HIV-1 Tat protein augments methamphetamine-induced impairment of dopamine transporter activity

Robert Steger1, Kustauv Saha1, Danielle Sambo1, Taiwo Alonge1, Brittany Butler1, Caetherina Apadoo1, James Goodwin2, Shilpa Buch3, Habibeh Khoshbouei1

1.University of Florida, Gainesville, Fl 2. University of Texas Medical Branch, Galveston, TX 3.University of Nebraska Medical Center, Omaha, NE

HIV-1 contributes to cognitive and behavioral decline in infected individuals, in part, through the production of HIV-1 transactivator of transcription (Tat) protein. While antiretroviral agents reduce viral load, they do not eliminate the production of Tat and thus are ineffective against neuropathologies caused by HIV-1. Specifically, the Tat protein has been shown to play a critical role in perturbations of the dopamine neurotransmission in the brain. In addition, there also exists a high comorbidity between HIV-1 infection and methamphetamine (METH) abuse. Both the Tat protein and METH exert their effects through the dopamine transporter (DAT). DAT is a major regulator of extracellular dopamine and thus dopamine neurotransmission in the brain and it is implicated in neurological and neuropsychiatric disorders such as Parkinson’s disease and drug addiction. Recent reports suggest the Tat protein enhances METH-induced impairment of dopamine transmission, albeit with a less understood mechanism. Under control conditions, METH increases the DAT-mediated inward current, increases the spontaneous firing activity of dopaminergic neurons and increases DAT-mediated DA efflux. However, we found that intracellular Tat-101(200ng/ml) protein attenuated basal and METH (10µM) induced DAT-dependent DA efflux, in both bath solution or delivered directly into the dopamine neurons via the patch electrode. Furthermore, we found intercellular Tat-101 significantly decreases METH-induced inward current, supporting the hypothesis that Tat-101 might affect ionic permeability of DAT regulating dopamine neurotransmission. Consistent with previous reports we found methamphetamine or Tat-101 alone decreases dopamine uptake, while pretreatment of DAT cells with 100 nM Tat for 15 minutes synergistically decreases dopamine uptake. Previously we have shown DA efflux via DAT is regulated by intracellular calcium and its associated kinases including Calcium calmodulin-dependent protein kinase II (CaMKII) and Protein Kinase C (PKC). Currently we are examining the hypothesis that Tat-101 regulates DAT activity by regulating intracellular calcium homeostasis and signaling molecules such as PKC and CaMKII. These preliminary findings provide mechanistic insight involved in the combined deleterious effects of HIV-1 Tat and methamphetamine in the brain with the ultimate goal of testing novel therapeutics.



Etienne Cartier SFN 2013 Abstract

Sumoylation modulates Alpha synuclein interaction with the Dopamine Transporter

Etienne Cartier1 and Habibeh Khoshbouei1 ,1McKnight Brain Institute, Department of Neuroscience, University of Florida. Gainesville, FL                    

Sumoylation is a posttranslational modification by which small ubiquitin-like modifiers (SUMO) are covalently conjugated to target proteins. Sumoylation is highly dynamic and reversible as SUMO specific proteases can rapidly remove SUMO from their substrates. The specific SUMO conjugase, UBC9, sumoylates target proteins. Conversely, the SUMO Interacting Domains (SIM) of non-sumoylated proteins can recognize conjugated SUMO, and SUMO modification of a given protein has been shown to create or abolish binding interfaces that can regulate trafficking, protein-protein interactions, protein aggregation and solubility. Recent reports suggest that alpha-synuclein, a synaptic protein implicated in multiple neurodegenerative diseases and drug addiction, contains SUMO binding domain, and is sumoylated. Alpha-synuclein is one of DAT’s protein partners that directly interact with the C-terminus of the transporter. Confocal microscopy and biochemical data from our lab have demonstrated that alpha-synuclein and dopamine transporter interact at the plasma membrane. Whole-cell patch clamp electrophysiology results suggest that alpha-synuclein over-expression induces a transporter mediated inward chloride current and decreases DAT-mediated substrate uptake. DAT molecule contains five putative SUMO interacting domains. In this study we used co-immunoprecipitation (Co-IP) and biotinylation assays to examine whether DAT interacts with SUMO (SUMO-1 and/or 2), and whether overexpression of alpha-synuclein affects DAT/SUMO interaction. Our preliminary data suggest that DAT forms a complex with the SUMO conjugase UBC9 and appears to be sumoylated. In addition, in the striatum, HEK and MN9D cells expressing DAT, we found a strong interaction between DAT and SUMO-1 which is consistent with the exsitance of one or more SIM in the DAT structure. Moreover, sumoylation appears to reduce the association between DAT and alpha-synuclein in immunoprecipitation assays, inducing the expression of DAT at the plasma membrane demonstrated by biotinylation. Our ongoing confocal microscopy will examine whether sumoylation regulates DAT/alpha synuclein interaction in living cells. The functional assays will demonstrate the functional significance of these observations in live cells.