Abstract
Altered serotonin (5-HT) signaling is associated with multiple brain disorders, including major depressive disorder (MDD), obsessive-compulsive disorder (OCD), and autism spectrum disorder (ASD). The presynaptic, high-affinity 5-HT transporter (SERT) tightly regulates 5-HT clearance after release from serotonergic neurons in the brain and enteric nervous systems, among other sites. Accumulating evidence suggests that SERT is dynamically regulated in distinct activity states as a result of environmental and intracellular stimuli, with regulation perturbed by disease-associated coding variants. Our lab identified a rare, hypermorphic SERT coding substitution, Gly56Ala, in subjects with ASD, finding that the Ala56 variant stabilizes a high-affinity outward-facing conformation (SERT∗) that leads to elevated 5-HT uptake in vitro and in vivo. Hyperactive SERT Ala56 appears to preclude further activity enhancements by p38α mitogen-activated protein kinase (MAPK) and can be normalized by pharmacological p38α MAPK inhibition, consistent with SERT Ala56 mimicking, constitutively, a high-activity conformation entered into transiently by p38α MAPK activation. We hypothesize that changes in SERT-interacting proteins (SIPs) support the shift of SERT into the SERT∗ state which may be captured by comparing the composition of SERT Ala56 protein complexes with those of wildtype (WT) SERT, defining specific interactions through comparisons of protein complexes recovered using preparations from SERT–/– (knockout; KO) mice. Using quantitative proteomic-based approaches, we identify a total of 459 SIPs, that demonstrate both SERT specificity and sensitivity to the Gly56Ala substitution, with a striking bias being a loss of SIP interactions with SERT Ala56 compared to WT SERT. Among this group are previously validated SIPs, such as flotillin-1 (FLOT1) and protein phosphatase 2A (PP2A), whose functions are believed to contribute to SERT microdomain localization and regulation. Interestingly, our studies nominate a number of novel SIPs implicated in ASD, including fragile X mental retardation 1 protein (FMR1) and SH3 and multiple ankyrin repeat domains protein 3 (SHANK3), of potential relevance to long-standing evidence of serotonergic contributions to ASD. Further investigation of these SIPs, and the broader networks they engage, may afford a greater understanding of ASD as well as other brain and peripheral disorders associated with perturbed 5-HT signaling.
Highlights
Disruptions in serotonergic signaling have been implicated in a wide array of neurological disorders, including major depressive disorder (MDD) (Plaznik et al, 1989; Meltzer, 1990), autism spectrum disorder (ASD) (Cook et al, 1997; Muller et al, 2016) and obsessive–compulsive disorder (OCD) (Sinopoli et al, 2017)
Single particle labeling studies reveal that regulation of SERT by protein kinase G (PKG)/p38α MAPK pathways leads to changes in SERT surface mobility mobility, with functional and mobility effects mimicked by treatment with the actin cytoskeleton destabilizer cytochalasin D (Chang et al, 2012), suggesting surface transporter anchoring by one or more actin-associated proteins
We limited our studies to male mice since past studies of SERT Ala56 mice have been restricted to males (Veenstra-VanderWeele et al, 2012; Robson et al, 2018; Quinlan et al, 2019), primarily due to the 4:1 male bias found in ASD (Baio et al, 2018)
Summary
Disruptions in serotonergic signaling have been implicated in a wide array of neurological disorders, including major depressive disorder (MDD) (Plaznik et al, 1989; Meltzer, 1990), autism spectrum disorder (ASD) (Cook et al, 1997; Muller et al, 2016) and obsessive–compulsive disorder (OCD) (Sinopoli et al, 2017). Pharmacological activation of intracellular signaling pathways can be mimicked by stimulation of cell surface receptors in transfected and natively expressing preparations, including the A3 adenosine receptor (A3AR) (Miller and Hoffman, 1994; Zhu et al, 2004, 2007), kappa opiate receptors (Sundaramurthy et al, 2017), integrin b3 (ITGB3) receptor (Carneiro et al, 2008), and interleukin 1 receptor (IL-1R) (Zhu et al, 2010), with A3ARs and ITGB3 receptors reported to be SIPs (Carneiro et al, 2008; Zhu et al, 2011) It is hypothesized SERT activity is defined and regulated by distinct SIP complexes that can include receptors as well as intracellular signaling machinery. More subtle therapeutics that alter or overcome the actions of SIPs may allow for a restoration of normal, physiological 5-HT signaling with the appropriate control mechanisms intact
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