Publications by authors named "Randy D Blakely"

194 Publications

Serotonin Transporter Ala276 Mouse: Novel Model to Assess the Neurochemical and Behavioral Impact of Thr276 Phosphorylation In Vivo.

Neurochem Res 2021 Apr 8. Epub 2021 Apr 8.

Department of Biomedical Science, Charles E. Schmidt College of Medicine, Florida Atlantic University, Jupiter, FL, USA.

The serotonin (5-HT) transporter (SERT) is a key regulator of 5-HT signaling and is a major target for antidepressants and psychostimulants. Human SERT coding variants have been identified in subjects with obsessive-compulsive disorder (OCD) and autism spectrum disorder (ASD) that impact transporter phosphorylation, cell surface trafficking and/or conformational dynamics. Prior to an initial description of a novel mouse line expressing the non-phosphorylatable SERT substitution Thr276Ala, we review efforts made to elucidate the structure and conformational dynamics of SERT with a focus on research implicating phosphorylation at Thr276 as a determinant of SERT conformational dynamics. Using the high-resolution structure of human SERT in inward- and outward-open conformations, we explore the conformation dependence of SERT Thr276 exposure, with results suggesting that phosphorylation is likely restricted to an inward-open conformation, consistent with prior biochemical studies. Assessment of genotypes from SERT/Ala276 heterozygous matings revealed a deviation from Mendelian expectations, with reduced numbers of Ala276 offspring, though no genotype differences were seen in growth or physical appearance. Similarly, no genotype differences were evident in midbrain or hippocampal 5-HT levels, midbrain and hippocampal SERT mRNA or midbrain protein levels, nor in midbrain synaptosomal 5-HT uptake kinetics. Behaviorally, SERT Ala276 homozygotes appeared normal in measures of anxiety and antidepressant-sensitive stress coping behavior. However, these mice displayed sex-dependent alterations in repetitive and social interactions, consistent with circuit-dependent requirements for Thr276 phosphorylation underlying these behaviors. Our findings indicate the utility of SERT Ala276 mice in evaluation of developmental, functional and behavioral consequences of regulatory SERT phosphorylation in vivo.
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http://dx.doi.org/10.1007/s11064-021-03299-wDOI Listing
April 2021

Gαo is a major determinant of cAMP signaling in the pathophysiology of movement disorders.

Cell Rep 2021 Feb;34(5):108718

Department of Neuroscience, The Scripps Research Institute, Jupiter, FL 33458, USA. Electronic address:

The G protein alpha subunit o (Gαo) is one of the most abundant proteins in the nervous system, and pathogenic mutations in its gene (GNAO1) cause movement disorder. However, the function of Gαo is ill defined mechanistically. Here, we show that Gαo dictates neuromodulatory responsiveness of striatal neurons and is required for movement control. Using in vivo optical sensors and enzymatic assays, we determine that Gαo provides a separate transduction channel that modulates coupling of both inhibitory and stimulatory dopamine receptors to the cyclic AMP (cAMP)-generating enzyme adenylyl cyclase. Through a combination of cell-based assays and rodent models, we demonstrate that GNAO1-associated mutations alter Gαo function in a neuron-type-specific fashion via a combination of a dominant-negative and loss-of-function mechanisms. Overall, our findings suggest that Gαo and its pathological variants function in specific circuits to regulate neuromodulatory signals essential for executing motor programs.
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http://dx.doi.org/10.1016/j.celrep.2021.108718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7903328PMC
February 2021

Serotonin 5-HT receptor-mediated behavior and binding in mice with the overactive and dysregulated serotonin transporter Ala56 variant.

Psychopharmacology (Berl) 2021 Apr 29;238(4):1111-1120. Epub 2021 Jan 29.

Department of Psychiatry, New York State Psychiatric Institute, Columbia University, 1051 Riverside Dr., Unit 78, New York, NY, 10032, USA.

Rationale: Elevated whole-blood serotonin (5-HT) is a robust biomarker in ~ 30% of patients with autism spectrum disorders, in which repetitive behavior is a core symptom. Furthermore, elevated whole-blood 5-HT has also been described in patients with pediatric obsessive-compulsive disorder. The 5-HT receptor is associated with repetitive behaviors seen in both disorders. Chronic blockade of serotonin transporter (SERT) reduces 5-HT receptor levels in the orbitofrontal cortex (OFC) and attenuates the sensorimotor deficits and hyperactivity seen with the 5-HT agonist RU24969. We hypothesized that enhanced SERT function would increase 5-HT receptor levels in OFC and enhance sensorimotor deficits and hyperactivity induced by RU24969.

