University of Massachusetts Medical School Faculty Publications

UMMS Affiliation

Brudnick Neuropsychiatric Research Institute; Department of Psychiatry; Biomedical Imaging Group, Program in Molecular Medicine; Melikian Lab; Graduate School of Biomedical Sciences, Program in Neuroscience

Publication Date

9-27-2017

Document Type

Article

Disciplines

Molecular and Cellular Neuroscience

Abstract

Presynaptic reuptake, mediated by the dopamine (DA) transporter (DAT), terminates DAergic neurotransmission and constrains extracellular DA levels. Addictive and therapeutic psychostimulants inhibit DA reuptake and multiple DAT coding variants have been reported in patients with neuropsychiatric disorders. These findings underscore that DAT is critical for DA neurotransmission and homeostasis. DAT surface availability is regulated acutely by endocytic trafficking, and considerable effort has been directed toward understanding mechanisms that govern DAT's plasma membrane expression and postendocytic fate. Multiple studies have demonstrated DAT endocytic recycling and enhanced surface delivery in response to various stimuli. Paradoxically, imaging studies have not detected DAT targeting to classic recycling endosomes, suggesting that internalized DAT targets to either degradation or an undefined recycling compartment. Here, we leveraged PRIME (PRobe Incorporation Mediated by Enzyme) labeling to couple surface DAT directly to fluorophore, and tracked DAT's postendocytic itinerary in immortalized mesencephalic cells. Following internalization, DAT robustly targeted to retromer-positive endosomes, and DAT/retromer colocalization was observed in male mouse dopaminergic somatodendritic and terminal regions. Short hairpin RNA-mediated Vps35 knockdown revealed that DAT endocytic recycling requires intact retromer. DAT also targeted rab7-positive endosomes with slow, linear kinetics that were unaffected by either accelerating DAT internalization or binding a high-affinity cocaine analog. However, cocaine increased DAT exit from retromer-positive endosomes significantly. Finally, we found that the DAT carboxy-terminal PDZ-binding motif was required for DAT recycling and exit from retromer. These results define the DAT recycling mechanism and provide a unifying explanation for previous, seemingly disparate, DAT endocytic trafficking findings.

SIGNIFICANCE STATEMENT The neuronal dopamine (DA) transporter (DAT) recaptures released DA and modulates DAergic neurotransmission, and a number of DAT coding variants have been reported in several DA-related disorders, including infantile parkinsonism, attention-deficit/hyperactivity disorder and autism spectrum disorder. DAT is also competitively inhibited by psychostimulants with high abuse potential. Therefore, mechanisms that acutely affect DAT availability will likely exert significant impact on both normal and pathological DAergic homeostasis. Here, we explore the cellular mechanisms that acutely control DAT surface expression. Our results reveal the intracellular mechanisms that mediate DAT endocytic recycling following constitutive and regulated internalization. In addition to shedding light on this critical process, these findings resolve conflict among multiple, seemingly disparate, previous reports on DAT's postendocytic fate.

Keywords

cocaine, dopamine, endocytosis, retromer, reuptake, trafficking

Rights and Permissions

Copyright © 2017 the authors. Publisher PDF posted after 6 months as allowed by the publisher's author rights policy at http://www.jneurosci.org/sites/default/files/files/JN_License_to_Publish.pdf.

DOI of Published Version

10.1523/JNEUROSCI.3885-16.2017

Source

J Neurosci. 2017 Sep 27;37(39):9438-9452. doi: 10.1523/JNEUROSCI.3885-16.2017. Epub 2017 Aug 28. Link to article on publisher's site

Related Resources

Link to Article in PubMed

Journal/Book/Conference Title

The Journal of neuroscience : the official journal of the Society for Neuroscience

PubMed ID

28847807

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