Epitope‐tagged dopamine transporter knock‐in mice reveal rapid endocytic trafficking and filopodia targeting of the transporter in dopaminergic axons

Rao, A. G. Appu; Richards, Toni L.; Simmons, Diana; Zahniser, Nancy R.; Sorkin, Alexander

doi:10.1096/fj.11-196113

Cited by 42 publications

(69 citation statements)

References 36 publications

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“…Furthermore, PM delivery (recycling) and/or endocytosis can be enhanced by a variety of stimuli, including cocaine, amphetamine, and the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate, findings that are supported by biochemical studies with primary cultured neurons and ex vivo tissue preparations, such as synaptosomes or brain slices (Huff et al, 1997;Melikian and Buckley, 1999;Hoover et al, 2007;Cremona et al, 2011;Gabriel et al, 2013). In contrast, PKC-mediated endocytosis of DAT has not been observed in several studies using primary neuronal cultures Rao et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

“…Studies using primary neuronal cultures suggest that DAT passes through the biosynthetic pathway to the PM and undergoes constitutive endocytosis Miranda et al, 2004;Sorkina et al, 2006;. However, there is disagreement on whether endocytosed DAT is preferentially targeted to lysosomes for degradation or recycled back to the PM Chen et al, 2010;Eriksen et al, 2010;Rao et al, 2011). Furthermore, PM delivery (recycling) and/or endocytosis can be enhanced by a variety of stimuli, including cocaine, amphetamine, and the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate, findings that are supported by biochemical studies with primary cultured neurons and ex vivo tissue preparations, such as synaptosomes or brain slices (Huff et al, 1997;Melikian and Buckley, 1999;Hoover et al, 2007;Cremona et al, 2011;Gabriel et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

“…For the present studies, we have examined DAT subcellular localization and regulation using a knock-in mouse that expresses a DAT with a hemagglutinin (HA) epitope inserted into the second extracellular loop (HA-DAT; Rao et al, 2012). The HA epitope is highly efficient for EM localization studies, permitting quantitative analysis and the identification of intracellular DAT pools.…”

Section: Introductionmentioning

confidence: 99%

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Brain Region-Specific Trafficking of the Dopamine Transporter

et al. 2015

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The dopamine (DA) transporter (DAT) controls dopaminergic neurotransmission by removing extracellular DA. Although DA reuptake is proposed to be regulated by DAT traffic to and from the cell surface, the membrane trafficking system involved in the endocytic cycling of DAT in the intact mammalian brain has not been characterized. Hence, we performed immunolabeling and quantitative analysis of the subcellular and regional distribution of DAT using the transgenic knock-in mouse expressing hemagglutinin (HA) epitope-tagged DAT (HA-DAT) and by using a combination of electron microscopy and a novel method for immunofluorescence labeling of HA-DAT in acute sagittal brain slices. Both approaches demonstrated that, in midbrain somatodendritic regions, HA-DAT was present in the plasma membrane, endoplasmic reticulum, and Golgi complex, with a small fraction in early and recycling endosomes and an even smaller fraction in late endosomes and lysosomes. In the striatum and in axonal tracts between the midbrain and striatum, HA-DAT was detected predominantly in the plasma membrane, and quantitative analysis revealed increased DAT density in striatal compared with midbrain plasma membranes. Endosomes were strikingly rare and lysosomes were absent in striatal axons, in which there was little intracellular HA-DAT. Acute administration of amphetamine in vivo (60 min) or to slices ex vivo (10 -60 min) did not result in detectable changes in DAT distribution. Altogether, these data provide evidence for regional differences in DAT plasma membrane targeting and retention and suggest a surprisingly low level of endocytic trafficking of DAT in the striatum along with limited DAT endocytic activity in somatodendritic areas.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Brain Region-Specific Trafficking of the Dopamine Transporter

et al. 2015

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“…7A). Second, HA-tagged DAT was detected by immunofluorescence in striatum slices from knock-in mice (12). Again, there was overlap of DAT and CYAM (Fig.…”

Section: Resultsmentioning

confidence: 85%

“…Postnatal day 13 male SpragueDawley rat pups and their mothers were obtained from Hilltop or Harlan Laboratories. Knock-in mice with an HA tag on the external surface of the DAT (HA-DAT) have been previously described (12). Rats and mice were housed in the University of Pittsburgh vivarium, maintained on a 12:12-h light/dark cycle, and had ad libitum access to food and water.…”

Section: Methodsmentioning

confidence: 99%

Action potentials and amphetamine release antipsychotic drug from dopamine neuron synaptic VMAT vesicles

Tucker

Block

Levitan

2015

Proc. Natl. Acad. Sci. U.S.A.

