A PRIME Labeling Approach For Investigating Dopamine Transporter Endocytic Trafficking In Dopaminergic Cell Lines And Neurons

Abstract

The presynaptic dopamine transporter (DAT) facilitates synaptic DA reuptake that spatially and temporally constrains DA neurotransmission. DAT is inhibited by addictive psychostimulants, and the rewarding properties of those psychostimulants require their direct binding to DAT. More recently, aberrant DAT function has been identified in Attention Deficit/Hyperactivity Disorder, Autism Spectrum Disorder and Infantile Parkinsonism, emphasizing the critical role DAT plays in maintaining normal DA tone. DAT robustly traffics to and from the cell surface via the endocytic pathway. Numerous stimuli, including protein kinase C (PKC) activation and amphetamine (AMPH) exposure acutely decrease DAT surface levels in dopaminergic terminals by promoting DAT endocytosis. However, DAT's post‐endocytic fate is unclear and conflicting studies report that DAT targets to either recycling or degradative endocytic compartments, or that DAT may not traffic in bona fide DAergic neurons. These discrepancies may stem from the various approaches used examine DAT trafficking, which rely primarily on either antibody or inhibitory ligand‐based methods that could potentially mistarget internalized DAT. In the current study, we take advantage of a novel PRIME (Probe Incorporation Mediated by Enzyme) labeling strategy to track the DAT endocytic itinerary under basal and stimulated conditions. This approach covalently couples fluorophore to surface proteins that encode a ligase acceptor peptide (LAP), thereby facilitating direct protein visualization without the need for inhibitory ligands or bulky antibodies. We engineered the LAP peptide into the DAT 2nd extracellular loop (LAP‐DAT) and observed highly specific fluorophore labeling at the cell surface in cells expressing LAP‐DAT, but not in cells expressing wildtype DAT. LAP‐DAT expresses, functions, and exhibits PKC‐mediated downregulation comparable to wildtype DAT. Moreover, LAP‐DAT robustly internalized in the immortalized mesencephalic cell line, AN27, allowing us to generate a dynamic flow profile that tracks DAT's movement through the endocytic pathway. Ongoing studies using AAV2‐expressed LAP‐DAT in the striatum of TH‐Cre mice, will permit a rigorous examination of DAT endocytic trafficking in bona fide native dopaminergic neurons in ex vivo tissue samples.Support or Funding InformationNIH R01DA035224 to H.E.M.