title: Dopamine transporter endocytic trafficking in striatal dopaminergic neurons: differential dependence on dynamin and the actin cytoskeleton.
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.
authors.affiliation: Brudnick Neuropsychiatric Research Institute, Department of Psychiatry, Neuroscience Graduate Program, Graduate School of Biomedical Sciences, and Program in Molecular Medicine, Biomedical Imaging Group, University of Massachusetts Medical School, Worcester, Massachusetts 01655.
authors.forename: Luke R
authors.initials: LR
authors.lastname: Gabriel
authors.forename: Sijia
authors.initials: S
authors.lastname: Wu
authors.forename: Patrick
authors.initials: P
authors.lastname: Kearney
authors.forename: Karl D
authors.initials: KD
authors.lastname: Bellvé
authors.forename: Clive
authors.initials: C
authors.lastname: Standley
authors.forename: Kevin E
authors.initials: KE
authors.lastname: Fogarty
authors.forename: Haley E
authors.initials: HE
authors.lastname: Melikian
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003342:Corpus Striatum / Q000166:cytology / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism*; D059290:Dopaminergic Neurons / Q000166:cytology / Q000378:metabolism*; D034281:Dynamins / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D008297:Male; D051379:Mice; D021381:Protein Transport / Q000502:physiology
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D034281:Dynamins