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Based on lysotracker red imaging in cultured hippocampal neurons, antipsychotic drugs (APDs) were proposed to accumulate in synaptic vesicles by acidic trapping and to be released in response to action potentials. Because many APDs are dopamine (DA) D2 receptor (D2R) antagonists, such a mechanism would be particularly interesting if it operated in midbrain DA neurons. Here, the APD cyamemazine (CYAM) is visualized directly by two-photon microscopy in substantia nigra and striatum brain slices. CYAM accumulated slowly into puncta based on vacuolar H + -ATPase activity and dispersed rapidly upon dissipating organelle pH gradients. Thus, CYAM is subject to acidic trapping and released upon deprotonation. In the striatum, Ca 2+ -dependent reduction of the CYAM punctate signal was induced by depolarization or action potentials. Striatal CYAM overlapped with the dopamine transporter (DAT). Furthermore, parachloroamphetamine (pCA), acting via vesicular monoamine transporter (VMAT), and a charged VMAT, substrate 1-methyl-4-phenylpyridinium (MPP + ), reduced striatal CYAM. In vivo CYAM administration and in vitro experiments confirmed that clinically relevant CYAM concentrations result in vesicular accumulation and pCA-dependent release. These results show that some CYAM is in DA neuron VMAT vesicles and suggests a new drug interaction in which amphetamine induces CYAM deprotonation and release as a consequence of the H + countertransport by VMAT that accompanies vesicular uptake, but not by inducing exchange or acting as a weak base. Therefore, in the striatum, APDs are released with DA in response to action potentials and an amphetamine. This synaptic corelease is expected to enhance APD antagonism of D2Rs where and when dopaminergic transmission occurs.M ost antipsychotic drugs (APDs) are competitive dopamine (DA) D2 receptor (D2R) antagonists (1, 2). The standard view is that APDs, by being weak bases that are in equilibrium with an uncharged form, cross the blood-brain barrier to access extracellular receptor binding sites from the circulation. However, because APDs are weak bases, they have been proposed to accumulate also in acidic intracellular organelles by acidic trapping (i.e., the less abundant uncharged basic hydrophobic form enters organelles passively and then is protonated to become membrane-impermeant). This hypothesis was first explored by examining a D2 antagonist with a covalently added fluorophore and APD displacement of acridine orange (3). More recently, APDs were found to be released from brain tissue by depolarization, which is expected for any positively charged drug. However, follow-up experiments in hippocampal neuron cultures showed that APDs displace the acidophilic dye lysotracker red, implying there is overlap in intracellular distribution between APDs and lysotracker red. Furthermore, lysotracker red was found to accumulate in hippocampal neuron synaptic vesicles by acidic trapping without displacing native neurotransmitter and to be released in response to electrical stimulation. Thus, b...

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MPTP‐induced dopamine neuron degeneration and glia activation is potentiated in MDMA‐pretreated mice

et al. 2013

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Clinical observations report a greater propensity to develop Parkinson's disease (PD) in amphetamine users. 3,4-Methylenedioxymethamphetamine (MDMA; "ecstasy") is an amphetamine-related drug that is largely consumed by adolescents and young adults, which may have neuroinflammatory and neurotoxic effects. Here, the objective was to evaluate in mice whether consumption of MDMA during adolescence might influence the neuroinflammatory and neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a toxin known to induce PD in humans. The activation of astroglia and microglia by glial fibrillary acidic protein (GFAP) and complement receptor type 3 (CD11b) immunohistochemistry and the degeneration of dopaminergic neurons by tyrosine hydroxylase (TH) immunohistochemistry were evaluated. MPTP (20 mg/kg × 4) was administered to mice treated from ages 8 weeks to 17 weeks with MDMA (10 mg/kg twice daily, two times a week). In mice that were chronically treated with MDMA, administration of MPTP induced a higher microglial and astroglial response in both the striatum and the substantia nigra pars compacta (SNc) compared with vehicle-treated or vehicle + MPTP-treated mice. Inflammatory changes were associated with a decrease in TH immunoreactivity in the SNc of MDMA-treated mice and with a further decrease in the striatum and the SNc of MDMA + MPTP-treated mice compared with vehicle-treated, MDMA-treated, and MPTP-treated mice. The results demonstrate that chronic administration of MDMA during late adolescence in mice exacerbates the neurodegeneration and neuroinflammation caused by MPTP, suggesting that MDMA may constitute a risk factor for dopaminergic neuron degeneration.

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Epitope‐tagged dopamine transporter knock‐in mice reveal rapid endocytic trafficking and filopodia targeting of the transporter in dopaminergic axons

Cited by 42 publications

References 36 publications

Brain Region-Specific Trafficking of the Dopamine Transporter

Brain Region-Specific Trafficking of the Dopamine Transporter

Action potentials and amphetamine release antipsychotic drug from dopamine neuron synaptic VMAT vesicles

MPTP‐induced dopamine neuron degeneration and glia activation is potentiated in MDMA‐pretreated mice

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