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
2013-11-06
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
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.
DOI of Published Version
10.1523/JNEUROSCI.3284-13.2013
Source
J Neurosci. 2013 Nov 6;33(45):17836-46. doi: 10.1523/JNEUROSCI.3284-13.2013. Link to article on publisher's site
Related Resources
Journal/Book/Conference Title
The Journal of neuroscience : the official journal of the Society for Neuroscience
PubMed ID
24198373
Repository Citation
Gabriel, Luke; Wu, Sijia; Kearney, Patrick; Bellve, Karl D.; Standley, Clive; Fogarty, Kevin E.; and Melikian, Haley, "Dopamine transporter endocytic trafficking in striatal dopaminergic neurons: differential dependence on dynamin and the actin cytoskeleton" (2013). University of Massachusetts Medical School Faculty Publications. 809.
https://escholarship.umassmed.edu/faculty_pubs/809
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.
Comments
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.