The norepinephrine transporter (NET) is a presynaptic plasma membrane protein that mediates reuptake of synaptically released norepinephrine. NET is also a major target for medications used for the treatment of depression, attention deficit/hyperactivity disorder, narcolepsy, and obesity. NET is regulated by numerous mechanisms, including catalytic activation and membrane trafficking. Amphetamine (AMPH), a psychostimulant and NET substrate, has also been shown to induce NET trafficking. However, neither the molecular basis nor the nature of the relevant membrane compartments of AMPH-modulated NET trafficking has been defined. Indeed, direct visualization of drug-modulated NET trafficking in neurons has yet to be demonstrated. In this study, we used a recently developed NET antibody and the presence of large presynaptic boutons in sympathetic neurons to examine basal and AMPH-modulated NET trafficking. Specifically, we establish a role for Rab11 in AMPHinduced NET trafficking. First, we found that, in cortical slices, AMPH induces a reduction in surface NET. Next, we observed AMPH-induced accumulation and colocalization of NET with Rab11a and Rab4 in presynaptic boutons of cultured neurons. Using tagged proteins, we demonstrated that NET and a truncated Rab11 effector (FIP2⌬C2) do not redistribute in synchrony, whereas NET and wild-type Rab11a do. Analysis of various Rab11a/b mutants further demonstrates that Rab11 regulates NET trafficking. Expression of the truncated Rab11a effector (FIP2⌬C2) attenuates endogenous Rab11 function and prevented AMPH-induced NET internalization as does GDP-locked Rab4 S22N. Our data demonstrate that AMPH leads to an increase of NET in endosomes of single boutons and varicosities in a Rab11-dependent manner.

The segregation of homologous chromosomes from one another is the essence of meiosis. In many organisms, accurate segregation is ensured by the formation of chiasmata resulting from crossing over. Drosophila melanogaster females use this type of recombination-based system, but they also have mechanisms for segregating achiasmate chromosomes with high fidelity. We describe a P-element mutagenesis and screen in a sensitized genetic background to detect mutations that impair meiotic chromosome pairing, recombination, or segregation. Our screen identified two new recombination-deficient mutations: mei-P22, which fully eliminates meiotic recombination, and mei-P26, which decreases meiotic exchange by 70% in a polar fashion. We also recovered an unusual allele of the ncd gene, whose wild-type product is required for proper structure and function of the meiotic spindle. However, the screen yielded primarily mutants specifically defective in the segregation of achiasmate chromosomes. Although most of these are alleles of previously undescribed genes, five were in the known genes αTubulin67C, CycE, push, and Trl. The five mutations in known genes produce novel phenotypes for those genes.

No abstract

Parkinson's disease (PD) is a neurodegenerative disorder affecting over 6.1 million people worldwide. Studies of highly-penetrant mutations identified in early-onset familial parkinsonism have contributed to our understanding of the mechanisms underlying PD. Dopamine (DA) transporter (DAT) deficiency syndrome (DTDS) is a type of infantile parkinsonism-dystonia that shares clinical features with PD. Here, we define structural, functional, and behavioral consequences of a Cys substitution at R445 in human DAT (hDAT R445C), identified in a patient with DTDS. This R445 substitution disrupts a phylogenetically conserved intracellular (IC) network of interactions that compromise the hDAT IC gate. The disruption of this IC network supported a channel-like intermediate of hDAT and compromised hDAT function. Drosophila melanogaster expressing hDAT R445C show impaired hDAT activity, DA dysfunction in isolated brains and abnormal motor behaviors monitored at high-speed time resolution. These behaviors are linked with altered dopaminergic signaling, loss of DA neurons and decreased DA availability.

Phosphatidylinositol (4,5)‐bisphosphate (PIP2) is known to regulate the function of ion channels and transporters. The human dopamine (DA) transporter (hDAT) is a key regulator of DA homeostasis and a target of the psychostimulant amphetamine (AMPH). AMPH’s addictive properties are mediated, at least in part, through elevation of extracellular DA by inducing DA efflux through the DAT. Thus, the objective of our study is to understand how to precisely manipulate the DAT to prevent DA efflux without altering its physiological function of DA uptake. This understanding is paramount to the development of pharmacological therapies for AMPH abuse. Here, we demonstrate that PIP2 directly binds to the hDAT. This binding occurs through electrostatic interactions with positively charged hDAT N‐terminal residues and is shown to facilitate AMPH‐induced, DAT‐mediated DA efflux and the psychomotor properties of AMPH. Substitution of these residues with uncharged amino acids reduces hDAT‐PIP2 interactions and AMPH‐induced DA efflux, without altering the hDAT physiological function of DA uptake. We evaluated, for the first time, the significance of this interaction in vivo using locomotion as a behavioral assay in Drosophila melanogaster. Expression of mutated hDAT with reduced PIP2 interaction in Drosophila DA neurons impairs AMPH‐induced locomotion without altering basal locomotion. We present the first demonstration of how PIP2 interactions with a membrane protein can regulate organismal behaviors, such as locomotion. Grant Funding Source: Supported by DGE0909667 & F31 DA 035535‐01(PJH), P22893(HHS), DA13975(AG), P01 DA12408(AG, HW, JAJ)