Regulation of the norepinephrine transporter by α‐synuclein‐mediated interactions with microtubules

Abstract

alpha-Synuclein (alpha-Syn) regulates catecholaminergic neurotransmission. We demonstrate that alpha-Syn regulates the activity and surface expression of the norepinephrine transporter (NET), depending on its expression levels. In cells co-transfected with NET and low amounts of alpha-Syn, NET activity and cell surface expression were increased and protein interactions with alpha-Syn decreased, compared with cells transfected with NET alone. Converse effects were observed at higher levels of alpha-Syn expression. Treatment with nocodazole and other microtubule (MT) destabilizers abolished the expression-dependent bimodal regulation of NET by alpha-Syn. At low alpha-Syn levels, nocodazole had no effect on NET surface expression or protein interactions, while inducing increases in these measures at higher levels. Cells that were transfected with NET alone displayed no sensitivity to nocodazole, indicating that alpha-Syn expression was necessary for the MT-dependent changes in NET activity. MT destabilizers also caused a significant increase in [(3)H]-NE uptake in brainstem primary neurons and synaptosomes from the frontal cortex, but not striatal synaptosomes. These findings suggest that the surface localization and activity of NET is modulated by alpha-Syn in a manner that is both dependent on interactions with the MT cytoskeleton and varies across brain regions.

SLC6 Neurotransmitter Transporters: Structure, Function, and Regulation

“…␣-Synuclein. DAT, NET, and SERT have all been found to interact with ␣-synuclein, a presynaptic protein that is implicated in the pathogenesis of Parkinson's disease Wersinger and Sidhu, 2005;Wersinger et al, 2006a,b;Jeannotte and Sidhu, 2007). Coexpression studies revealed that ␣-synuclein modulated transporter activity by regulating cell surface expression; however, it is not clear whether the regulation is stimulatory or inhibitory.…”

Desipramine Modulation of α-, γ-Synuclein, and the Norepinephrine Transporter in an Animal Model of Depression

“…Synaptosomes were prepared from freshly killed animals as described previously (Jeannotte and Sidhu, 2007). Briefly, the tissue was suspended in 10% w/v synaptosome homogenizing buffer, homogenized on ice, and centrifuged at 500 g for 10 min at 41C.…”

“…Uptake assays were performed on intact and adherent transiently transfected cells or freshly prepared synaptoa-and c-Synuclein modulation by desipramine in depression AM Jeannotte et al somes as described previously (Jeannotte and Sidhu, 2007). Briefly, uptake into cells was performed using room temperature uptake buffer (Krebs-Ringer-Hepes) and a final concentration of 20 nM [ 3 H]-NE.…”

“…NET DNA was a gift from Dr Randy Blakely at Vanderbilt University, a-Syn from Dr Ted Dawson at Johns Hopkins University, and b-Syn and g-Syn from Dr Vladimir Buchmann at Cardiff University. All constructs were subcloned into a pcDNA3.1 vector (Invitrogen), transiently transfected using Fugene (Roche) according to the manufacturer's instructions, and plated as described previously (Jeannotte and Sidhu, 2007).…”

“…Chronic treatment with the NETselective antidepressant, desipramine (DMI), not only decreased NET activity (Benmansour et al, 2004), but also downregulated the binding and expression of the transporter (Bauer and Tejani-Butt, 1992;Hebert et al, 2001;Zhu et al, 2002). Previously, we determined that the trafficking, cellular localization, and activity of NET is regulated by a-synuclein (a-Syn) in a manner that is dependent on the expression levels of a-Syn and an intact microtubule (MT) network (Jeannotte and Sidhu, 2007;Wersinger et al, 2006a). Specifically, a-Syn aids in trafficking NET away from the cell surface by tethering the transporter to MTs, reducing its expression at the cell surface, whereas simultaneously decreasing its reuptake activity.…”