Objectives: We examined the impact of the SERT Ala56 mutation, which leads to enhanced SERT function, on 5-HT receptor binding and 5-HT-mediated sensorimotor deficits.

Methods: Specific binding to 5-HT receptors was measured in OFC and striatum of naïve SERT Ala56 or wild-type mice. The impact of the 5-HT receptor agonist RU24969 on prepulse inhibition (PPI) of startle, hyperactivity, and expression of cFos was examined.

Results: While enhanced SERT function increased 5-HT receptor levels in OFC of Ala56 mice, RU24969-induced PPI deficits and hyperlocomotion were not different between genotypes. Baseline levels of cFos expression were not different between groups. RU24969 increased cFos expression in OFC of wild-types and decreased cFos in the striatum.

Conclusions: While reducing 5-HT receptors may attenuate sensorimotor gating deficits, increased 5-HT levels in SERT Ala56 mice do not necessarily exacerbate these deficits, potentially due to compensations during neural circuit development in this model system.
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http://dx.doi.org/10.1007/s00213-020-05758-8DOI Listing
April 2021

There's no place like home? Return to the home cage triggers dopamine release in the mouse nucleus accumbens.

Neurochem Int 2021 01 5;142:104894. Epub 2020 Nov 5.

Department of Biomedical Science, Florida Atlantic University, Jupiter, FL, USA; Brain Institute, Florida Atlantic University, Jupiter, FL, USA. Electronic address:

Various stimuli have been employed as reinforcers in preclinical rodent models to elucidate the underpinnings of reward at a molecular and circuit level, with the release of dopamine (DA) in the nucleus accumbens (NAc) as a well-replicated, physiological correlate. Many factors, however, including strain differences, sex, prior stress, and reinforcer administration protocols can influence reward responding and DA release. Although previous evidence indicates that access to the home cage can be an effective reinforcer in behavioral tasks, whether this simple environmental manipulation can trigger DA release in the NAc has not been demonstrated. Here, using fiber photometric recordings of in vivo NAc dopamine release from a genetically-encoded DA sensor, we show that the movement of animals from the home cage to a clear, polycarbonate recording chamber evokes little to no DA release following initial exposure whereas returning animals from the recording chamber to a clean, home-like cage or to the home cage robustly triggers the release of DA, comparable in size to that observed with a 10 mg/kg i.p. Cocaine injection in the recording chamber. Although DA release can be evoked in moving mice to a clean cage, this release was significantly augmented when moving animals from the clean cage to the home cage. Our data provide direct evidence that home cage return from a foreign environment results in a biochemical change consistent with that of a rewarding stimulus. This simple environmental manipulation provides a minimally invasive approach to study the reward circuitry underlying an ethologically relevant reinforcer, return to the safe confines of "home". The home cage - DA release paradigm may also represent a biomarker-driven paradigm for the evaluation of genetic and experiential events that underlie anhedonic states, characteristic of major mood disorders, and to present new opportunities to identify their treatments.
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http://dx.doi.org/10.1016/j.neuint.2020.104894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7779706PMC
January 2021

Regulation of autism-relevant behaviors by cerebellar-prefrontal cortical circuits.

Nat Neurosci 2020 09 13;23(9):1102-1110. Epub 2020 Jul 13.

Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Cerebellar dysfunction has been demonstrated in autism spectrum disorders (ASDs); however, the circuits underlying cerebellar contributions to ASD-relevant behaviors remain unknown. In this study, we demonstrated functional connectivity between the cerebellum and the medial prefrontal cortex (mPFC) in mice; showed that the mPFC mediates cerebellum-regulated social and repetitive/inflexible behaviors; and showed disruptions in connectivity between these regions in multiple mouse models of ASD-linked genes and in individuals with ASD. We delineated a circuit from cerebellar cortical areas Right crus 1 (Rcrus1) and posterior vermis through the cerebellar nuclei and ventromedial thalamus and culminating in the mPFC. Modulation of this circuit induced social deficits and repetitive behaviors, whereas activation of Purkinje cells (PCs) in Rcrus1 and posterior vermis improved social preference impairments and repetitive/inflexible behaviors, respectively, in male PC-Tsc1 mutant mice. These data raise the possibility that these circuits might provide neuromodulatory targets for the treatment of ASD.
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http://dx.doi.org/10.1038/s41593-020-0665-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483861PMC
September 2020

Quantitative Proteomic Analysis of Serotonin Transporter Interactome: Network Impact of the SERT Ala56 Coding Variant.

