Abstract
The dopamine transporter (DAT) is a plasma membrane protein responsible for the uptake of released dopamine back to the presynaptic terminal and ending dopamine neurotransmission. The DAT is the molecular target for cocaine and amphetamine as well as a number of pathological conditions including autism spectrum disorders, attention-deficit hyperactivity disorder (ADHD), dopamine transporter deficiency syndrome (DTDS), and Parkinson’s disease. The DAT uptake capacity is dependent on its level in the plasma membrane. In vitro studies show that DAT functional expression is regulated by a balance of endocytosis, recycling, and lysosomal degradation. However, recent reports suggest that DAT regulation by endocytosis in neurons is less significant than previously reported. Therefore, additional mechanisms appear to determine DAT steady-state level and functional expression in the neuronal plasma membrane. Here, we hypothesize that the ubiquitin-like protein small ubiquitin-like modifier 1 (SUMO1) increases the DAT steady-state level in the plasma membrane. In confocal microscopy, fluorescent resonance energy transfer (FRET), and Western blot analyses, we demonstrate that DAT is associated with SUMO1 in the rat dopaminergic N27 and DAT overexpressing Human Embryonic Kidney cells (HEK)-293 cells. The overexpression of SUMO1 and the Ubc9 SUMO-conjugase induces DAT SUMOylation, reduces DAT ubiquitination and degradation, enhancing DAT steady-state level. In addition, the Ubc9 knock-down by interference RNA (RNAi) increases DAT degradation and reduces DAT steady-state level. Remarkably, the Ubc9-mediated SUMOylation increases the expression of DAT in the plasma membrane and dopamine uptake capacity. Our results strongly suggest that SUMOylation is a novel mechanism that plays a central role in regulating DAT proteostasis, dopamine uptake, and dopamine signaling in neurons. For that reason, the SUMO pathway including SUMO1, SUMO2, Ubc9, and DAT SUMOylation, can be critical therapeutic targets in regulating DAT stability and dopamine clearance in health and pathological states.
Highlights
We demonstrated by immunofluorescence and confocal microscopy that dopamine transporter (DAT) was endogenously expressed in fixed N27 cells which express tyrosine hydroxylase (TH) to synthesize dopamine (Figure 1B)
Once the optimal RNAi conditions for knocking down the endogenous Ubc9 were established, we identified that SUMOylated DAT (SUMO-DAT) was significantly reduced in N27 cells transfected with the RNAi-Ubc9 mix
The main goal of this study was to demonstrate that the Small Ubiquitin-like Modifier (SUMO) pathway, by the action of Ubc9, increases the DAT steady-state level
Summary
The dopamine transporter (DAT) is a plasma membranespanning protein responsible for the termination of dopaminergic neurotransmission via reuptake of released dopamine from the presynaptic terminals in the central nervous system, which is the main mechanism for terminating dopamine transmission in the brain (Hong and Amara, 2013; Rudnick et al, 2014; German et al, 2015). The dopamine uptake capacity by DAT is dependent on its availability in the plasma membrane (Sakrikar et al, 2012; Hong and Amara, 2013; Bowton et al, 2014; Rudnick et al, 2014; Cartier et al, 2015; German et al, 2015). Recent studies have linked mutations in the human DAT gene causing DTDS, to be responsible for DAT misfolding, degradation, and reduced level in the plasma membrane (Kurian et al, 2009, 2011; Hetz and Mollereau, 2014; Ng et al, 2014; Ciechanover and Kwon, 2015; Beerepoot et al, 2016; Kasture et al, 2016). Comprehending the regulatory mechanisms that modulate DAT folding and degradation may have a profound therapeutic impact
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