Nancy R. Zahniser

RNA Interference Screen Reveals an Essential Role of Nedd4–2 in Dopamine Transporter Ubiquitination and Endocytosis

The function of the dopamine transporter (DAT) to terminate dopamine neurotransmission is regulated by endocytic trafficking of DAT. To elucidate the mechanisms of DAT endocytosis, we generated a fully functional mutant of the human DAT in which a hemagglutinin epitope (HA) was incorporated into the second extracellular loop. The endocytosis assay, based on the uptake of an HA antibody, was designed to study constitutive- and protein kinase C (PKC)-dependent internalization of HA-DAT expressed in non-neuronal cells and rat dopaminergic neurons. Large-scale RNA interference analysis of PKC-dependent endocytosis of HA-DAT revealed the essential and specific role of an E3 ubiquitin ligase, Nedd4–2 (neural precursor cell expressed, developmentally downregulated 4–2), as well as the involvement of adaptor proteins present in clathrin-coated pits, such as epsin, Eps15 (epidermal growth factor pathway substrate clone 15), and Eps15R (Eps15-related protein). Depletion of Nedd4–2 resulted in a dramatic reduction of PKC-dependent ubiquitination of DAT. Endogenous Nedd4–2, epsin, and Eps15 were coimmunoprecipitated with heterologously expressed human HA-DAT and endogenous DAT isolated from rat striatum. A new mechanistic model of DAT endocytosis is proposed whereby the PKC-induced ubiquitination of DAT mediated by Nedd4–2 leads to interaction of DAT with adaptor proteins in coated pits and acceleration of DAT endocytosis.

Binding characteristics of the dopamine uptake inhibitor [3H]nomifensine to striatal membranes.

Binding of the radiolabeled antidepressant [3H]nomifensine to rat and rabbit striatal membranes has been characterized. The specific binding of [3H]nomifensine to striatal membranes was stable, reversible and saturable. Saturation experiments indicated that [3H]nomifensine labeled a single site with an affinity (Kd) of 80 nM and a total number of binding sites (Bmax) of 6.5 pmoles/mg protein both in rat and rabbit striatal membranes. The affinity constants obtained from kinetic analyses and competition experiments were in fairly good agreement with those obtained in saturation experiments. Compounds known to inhibit [3H]dopamine uptake in vitro, such as nomifensine, 4-hydroxy-nomifensine, mazindol, amfonelic acid and benztropine, were the most potent competitors of nomifensine binding. Additionally, the absolute potencies of various drugs in competing for [3H]nomifensine binding to rat and rabbit striatal membranes correlated closely with their potencies in inhibiting [3H]dopamine uptake into striatal synaptosomes. Specific [3H]nomifensine binding was dependent on the presence of NaCl which is also consistent with its association with the dopamine uptake pump. The number, but not the affinity, of striatal [3H]nomifensine binding sites was reduced significantly following in vivo lesions with 6-hydroxydopamine. The number of [3H]nomifensine binding sites was found to be highest in areas rich in dopamine nerve terminals such as the striatum and olfactory tubercle. These results suggest that [3H]nomifensine binds to a site on dopaminergic nerve terminals associated with the dopamine uptake pump.

Direct reprogramming of human fibroblasts into dopaminergic neuron-like cells

Transplantation of exogenous dopaminergic neuron (DA neurons) is a promising approach for treating Parkinsons disease (PD). However, a major stumbling block has been the lack of a reliable source of donor DA neurons. Here we show that a combination of five transcriptional factors Mash1, Ngn2, Sox2, Nurr1, and Pitx3 can directly and effectively reprogram human fibroblasts into DA neuron-like cells. The reprogrammed cells stained positive for various markers for DA neurons. They also showed characteristic DA uptake and production properties. Moreover, they exhibited DA neuron-specific electrophysiological profiles. Finally, they provided symptomatic relief in a rat PD model. Therefore, our directly reprogrammed DA neuron-like cells are a promising source of cell-replacement therapy for PD.