Front Mol Neurosci 2020 8;13:89. Epub 2020 Jun 8.

Department of Pharmacology, Vanderbilt University, Nashville, TN, United States.

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 and . 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.
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http://dx.doi.org/10.3389/fnmol.2020.00089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295033PMC
June 2020

A social encounter drives gene expression changes linked to neuronal function, brain development, and related disorders in mice expressing the serotonin transporter Ala56 variant.

Neurosci Lett 2020 06 11;730:135027. Epub 2020 May 11.

Department of Psychiatry, Columbia University, New York State Psychiatric Institute, 1051 Riverside Dr, Unit 78, New York, NY 10032, United States; Vanderbilt University, Nashville, TN 37232, United States. Electronic address:

Multiple lines of evidence implicate the serotonin (5-HT) system in social function, including biomarker findings in autism spectrum disorder. In mice, knock-in of a rare Gly56Ala substitution in the serotonin transporter (SERT) causes elevated whole blood 5-HT levels, increased 5-HT clearance in the brain, and altered social and repetitive behavior. To further examine the molecular impact of this variant on social response, SERT Ala56 mutant mice and wildtype littermate controls were exposed to a social or non-social stimulus. We examined the differential activation of the prefrontal cortex, lateral amygdala, and medial amygdala, to social stimuli through RNA sequencing. Differentially expressed genes were enriched in axonal guidance signaling pathways, networks related to nervous system development and function, neurological and psychiatric disorders, and behavior. These identified pathways and networks may shed light on the molecular cascades underlying the impact of altered SERT function on social behavior.
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http://dx.doi.org/10.1016/j.neulet.2020.135027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7440397PMC
June 2020

Neurobehavioral changes arising from early life dopamine signaling perturbations.

Neurochem Int 2020 07 20;137:104747. Epub 2020 Apr 20.

Department of Biomedical Science, Florida Atlantic University, Jupiter, FL, 33458, USA; Brain Institute, Florida Atlantic University, Jupiter, FL, 33458, USA. Electronic address:

Dopamine (DA) signaling is critical to the modulation of multiple brain functions including locomotion, reinforcement, attention and cognition. The literature provides strong evidence that altered DA availability and actions can impact normal neurodevelopment, with both early and enduring consequences on anatomy, physiology and behavior. An appreciation for the developmental contributions of DA signaling to brain development is needed to guide efforts to preclude and remedy neurobehavioral disorders, such as attention-deficit/hyperactivity disorder, addiction, bipolar disorder, schizophrenia and autism spectrum disorder, each of which exhibits links to DA via genetic, cellular and/or pharmacological findings. In this review, we highlight research pursued in preclinical models that use genetic and pharmacological approaches to manipulate DA signaling at sensitive developmental stages, leading to changes at molecular, circuit and/or behavioral levels. We discuss how these alterations can be aligned with traits displayed by neuropsychiatric diseases. Lastly, we review human studies that evaluate contributions of developmental perturbations of DA systems to increased risk for neuropsychiatric disorders.
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http://dx.doi.org/10.1016/j.neuint.2020.104747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261509PMC
July 2020

Dopamine-dependent, swimming-induced paralysis arises as a consequence of loss of function mutations in the RUNX transcription factor RNT-1.

PLoS One 2019 13;14(5):e0216417. Epub 2019 May 13.

Department of Biomedical Science, Charles E. Schmidt College of Science, Florida Atlantic University, Jupiter, FL United States of America.

