Summary: SERT Ala56 impacts the structure of the SERT protein cells, increasing the activity of the transporter to abnormally high levels. The high-activity state results in the removal of too much serotonin from brain sites where serotonin is needed, both during development and in adults.
Source: Florida Atlantic University
Recent estimates indicate a prevalence of autism spectrum disorder (ASD) in the United States of 1 in 59 children with a well-established 4:1 male predominance. Individual costs for care are estimated at about $2.4 million, yielding a societal burden that is expected to exceed $400 billion by 2025. Currently, there are no FDA-approved medications that improve the core symptoms of ASD.
Neuroscientists in the laboratory of Randy D. Blakely, Ph.D., professor of biomedical science in Florida Atlantic University’s Schmidt College of Medicine and executive director of the FAU Brain Institute, are focused on serotonin, a mood-regulating molecule in the brain that regulates many brain synapses — the gaps between nerve cells where signals are sent and received. The supply of serotonin is tightly regulated by a protein called the serotonin transporter (SERT), which sweeps away serotonin from synapses to limit its action. Shifts in the transporter’s activity can significantly impact the ability of serotonin to act in the brain.
Changes in signaling by the neurotransmitter serotonin has been connected to autism for more than 50 years, dating back to findings of elevated serotonin levels in the blood of some people with the disorder, a feature termed hyperserotonemia. Since this time, multiple rare changes in the genetic code that impact SERT have been observed.
Recent studies of mice expressing the most common of these gene variants, called SERT Ala56, induces behavioral changes in animals that scientists connect to alterations in people with autism – repetitive behavior, altered communication, and atypical social interactions, as well as hyperserotonemia. Although the effects of the SERT Ala56 mutation are paralleled by other mutations in the SERT gene, none of these mutations are common enough to account for the number of individuals with autism demonstrating hyperserotonemia.
Blakely and collaborators have published a study in the journal Frontiers in Molecular Neuroscience, which now offers a possible explanation for this puzzle.
“We have known for some time that the activity and regulation of the SERT protein is critically dependent on a number of other proteins that tell the protein where to locate on nerve cells and how to act,” said Blakely, lead author.
It turns out that the SERT Ala56 mutation changes the structure of the transporter in ways that keep these partner proteins from interacting with the transporter, a protein that Blakely’s laboratory identified thirty years ago.
Last year, Blakely’s former graduate student Meagan Quinlan, Ph.D., first author and a post-doctoral fellow at the University of Washington, demonstrated that the SERT Ala56 mutation impacts the structure of the SERT protein in cells in culture, revving up the transporter into a state of abnormally high activity.
“We think that this high-activity state results in removal of too much serotonin from sites in the brain where serotonin is needed, both during development and in adults,” said Quinlan. “I suspect that the structural changes we have observed likely reflected changes in the regulatory ability of SERT-associated proteins to maintain serotonin inactivation at normal levels.”
To test this concept, Quinlan purified the mutant transporter from the brain of SERT Ala56 expressing mice, as well as from normal mice, and then determined whether proteins normally bound to SERT were still attached.
“We found that only a few proteins seemed to interact with SERT Ala56 more than they should. In contrast, many more proteins appeared no longer tethered to the transporter, including some proteins we already knew to be SERT regulators and others that have been linked to ASD,” said Quinlan.
Blakely was actually quite surprised at the findings. “It is quite amazing that one of the smallest changes you can make in SERT can alter its interactions with partners,” said Blakely.
According to Blakely, further work could reveal more extensive links between SERT and other genes known to associate with autism, broadening the impact of SERT Ala56 to other neurotransmitter systems.
“Following the scent of serotonin may well take us to molecules and medications we had no clue about just a few years ago,” said Blakely.
Blakely’s team recently reported a new small molecule that can tone down SERT activity and normalize behaviors in the SERT Ala56 mouse, a molecule that he hopes may lead to a new therapeutic.
“A goal moving forward with this drug is to see if it can reverse the changes in SERT Ala56 protein associations, tying the molecular changes we see with the mutant to how the drug may work,” said Blakely.
ASD is a serious developmental disorder with increased prevalence that impairs the ability to communicate and interact and impacts the nervous system. The range and severity of symptoms can vary widely and common symptoms can include difficulty with communication and social interactions, obsessive interests, and repetitive behaviors. No current medications treat the core features of ASD.
Study co-authors are Matthew J. Robson, Ph.D., Division of Pharmaceutical Sciences, James L. Winkle College of Pharmacy, University of Cincinnati; Ran Ye, Ph.D., Department of Pharmacology, Vanderbilt University; and Kristie L. Rose, Ph.D., and Kevin L. Schey, Ph.D., both within the Department of Biochemistry, Vanderbilt University.
Funding: Financial support for the studies derives from Vanderbilt Pharmacology T32 (GM07628), an Elaine Sanders-Bush Scholar’s Award from the Vanderbilt Silvio O. Conte Center for Neuroscience Research (Quinlan), Vanderbilt Digestive Disease Research Center (P30DK058404), PhRMA Foundation Award and NARSAD YI Award 25230 (Robson), and National Institutes of Health grants MH096972 (Blakely) and MH094527 (Blakely).
About this neurodevelopment research article
Source:
Florida Atlantic University
Contacts:
Gisele Galoustian – Florida Atlantic University
Image Source:
The image is credited to Randy D. Blakely, Ph.D.
Original Research: Open access
“Ex vivo Quantitative Proteomic Analysis of Serotonin Transporter Interactome: Network Impact of the SERT Ala56 Coding Variant” by Randy D. Blakely et al. Frontiers in Molecular Neuroscience.
Abstract
Ex vivo Quantitative Proteomic Analysis of Serotonin Transporter Interactome: Network Impact of the SERT Ala56 Coding Variant
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.
