Insights in how amphetamine ROCKs (Rho-associated containing kinase) membrane protein trafficking

Amphetamine (AMPH) is a psychostimulant that induces efflux of dopamine (DA) within dopaminergic nodes, a phenomenon that has been recognized since the late 1950s (1). However, the ability of AMPH to alter DA transporter (DAT) cell surface expression did not emerge from the literature until the late 1990s (2). DAT, a key target of AMPH actions, is an important regulator of synaptic DA levels. Fleckenstein et al. (3) first hypothesized that AMPH not only induces transporter efflux but also may regulate transporter surface expression levels. This hypothesis was based on their observation that in rats, a single, high-dose injection of AMPH results in a decrease of DAT function 1 h later (3). Since then, initiated by the work of Saunders et al. (2), AMPH-induced regulation of DAT trafficking has been demonstrated in numerous studies, and the investigation into the mechanisms underlying this phenomenon has become an area of intense research (4–6). In PNAS, Wheeler et al. (7) shed light on the mechanism by which AMPH causes trafficking of DAT as well as on the behavioral impact of this mechanism.

AMPH-Induced DAT Trafficking

The first demonstration of AMPH-induced trafficking of DAT in heterologous systems surfaced a few years after Fleckenstein’s original proposal (3). In that study, acute treatment with the DAT substrates AMPH and DA not only reduced [³H]DA uptake and AMPH-induced currents but also clearly decreased DAT cell surface expression (1). By using a dominant-negative mutant of dynamin I (K44A) to prevent substrate-induced trafficking, the authors also provided preliminary evidence to suggest that AMPH-stimulated DAT endocytosis occurs via a dynamin-dependent pathway. Following these experiments, several investigations supported these results in other heterologous systems (Xenopus oocytes); rat synaptosomal preparations; and, finally, indirectly in vivo via high-speed chronoamperometry (8–10). Application of DAT inhibitors, such as cocaine and mazindol, was sufficient to prevent AMPH-induced DAT trafficking, implying that transport of AMPH into the cell, as well as transporter function, per se, may be important components of this regulation (2). This hypothesis was partially addressed experimentally using the DAT mutant (Y335A), which is capable of binding substrate, albeit with impaired transport function. Upon

Wheeler et al. shed light on the mechanism by which AMPH causes trafficking of DAT as well as on the behavioral impact of this mechanism.

exposure to AMPH, this mutant was analyzed for DAT redistribution from the cell surface to the cytosol. Interestingly, extracellular application of AMPH did not induce internalization of the uptake-impaired DAT; however, when applied directly into the intracellular milieu, AMPH was capable of inducing trafficking of the DAT mutant (6). From this result, researchers hypothesized that although the DAT transport cycle is unnecessary for AMPH-induced DAT trafficking, an increase in intracellular AMPH is an essential component of this regulation. However, only partial clues were offered for understanding through which mechanism AMPH causes DAT trafficking, and of the specific role of intracellular AMPH in this trafficking phenomenon.

In the study by Wheeler et al. (7), Amara and her collaborators advance the neuroscience community in its understanding of how the psychostimulant AMPH functions. They demonstrate that the ability of AMPH to cause DAT trafficking is a dynamin-dependent and clathrin-independent phenomenon, and is mediated by the activation of the small GTPase RhoA. Wheeler et al. (7) point out that the downstream effects of Rho-family GTPase signaling pathways on cellular functions, such as actin remodeling and membrane protein trafficking, may explain some of the unique features of how AMPH works compared with cocaine. Indeed, activation of Rho modulates the actin cytoskeleton and neurite extension, both of which have been linked to the plasticity and alterations in dendritic spine morphology. These phenomena have been observed in models of addiction. Furthermore, the authors involve the Rho-associated coiled-coil containing kinase (ROCK) in the AMPH actions, because ROCK inhibition blocks the effects of AMPH pretreatment on DA uptake. These data support previous studies, suggesting a role for ROCK in AMPH’s behavioral effects.