Dopamine (DA) is a neurotransmitter with actions across phylogeny that modulate core behaviors such as motor activity, reward, attention, and cognition. Perturbed DA signaling in humans is associated with multiple disorders, including addiction, ADHD, schizophrenia, and Parkinson's disease. The presynaptic DA transporter exerts powerful control on DA signaling by efficient clearance of the neurotransmitter following release. As in vertebrates, Caenorhabditis elegans DAT (DAT-1) constrains DA signaling and loss of function mutations in the dat-1 gene result in slowed crawling on solid media and swimming-induced paralysis (Swip) in water. Previously, we identified a mutant line, vt34, that exhibits robust DA-dependent Swip. vt34 exhibits biochemical and behavioral phenotypes consistent with reduced DAT-1 function though vt34; dat-1 double mutants exhibit an enhanced Swip phenotype, suggesting contributions of the vt34-associated mutation to additional mechanisms that lead to excess DA signaling. SNP mapping and whole genome sequencing of vt34 identified the site of the molecular lesion in the gene B0412.2 that encodes the Runx transcription factor ortholog RNT-1. Unlike dat-1 animals, but similar to other loss of function rnt-1 mutants, vt34 exhibits altered male tail morphology and reduced body size. Deletion mutations in both rnt-1 and the bro-1 gene, which encodes a RNT-1 binding partner also exhibit Swip. Both vt34 and rnt-1 mutations exhibit reduced levels of dat-1 mRNA as well as the tyrosine hydroxylase ortholog cat-2. Although reporter studies indicate that rnt-1 is expressed in DA neurons, its re-expression in DA neurons of vt34 animals fails to fully rescue Swip. Moreover, as shown for vt34, rnt-1 mutation exhibits additivity with dat-1 in generating Swip, as do rnt-1 and bro-1 mutations, and vt34 exhibits altered capacity for acetylcholine signaling at the neuromuscular junction. Together, these findings identify a novel role for rnt-1 in limiting DA neurotransmission and suggest that loss of RNT-1 may disrupt function of both DA neurons and body wall muscle to drive Swip.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0216417PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6513266PMC
January 2020

Repetitive mild concussion in subjects with a vulnerable cholinergic system: Lasting cholinergic-attentional impairments in CHT+/- mice.

Behav Neurosci 2019 Aug 21;133(4):448-459. Epub 2019 Mar 21.

Department of Psychology and Neuroscience Program.

Previous research emphasized the impact of traumatic brain injury on cholinergic systems and associated cognitive functions. Here we addressed the converse question: Because of the available evidence indicating cognitive and neuronal vulnerabilities in humans expressing low-capacity cholinergic systems or with declining cholinergic systems, do injuries cause more severe cognitive decline in such subjects, and what cholinergic mechanisms contribute to such vulnerability? Using mice heterozygous for the choline transporter (CHT+/- mice) as a model for a limited cholinergic capacity, we investigated the cognitive and neuronal consequences of repeated, mild concussion injuries (rmCc). After five rmCc, and compared with wild type (WT) mice, CHT+/- mice exhibited severe and lasting impairments in sustained attention performance, consistent with effects of cholinergic losses on attention. However, rmCc did not affect the integrity of neuronal cell bodies and did not alter the density of cortical synapses. As a cellular mechanism potentially responsible for the attentional impairment in CHT+/- mice, we found that rmCc nearly completely attenuated performance-associated, CHT-mediated choline transport. These results predict that subjects with an already vulnerable cholinergic system will experience severe and lasting cognitive-cholinergic effects after even relatively mild injuries. If confirmed in humans, such subjects may be excluded from, or receive special protection against, activities involving injury risk. Moreover, the treatment and long-term outcome of traumatic brain injuries may benefit from determining the status of cholinergic systems and associated cognitive functions. (PsycINFO Database Record (c) 2019 APA, all rights reserved).
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http://dx.doi.org/10.1037/bne0000310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6625848PMC
August 2019

The SERT Met172 Mouse: An Engineered Model To Elucidate the Contributions of Serotonin Signaling to Cocaine Action.

ACS Chem Neurosci 2019 07 13;10(7):3053-3060. Epub 2019 Mar 13.

Department of Biomedical Science, Charles E. Schmidt College of Medicine and Brain Institute , Florida Atlantic University , Jupiter , Florida 33458 , United States.

Cocaine abuse and addiction remain highly prevalent and, unfortunately, poorly treated. It is well-known that essential aspects of cocaine's addictive actions involve the drug's ability to block the presynaptic dopamine (DA) transporter (DAT), thereby elevating extracellular levels of DA in brain circuits that subserve reward, reinforcement, and habit. Less well appreciated are the multiple DA-independent actions of cocaine, activities that we and others believe contribute key pieces to the puzzle of cocaine addiction, treatment, and relapse. In particular, a significant body of work points to altered serotonin (5-HT) signaling as one such component, not surprising given that, relative to DAT, cocaine acts as potently to block the 5-HT transporter (SERT) as to block DAT, and thereby elevates extracellular 5-HT levels throughout the brain when reward-eliciting DA elevations occur. To elucidate the contribution of SERT antagonism to the actions of cocaine, we engineered a mouse model that significantly reduces cocaine potency at SERT without disrupting the expression or function of SERT in vivo. In this short Perspective, we review the rationale for development of the SERT Met172 model, the studies that document the pharmacological impact of the Ile172Met substitution in vitro and in vivo, and our findings with the model that demonstrate serotonergic contributions to the genetic, physiological, and behavioral actions of cocaine.
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http://dx.doi.org/10.1021/acschemneuro.9b00005DOI Listing
July 2019

Cell-Type-Specific Interleukin 1 Receptor 1 Signaling in the Brain Regulates Distinct Neuroimmune Activities.

