substrates of PKC and MAPK, as investigated by western blotting with kinase specific phospho-protein antibodies. However, there did not appear to be phosphoproteins expressed specifically in response to DMI. To study the regulation of the NAT, a stable human noradrenergic neuroblastoma cell line, SY5Y cells transfected to express a GFP-NAT fusion protein, was produced for this thesis work. SY5Y-GFP-NAT cells displayed a 3-5 fold increase in [3H]noradrenaline uptake with GFP-fluorescence localized to the plasma membrane. The level of GFP-NAT expression was sufficient for biotinylation analysis of surface NAT expression. SY5Y-GFP-NAT cells treated with the PKC activator β-PMA showed a significant reduction in both [3H]noradrenaline uptake and surface GFP-NAT expression, confirming that the cell line was suitable for studies of NAT regulation. Treatment with muscarinic receptor agonists was found to reduce NAT activity. This effect was abolished by the depletion of PKC activity, suggesting that the signalling pathway involved PKC. Unlike the effect of β-PMA, muscarinic agonists did not affect cell surface NAT expression. Regulation of the NAT by the insulin signalling pathway was investigated in SY5Y-GFPNAT cells. Insulin treatment robustly increased [3H]noradrenaline uptake, an effect blocked by the tyrosine kinase inhibitor, genistein. Genistein treatment alone significantly reduced noradrenaline uptake, suggesting insulin receptor tyrosine kinase regulated basal NAT activity. Further investigations suggested that insulin-dependent regulation of the NAT involved cross-talk between PKC and PI3K signalling pathways. DMI treatment of 1 to 3 days was shown to reduce cell surface NAT expression in the SY5Y-GFP-NAT cells. This was detected both by GFP fluorescence and surface protein biotinylation. Increased intracellular GFP-NAT was also observed in DMI-treated cells. GFP-NAT associated mostly with recycling endosomes after 1-day of DMI treatment, as shown by co-localization with transferrin conjugated to Alexa-568. GFP-NAT became associated with late endosomes and lysosomes after 2 – 3 days of DMI treatment. A model was developed to explain the effect of DMI on the cellular trafficking of the NAT. This research clearly demonstrated a novel finding, that the tricyclic antidepressant drug DMI, affects the cellular distribution of the NAT in the SY5Y-GFP-NAT model cell-line. While specific changes in protein expression in response to DMI were not found, it appears likely that regulation of the NAT by DMI treatment involves cell signalling pathways and post-translational protein modification. The SY5Y-GFP-NAT cell, developed during this research, is a novel cell-line that has proved its value for studies of the regulation and trafficking of the NAT. This cell line should be useful for future studies of potential transporter-protein interactions and additional studies of the cellular actions of DMI.