Amphetamine and Methamphetamine Differentially Affect Dopamine Transporters in Vitro and in Vivo

The psychostimulants d-amphetamine (AMPH) and methamphetamine (METH) release excess dopamine (DA) into the synaptic clefts of dopaminergic neurons. Abnormal DA release is thought to occur by reverse transport through the DA transporter (DAT), and it is believed to underlie the severe behavioral effects of these drugs. Here we compare structurally similar AMPH and METH on DAT function in a heterologous expression system and in an animal model. In the in vitro expression system, DAT-mediated whole-cell currents were greater for METH stimulation than for AMPH. At the same voltage and concentration, METH released five times more DA than AMPH and did so at physiological membrane potentials. At maximally effective concentrations, METH released twice as much [Ca2+]i from internal stores compared with AMPH. [Ca2+]i responses to both drugs were independent of membrane voltage but inhibited by DAT antagonists. Intact phosphorylation sites in the N-terminal domain of DAT were required for the AMPH- and METH-induced increase in [Ca2+]i and for the enhanced effects of METH on [Ca2+]i elevation. Calmodulin-dependent protein kinase II and protein kinase C inhibitors alone or in combination also blocked AMPH- or METH-induced Ca2+ responses. Finally, in the rat nucleus accumbens, in vivo voltammetry showed that systemic application of METH inhibited DAT-mediated DA clearance more efficiently than AMPH, resulting in excess external DA. Together these data demonstrate that METH has a stronger effect on DAT-mediated cell physiology than AMPH, which may contribute to the euphoric and addictive properties of METH compared with AMPH.

Characterization and Regulation of Insulin Receptors in Rat Brain

Abstract: An in vitro receptor binding assay, using filtration to separate bound from free [125I]insulin, was developed and used to characterize insulin receptors on membranes isolated from specific areas of rat brain. The kinetic and equilibrium binding properties of central receptors were similar to those of hepatic receptors. The binding profiles in all tissues were complex and were consistent with binding in multiple steps or to multiple sites. Similar binding properties were found among receptors in olfactory tubercle/bulb, cerebral cortex, hippocampus, striatum, hypothalamus, and cerebellum. High affinity [125I]insulin binding sites (KD= 3–11 nM) were distributed evenly between membranes isolated from P1 and P2 fractions of these brain areas, with the exception of the olfactory tubercle in which binding to P2 membranes was four‐fold greater (Bmax= 150 fmol/mg protein). One difference between insulin receptors in brain and peripheral target tissues, however, was observed. Following exposure to 0.17 μM insulin for 3 h at 37°C, the number of specific [125I]insulin binding sites on adipocytes decreased by 40%, while the number of binding sites on minces of cerebral cortex/olfactory tubercle remained constant. The results suggest that although the binding characteristics of central and peripheral insulin receptors are similar, these receptors do not appear to be regulated in the same manner.

Trafficking of dopamine transporters in psychostimulant actions.

Brain dopamine (DA) plays a pivotal role in drug addiction. Since the plasma membrane DA transporter (DAT) is critical for terminating DA neurotransmission, it is important to understand how DATs are regulated and this regulation impacts drug addiction. The number of cell surface DATs is controlled by constitutive and regulated endocytic trafficking. Psychostimulants impact this trafficking. Amphetamines, DAT substrates, cause rapid up-regulation and slower down-regulation of DAT whereas cocaine, a DAT inhibitor, increases surface DATs. Recent reports have begun to elucidate the molecular mechanisms of these psychostimulant effects and link changes in DAT trafficking to psychostimulant-induced reward/reinforcement in animal models.

Low and high locomotor responsiveness to cocaine predicts intravenous cocaine conditioned place preference in male Sprague-Dawley rats.

Outbred, male Sprague-Dawley rats can be classified as either low or high cocaine responders (LCRs or HCRs, respectively) based on cocaine-induced locomotor activity in an open-field arena. This difference reflects cocaines ability to inhibit the striatal dopamine transporter and predicts development of sensitization. To investigate the relationship between initial cocaine locomotor responsiveness and cocaine reward, here we first classified rats as either LCRs or HCRs in a conditioned place preference (CPP) apparatus. Subsequently, we conducted cocaine conditioning trials, twice-daily over 4 days with vehicle and cocaine (10 mg/kg, i.p. or 1 mg/kg, i.v.). When cocaine was administered by the i.p. route, similar to previous findings in the open-field, LCRs and HCRs were readily classified and locomotor sensitization developed in LCRs, but not HCRs. However, cocaine CPP was not observed. In contrast, when cocaine was administered by the i.v. route, the LCR/HCR classification not only predicted sensitization, but also CPP, with only LCR rats exhibiting sensitization and cocaine conditioning. Our findings show that the initial locomotor response to cocaine can predict CPP in male Sprague-Dawley rats under conditions when place conditioning develops, and that LCRs may be more prone to develop conditioning in the context of cocaine reward.