Immunity 2019 02 22;50(2):317-333.e6. Epub 2019 Jan 22.

Institute for Behavioral Medicine Research, College of Medicine, The Ohio State University, Columbus, OH 43210, USA; Division of Biosciences, College of Dentistry, The Ohio State University, Columbus, OH 43210, USA. Electronic address:

Interleukin-1 (IL-1) signaling is important for multiple potentially pathogenic processes in the central nervous system (CNS), but the cell-type-specific roles of IL-1 signaling are unclear. We used a genetic knockin reporter system in mice to track and reciprocally delete or express IL-1 receptor 1 (IL-1R1) in specific cell types, including endothelial cells, ventricular cells, peripheral myeloid cells, microglia, astrocytes, and neurons. We found that endothelial IL-1R1 was necessary and sufficient for mediating sickness behavior and drove leukocyte recruitment to the CNS and impaired neurogenesis, whereas ventricular IL-1R1 was critical for monocyte recruitment to the CNS. Although microglia did not express IL-1R1, IL-1 stimulation of endothelial cells led to the induction of IL-1 in microglia. Together, these findings describe the structure and functions of the brain's IL-1R1-expressing system and lay a foundation for the dissection and identification of IL-1R1 signaling pathways in the pathogenesis of CNS diseases.
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http://dx.doi.org/10.1016/j.immuni.2018.12.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759085PMC
February 2019

Human Serotonin Transporter Coding Variation Establishes Conformational Bias with Functional Consequences.

ACS Chem Neurosci 2019 07 5;10(7):3249-3260. Epub 2019 Feb 5.

Department of Biomedical Sciences , University of North Dakota School of Medicine and Health Sciences , Grand Forks , North Dakota 58202 , United States.

The antidepressant-sensitive serotonin (5-HT) transporter (SERT) dictates rapid, high-affinity clearance of the neurotransmitter in both the brain and periphery. In a study of families with multiple individuals diagnosed with autism spectrum disorder (ASD), we previously identified several, rare, missense coding variants that impart elevated 5-HT transport activity, relative to wild-type SERT, upon heterologous expression as well as in ASD subject lymphoblasts. The most common of these variants, SERT Ala56, located in the transporter's cytosolic N-terminus, has been found to confer in transgenic mice hyperserotonemia, an ASD-associated biochemical trait, an elevated brain 5-HT clearance rate, and ASD-aligned behavioral changes. Hyperfunction of SERT Ala56 has been ascribed to a change in 5-HT , though the physical basis of this change has yet to be elucidated. Through assessments of fluorescence resonance energy transfer (FRET) between cytosolic N- and C-termini, sensitivity to methanethiosulfonates, and capacity for N-terminal tryptic digestion, we obtain evidence for mutation-induced conformational changes that support an open-outward 5-HT binding conformation and . Aspects of these findings were also evident with another naturally occurring C-terminal SERT coding variant identified in our ASD study, Asn605. We conclude that biased conformations of surface resident transporters that can impact transporter function and regulation are an unappreciated consequence of heritable and disease-associated SERT coding variation.
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http://dx.doi.org/10.1021/acschemneuro.8b00689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640095PMC
July 2019

Serotonin transporter inhibition and 5-HT receptor activation drive loss of cocaine-induced locomotor activation in DAT Val559 mice.

Neuropsychopharmacology 2019 04 21;44(5):994-1006. Epub 2018 Dec 21.

Department of Biomedical Science, Florida Atlantic University, Jupiter, FL, USA.