As AMPH-induced DAT trafficking became more established, researchers shifted their focus toward identifying underlying key molecular players in the phenomenon. Clearly, the study by Wheeler et al. (7) adds important information on how AMPH triggers DAT trafficking, enhancing the significance of numerous previous studies demonstrating the role of kinase activation (e.g., PKC) for the rapid redistribution of DAT away from the cell surface in both heterologous and neuronal systems (11–13). Work from Yamamoto and coworkers (14) found that the protein flotillin-1 (Flot1; used as a marker for membrane rafts) is required for PKC-regulated internalization of members of two different neurotransmitter transporter families: the DAT and the glial glutamate transporter, EAAT2. Furthermore, this study revealed that Flot1 is also required to localize DAT within plasma membrane microdomains in stable cell lines, and is essential for AMPH-induced reverse transport of DA in neurons, but not for DA uptake. This result is important because Flot1 has been reported to delineate a discrete set of endocytic vesicles, consistent with reports describing a rapid, dynamin-dependent endocytic pathway involved in the formation of small, noncoated vesicles at the plasma membrane.

Cytoplasmic Actions of AMPH

Here, Wheeler et al. (7) demonstrate that when AMPH enters the cytoplasm, it rapidly stimulates DAT internalization through a dynamin-dependent, clathrin-independent process. They highlight the importance of AMPH needing to be cytoplasmic for DAT trafficking. As previously shown (2), they reinforce that cocaine inhibits the ability of AMPH to cause DAT surface redistribution, consistent with the idea the AMPH acts on an intracellular target to mediate its effects on DAT trafficking. The value of this observation is enhanced by the findings that AMPH-mediated DAT internalization is disrupted in DAT variants associated with attention deficit/hyperactivity disorder (ADHD) (15) as well as autism (16). Importantly, in a mutant associated with both ADHD and autism (DAT A559V), this trafficking phenomenon could be recovered by intracellular perfusion of AMPH (16), consistent with the mutant displaying impaired AMPH uptake (16). Although the precise nature and the pharmacological properties of the cytoplasmic target(s) of AMPH remain to be established, Wheeler at al. (7) hypothesize that a trace amine-associated receptor, TAAR1, expressed in dopamine neurons with a predominantly intracellular distribution might represent a potential target for AMPH. This hypothesis is compelling, presenting new experimental opportunities to further our understanding of how AMPH alters DAT surface expression.

In this elegant and thorough study (7), Amara and her collaborators identify multiple novel targets for intracellular AMPH. They demonstrate that cytoplasmic AMPH stimulates a secondary pathway of cAMP production, which leads to Rho inactivation by PKA-dependent phosphorylation. The authors provide a mechanism whereby RhoA-dependent and PKA signaling interact to regulate the timing and magnitude of AMPH’s effects on DAT internalization. The pivotal role of DAT trafficking in AMPH-induced behaviors was also tested in vivo. Wheeler et al. (7) show that preemptive D1/D5 receptor stimulation to activate PKA can reduce AMPH-evoked hyperlocomotion in mice without altering the increase in locomotion induced by cocaine. These results further support the idea that the direct activation of cytoplasmic signaling cascades by AMPH might contribute to the behavioral effects of acute AMPH exposure. As pointed out by the authors, there is a component of psychostimulant-induced hyperlocomotion that is not Rho-mediated, because the activation of D1/D5 receptors does not bring the AMPH-induced hyperlocomotion down to control levels. It is noteworthy to point out that in addition to its effects on Rho-mediated transporter trafficking, AMPH elevates extracellular DA through other mechanisms, such as facilitating efflux and inhibiting the DAT. Recent data demonstrate reduced AMPH-induced locomotion in flies expressing DATs resistant to effluxing DA in response to AMPH (17).

Although this beautiful and multifaceted study helps clarify how AMPH causes DAT trafficking in vitro and the behavioral consequences of this trafficking phenomenon in vivo, the role of DAT trafficking in physiological and pharmacological events clearly remains a complex model (18).