University of Massachusetts Medical School Faculty Publications

UMMS Affiliation

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

Publication Date

11-6-2013

Document Type

Article

Subjects

Animals; Cell Line, Tumor; Corpus Striatum; Cytoskeleton; Dopamine Plasma Membrane Transport Proteins; Dopaminergic Neurons; Dynamins; Endocytosis; Humans; Male; Mice; Protein Transport

Disciplines

Molecular and Cellular Neuroscience

Abstract

Dopaminergic signaling profoundly impacts rewarding behaviors, movement, and executive function. The presynaptic dopamine (DA) transporter (DAT) recaptures released DA, thereby limiting synaptic DA availability and maintaining dopaminergic tone. DAT constitutively internalizes and PKC activation rapidly accelerates DAT endocytosis, resulting in DAT surface loss. Longstanding evidence supports PKC-stimulated DAT trafficking in heterologous expression studies. However, PKC-stimulated DAT internalization is not readily observed in cultured dopaminergic neurons. Moreover, conflicting reports implicate both classic and nonclassic endocytic mechanisms mediating DAT trafficking. Prior DAT trafficking studies relied primarily upon chronic gene disruption and dominant-negative protein expression, or were performed in cell lines and cultured neurons, yielding results difficult to translate to adult dopaminergic neurons. Here, we use newly described dynamin inhibitors to test whether constitutive and PKC-stimulated DAT internalization are dynamin-dependent in adult dopaminergic neurons. Ex vivo biotinylation studies in mouse striatal slices demonstrate that acute PKC activation drives native DAT surface loss, and that surface DAT surprisingly partitions between endocytic-willing and endocytic-resistant populations. Acute dynamin inhibition reveals that constitutive DAT internalization is dynamin-independent, whereas PKC-stimulated DAT internalization is dynamin-dependent. Moreover, total internal reflection fluorescence microscopy experiments demonstrate that constitutive DAT internalization occurs equivalently from lipid raft and nonraft microdomains, whereas PKC-stimulated DAT internalization arises exclusively from lipid rafts. Finally, DAT endocytic recycling relies on a dynamin-dependent mechanism that acts in concert with the actin cytoskeleton. These studies are the first comprehensive investigation of native DAT trafficking in ex vivo adult neurons, and reveal that DAT surface dynamics are governed by complex multimodal mechanisms.

Rights and Permissions

Citation: J Neurosci. 2013 Nov 6;33(45):17836-46. doi: 10.1523/JNEUROSCI.3284-13.2013. Link to article on publisher's site

Comments

Copyright © 2013 the authors. Copyright of all material published in The Journal of Neuroscience remains with the authors. The authors grant the Society for Neuroscience an exclusive license to publish their work for the first 6 months. After 6 months the work becomes available to the public to copy, distribute, or display under a Creative Commons Attribution 4.0 International (CC BY 4.0) license.

Publisher PDF posted as allowed by the publisher's author rights policy at http://www.jneurosci.org/site/misc/ifa_policies.xhtml#copyright.

Co-authors Luke Gabriel and Sijia Wu are doctoral students in the Neuroscience Program in the Graduate School of Biomedical Sciences (GSBS) at UMass Medical School.

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

24198373

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

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