Dopamine (DA) signaling dysfunction is believed to contribute to multiple neuropsychiatric disorders including attention-deficit/hyperactivity disorder (ADHD). The rare DA transporter (DAT) coding substitution Ala559Val found in subjects with ADHD, bipolar disorder and autism, promotes anomalous DA efflux in vitro and, in DAT Val559 mice, leads to increased reactivity to imminent handling, waiting impulsivity, and enhanced motivation for reward. Here, we report that, in contrast to amphetamine and methylphenidate, which induce significant locomotor activation, cocaine administration to these mice elicits no locomotor effects, despite retention of conditioned place preference (CPP). Additionally, cocaine fails to elevate extracellular DA. Given that amphetamine and methylphenidate, unlike cocaine, lack high-affinity interactions with the serotonin (5-HT) transporter (SERT), we hypothesized that the lack of cocaine-induced hyperlocomotion in DAT Val559 mice arises from SERT blockade and augmented 5-HT signaling relative to cocaine actions on wildtype animals. Consistent with this idea, the SERT blocker fluoxetine abolished methylphenidate-induced locomotor activity in DAT Val559 mice, mimicking the effects seen with cocaine. Additionally, a cocaine analog (RTI-113) with greater selectivity for DAT over SERT retains locomotor activation in DAT Val559 mice. Furthermore, genetic elimination of high-affinity cocaine interactions at SERT in DAT Val559 mice, or specific inhibition of 5-HT receptors in these animals, restored cocaine-induced locomotion, but did not restore cocaine-induced elevations of extracellular DA. Our findings reveal a significant serotonergic plasticity arising in the DAT Val559 model that involves enhanced 5-HT signaling, acting independently of striatal DA release, capable of suppressing the activity of cocaine-sensitive motor circuits.
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http://dx.doi.org/10.1038/s41386-018-0301-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6462012PMC
April 2019

The brain in flux: Genetic, physiologic, and therapeutic perspectives on transporters in the CNS.

Neurochem Int 2019 02 17;123:1-6. Epub 2018 Dec 17.

Departments of Pediatrics and Systems Pharmacology and Translational Therapeutics, Children's Hospital of Philadelphia/University of Pennsylvania, Philadelphia, PA, 19104, United States. Electronic address:

The brain has specific properties that make it uniquely dependent upon transporters. This is the 3rd edition of a biennial special issue that originates from a scientific meeting devoted to studies of transporters and their relationship to brain function and to neurodevelopmental, neurologic, and psychiatric disorders. The field continues to rapidly evolve with advances in studies of structure that inform mechanism, with genetic analyses in humans revealing surprising aspects of biology, and with integrated cellular to whole animal analyses of the role of transporters in their control of physiology and pathophysiology. This special issue includes a sampling of review articles that address timely questions of the field followed by several primary research articles.
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http://dx.doi.org/10.1016/j.neuint.2018.12.006DOI Listing
February 2019

p38α MAPK signaling drives pharmacologically reversible brain and gastrointestinal phenotypes in the SERT Ala56 mouse.

Proc Natl Acad Sci U S A 2018 10 8;115(43):E10245-E10254. Epub 2018 Oct 8.

Department of Biomedical Science, Charles E. Schmidt College of Medicine, Florida Atlantic University, Jupiter, FL 33458;

Autism spectrum disorder (ASD) is a common neurobehavioral disorder with limited treatment options. Activation of p38 MAPK signaling networks has been identified in ASD, and p38 MAPK signaling elevates serotonin (5-HT) transporter (SERT) activity, effects mimicked by multiple, hyperfunctional SERT coding variants identified in ASD subjects. Mice expressing the most common of these variants (SERT Ala56) exhibit hyperserotonemia, a biomarker observed in ASD subjects, as well as p38 MAPK-dependent SERT hyperphosphorylation, elevated hippocampal 5-HT clearance, hypersensitivity of CNS 5-HT and 5-HT receptors, and behavioral and gastrointestinal perturbations reminiscent of ASD. As the α-isoform of p38 MAPK drives SERT activation, we tested the hypothesis that CNS-penetrant, α-isoform-specific p38 MAPK inhibitors might normalize SERT Ala56 phenotypes. Strikingly, 1-week treatment of adult SERT Ala56 mice with MW150, a selective p38α MAPK inhibitor, normalized hippocampal 5-HT clearance, CNS 5-HT and 5-HT receptor sensitivities, social interactions, and colonic motility. Conditional elimination of p38α MAPK in 5-HT neurons of SERT Ala56 mice restored 5-HT and 5-HT receptor sensitivities as well as social interactions, mirroring effects of MW150. Our findings support ongoing p38α MAPK activity as an important determinant of the physiological and behavioral perturbations of SERT Ala56 mice and, more broadly, supports consideration of p38α MAPK inhibition as a potential treatment for core and comorbid phenotypes present in ASD subjects.
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http://dx.doi.org/10.1073/pnas.1809137115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205438PMC
October 2018

Single Quantum Dot Imaging Reveals PKCβ-Dependent Alterations in Membrane Diffusion and Clustering of an Attention-Deficit Hyperactivity Disorder/Autism/Bipolar Disorder-Associated Dopamine Transporter Variant.

