P25α / TPPP expression increases plasma membrane presentation of the dopamine transporter and enhances cellular sensitivity to dopamine toxicity

Parkinson’s disease is characterized by preferential degeneration of the dopamine-producing neurons of the brain stem substantia nigra. Imbalances between mechanisms governing dopamine transport across the plasma membrane and cellular storage vesicles increase the level of toxic pro-oxidative cytosolic dopamine. The microtubule-stabilizing protein p25α accumulates in dopaminergic neurons in Parkinson’s disease. We hypothesized that p25α modulates the subcellular localization of the dopamine transporter via effects on sorting vesicles, and thereby indirectly affects its cellular activity. Here we show that co-expression of the dopamine transporter with p25α in HEK-293-MSR cells increases dopamine uptake via increased plasma membrane presentation of the transporter. No direct interaction between p25α and the dopamine transporter was demonstrated, but they co-fractionated during subcellular fractionation of brain tissue from striatum, and direct binding of p25α peptides to brain vesicles was demonstrated. Truncations of the p25α peptide revealed that the requirement for stimulating dopamine uptake is located in the central core and were similar to those required for vesicle binding. Co-expression of p25α and the dopamine transporter in HEK-293-MSR cells sensitized them to the toxicity of extracellular dopamine. Neuronal expression of p25α thus holds the potential to sensitize the cells toward dopamine and toxins carried by the dopamine transporter. Structured digital abstract • MINT-8055798: DAT (uniprotkb:Q01959) and p25 alpha (uniprotkb:O94811) colocalize (MI:0403) by fluorescence microscopy (MI:0416) • MINT-8054201: p25 alpha (uniprotkb:B1Q0K1), bip (refseq:GI:194033595), Synaptophysin (uniprotkb:Q62277), Alpha-synuclein (uniprotkb:Q3I5G7) and DAT (uniprotkb:C6KE31) colocalize (MI:0403) by cosedimentation in solution (MI:0028) • MINT-8055878: Synaptophysin (uniprotkb:Q62277), bip (refseq:GI:194033595) and p25-alpha (uniprotkb:O94811) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)