Dopamine transport by the serotonin transporter: a mechanistically distinct mode of substrate translocation

The serotonin transporter (SERT) is the principal mechanism for terminating serotonin (5-HT) signals in the nervous system and is a site of action for a variety of psychoactive drugs including antidepressants, amphetamines, and cocaine. Here we show that human SERTs (hSERTs) and rat SERTs are capable of robust dopamine (DA) uptake through a process that differs mechanistically from 5-HT transport in several unanticipated ways. DA transport by hSERT has a higher maximum velocity than 5-HT transport, requires significantly higher Na+ and Cl− concentrations to sustain transport, is inhibited noncompetitively by 5-HT, and is more sensitive to SERT inhibitors, including selective serotonin reuptake inhibitors. We use a thiol-reactive methane thiosulfonate (MTS) reagent to modify a conformationally sensitive cysteine residue to demonstrate that hSERT spends more time in an outward facing conformation when transporting DA than when transporting 5-HT. Cotransfection of an inactive or an MTS-sensitive SERT with wild-type SERT subunits reveals an absence of cooperative interactions between subunits during DA but not 5-HT transport. To establish the physiological relevance of this mechanism for DA clearance, we show using in vivo high-speed chronoamperometry that SERT has the capacity to clear extracellularly applied DA in the hippocampal CA3 region of anesthetized rats. Together, these observations suggest the possibility that SERT serves as a DA transporter in vivo and highlight the idea that there can be distinct modes of transport of alternative physiological substrates by SERT.

Individual differences in cocaine-induced locomotor activity in rats: behavioral characteristics, cocaine pharmacokinetics, and the dopamine transporter.

Outbred male Sprague–Dawley rats can be classified as either low or high cocaine responders (LCRs or HCRs, respectively) based on their locomotor response to acute cocaine. Concomitant measurement of dopamine clearance in these rats revealed that the differential behavioral responses are associated with the magnitude of dopamine transporter (DAT) inhibition by cocaine. Here, we investigated several factors that might contribute to cocaine-induced behavioral variability and its association with differential inhibition of DAT function. In rats classified as LCRs or HCRs after 10 mg/kg cocaine injection, we found no differences in (1) novelty-induced locomotion, (2) cocaine levels in dorsal striatum or nucleus accumbens (NAc), (3) DAT number or affinity in NAc, or (4) DAT affinity for cocaine in NAc. In rats given 20 mg/kg cocaine, behavior was more uniform across individuals, but still warranted separation into LCR/HCR categories. Additionally, we analyzed the stability of the LCR/HCR classification made during the first test with 10 or 20 mg/kg cocaine by retesting rats 7 days later with saline or cocaine (10 or 20 mg/kg). Before injection, HCRs were more active relative to LCRs and to their own behavior on the first test day. Following cocaine, LCRs and HCRs exhibited similar drug-induced changes in locomotion, but there were unique effects that depended on the cocaine dose given on the first and second test days. Our results argue against several likely explanations for individual differences in cocaine-induced behavior and highlight the influence of a single cocaine exposure on subsequent behavioral responses to the drug.

Negative Regulation of Dopamine Transporter Endocytosis by Membrane-Proximal N-Terminal Residues

The plasma membrane dopamine transporter (DAT) takes extracellular dopamine back up into dopaminergic neurons. Although the number of DATs at the cell surface is regulated by endocytosis and recycling, the molecular mechanisms that control this endocytic trafficking of DAT are not defined. To map the sequence motifs that are involved in constitutive DAT endocytosis, mutagenesis of human DAT tagged with yellow fluorescent protein (YFP) and an extracellular HA epitope was performed. Removal of the entire N terminus of DAT resulted in accumulation of the resulting DAT mutant (YFP-HA-ΔN-DAT) in early and recycling endosomes in HeLa and PAE cells, and in primary rat mesencephalic-striatal neuronal cocultures. This endosomal accumulation was due to rapid constitutive internalization of YFP-HA-ΔN-DAT by the clathrin-dependent pathway. Small deletions and multialanine substitutions in the N terminus revealed two molecular determinants within the membrane proximal residues 60–65 that are important for preventing rapid internalization of DAT. First, mutations of Arg60 or Trp63, leading to disruption of the “outward facing” DAT conformation, correlated with an increased pool of mobile DATs in the plasma membrane and accelerated constitutive internalization of the DAT mutants. Second, mutation of Lys65 also correlated with elevated endocytosis. While none of these mutations alone recapitulated the marked endocytic phenotype of YFP-HA-ΔN-DAT, simultaneous elimination of both the outward conformation of DAT and Lys65 resulted in DAT mutants that were rapidly internalized. Thus, our studies reveal a new link between DAT endocytosis and conformation-dependent uptake activity that represents a novel mode for regulating DAT function.