ACS Chem Neurosci 2019 01 11;10(1):460-471. Epub 2018 Sep 11.

The dopamine transporter (DAT) is a transmembrane protein that terminates dopamine signaling in the brain by driving rapid dopamine reuptake into presynaptic nerve terminals. Several lines of evidence indicate that DAT dysfunction is linked to neuropsychiatric disorders such as attention-deficit/hyperactivity disorder (ADHD), bipolar disorder (BPD), and autism spectrum disorder (ASD). Indeed, individuals with these disorders have been found to express the rare, functional DAT coding variant Val559, which confers anomalous dopamine efflux (ADE) in vitro and in vivo. To elucidate the impact of the DAT Val559 variant on membrane diffusion dynamics, we implemented our antagonist-conjugated quantum dot (QD) labeling approach to monitor the lateral mobility of single particle-labeled transporters in transfected HEK-293 and SK-N-MC cells. Our results demonstrate significantly higher diffusion coefficients of DAT Val559 compared to those of DAT Ala559, effects likely determined by elevated N-terminal transporter phosphorylation. We also provide pharmacological evidence that PKCβ-mediated signaling supports enhanced DAT Val559 membrane diffusion rates. Additionally, our results are complimented with diffusion rates of phosphomimicked and phosphorylation-occluded DAT variants. Furthermore, we show DAT Val559 has a lower propensity for membrane clustering, which may be caused by a mutation-derived shift out of membrane microdomains leading to faster lateral membrane diffusion rates. These findings further demonstrate a functional impact of DAT Val559 and suggest that changes in transporter localization and lateral mobility may sustain ADE and contribute to alterations in dopamine signaling underlying multiple neuropsychiatric disorders.
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http://dx.doi.org/10.1021/acschemneuro.8b00350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411462PMC
January 2019

Pancreatic deletion of the interleukin-1 receptor disrupts whole body glucose homeostasis and promotes islet β-cell de-differentiation.

Mol Metab 2018 08 6;14:95-107. Epub 2018 Jun 6.

Pennington Biomedical Research Center, Baton Rouge, LA, 70808, USA. Electronic address:

Objective: Pancreatic tissue, and islets in particular, are enriched in expression of the interleukin-1 receptor type I (IL-1R). Because of this enrichment, islet β-cells are exquisitely sensitive to the IL-1R ligands IL-1α and IL-1β, suggesting that signaling through this pathway regulates health and function of islet β-cells.

Methods: Herein, we report a targeted deletion of IL-1R in pancreatic tissue (IL-1R) in C57BL/6J mice and in db/db mice on the C57 genetic background. Islet morphology, β-cell transcription factor abundance, and expression of the de-differentiation marker Aldh1a3 were analyzed by immunofluorescent staining. Glucose and insulin tolerance tests were used to examine metabolic status of these genetic manipulations. Glucose-stimulated insulin secretion was evaluated in vivo and in isolated islets ex vivo by perifusion.

Results: Pancreatic deletion of IL-1R leads to impaired glucose tolerance, a phenotype that is exacerbated by age. Crossing the IL-1R with db/db mice worsened glucose tolerance without altering body weight. There were no detectable alterations in insulin tolerance between IL-1R mice and littermate controls. However, glucose-stimulated insulin secretion was reduced in islets isolated from IL-1R relative to control islets. Insulin output in vivo after a glucose challenge was also markedly reduced in IL-1R mice when compared with littermate controls. Pancreatic islets from IL-1R mice displayed elevations in Aldh1a3, a marker of de-differentiation, and reduction in nuclear abundance of the β-cell transcription factor MafA. Nkx6.1 abundance was unaltered.

Conclusions: There is an important physiological role for pancreatic IL-1R to promote glucose homeostasis by suppressing expression of Aldh1a3, sustaining MafA abundance, and supporting glucose-stimulated insulin secretion in vivo.
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http://dx.doi.org/10.1016/j.molmet.2018.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034063PMC
August 2018

Adrenal serotonin derives from accumulation by the antidepressant-sensitive serotonin transporter.

Pharmacol Res 2019 02 9;140:56-66. Epub 2018 Jun 9.

Department of Biomedical Sciences, Cooper Medical School of Rowan University, Camden, NJ, USA; Department of Anesthesiology, Vanderbilt University School of Medicine, Nashville TN, USA. Electronic address:

Adrenal chromaffin cells comprise the neuroendocrine arm of the sympathetic nervous system and secrete catecholamines to coordinate the appropriate stress response. Deletion of the serotonin (5-HT) transporter (SERT) gene in mice (SERT mice) or pharmacological block of SERT function in rodents and humans augments this sympathoadrenal stress response (epinephrine secretion). The prevailing assumption is that loss of CNS SERT alters central drive to the peripheral sympathetic nervous system. Adrenal chromaffin cells also prominently express SERT where it might coordinate accumulation of 5-HT for reuse in the autocrine control of stress-evoked catecholamine secretion. To help test this hypothesis, we have generated a novel mouse model with selective excision of SERT in the peripheral sympathetic nervous system (SERT), generated by crossing floxed SERT mice with tyrosine hydroxylase Cre driver mice. SERT expression, assessed by western blot, was abolished in the adrenal gland but not perturbed in the CNS of SERT mice. SERT-mediated [H] 5-HT uptake was unaltered in midbrain, hindbrain, and spinal cord synaptosomes, confirming transporter function was intact in the CNS. Endogenous midbrain and whole blood 5-HT homeostasis was unperturbed in SERT mice, contrasting with the depleted 5-HT content in SERT mice. Selective SERT excision reduced adrenal gland 5-HT content by ≈ 50% in SERT mice but had no effect on adrenal catecholamine content. This novel model confirms that SERT expressed in adrenal chromaffin cells is essential for maintaining wild-type levels of 5-HT and provides a powerful tool to help dissect the role of SERT in the sympathetic stress response.
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http://dx.doi.org/10.1016/j.phrs.2018.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286867PMC
February 2019

Global untargeted serum metabolomic analyses nominate metabolic pathways responsive to loss of expression of the orphan metallo β-lactamase, MBLAC1.

Mol Omics 2018 Jun;14(3):142-155

Department of Biomedical Science, Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, FL, USA.

The C. elegans gene swip-10 encodes an orphan metallo β-lactamase that genetic studies indicate is vital for limiting neuronal excitability and viability. Sequence analysis indicates that the mammalian gene Mblac1 is the likely ortholog of swip-10, with greatest sequence identity localized to the encoded protein's single metallo β-lactamase domain. The substrate for the SWIP-10 protein remains unknown and to date no functional roles have been ascribed to MBLAC1, though we have shown that the protein binds the neuroprotective β-lactam antibiotic, ceftriaxone. To gain insight into the functional role of MBLAC1 in vivo, we used CRISPR/Cas9 methods to disrupt N-terminal coding sequences of the mouse Mblac1 gene, resulting in a complete loss of protein expression in viable, homozygous knockout (KO) animals. Using serum from both WT and KO mice, we performed global, untargeted metabolomic analyses, resolving small molecules via hydrophilic interaction chromatography (HILIC) based ultra-performance liquid chromatography, coupled to mass spectrometry (UPLC-MS/MS). Unsupervised principal component analysis reliably segregated the metabolomes of MBLAC1 KO and WT mice, with 92 features subsequently nominated as significantly different by ANOVA, and for which we made tentative and putative metabolite assignments. Bioinformatic analyses of these molecules nominate validated pathways subserving bile acid biosynthesis and linoleate metabolism, networks known to be responsive to metabolic and oxidative stress. Our findings lead to hypotheses that can guide future targeted studies seeking to identify the substrate for MBLAC1 and how substrate hydrolysis supports the neuroprotective actions of ceftriaxone.
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http://dx.doi.org/10.1039/c7mo00022gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6015503PMC
June 2018

Blockade and reversal of swimming-induced paralysis in C. elegans by the antipsychotic and D2-type dopamine receptor antagonist azaperone.

Neurochem Int 2019 02 22;123:59-68. Epub 2018 May 22.

Department of Biomedical Science, Charles E. Schmidt College of Medicine, Florida Atlantic University, FL, USA; Brain Institute, Florida Atlantic University, Jupiter, FL, 33458, USA. Electronic address:

The catecholamine neurotransmitter dopamine (DA) exerts powerful modulatory control of physiology and behavior across phylogeny. Perturbations of DA signaling in humans are associated with multiple neurodegenerative and behavioral disorders, including Parkinson's disease, attention-deficit/hyperactivity disorder, addiction and schizophrenia. In the nematode C. elegans, DA signaling regulates mating behavior, learning, food seeking and locomotion. Previously, we demonstrated that loss of function mutations in the dat-1 gene that encodes the presynaptic DA transporter (DAT-1) results in a rapid cessation of movement when animals are placed in water, termed Swimming Induced Paralysis (Swip). Loss of function mutations in genes that support DA biosynthesis, DA vesicular packaging and DA action at the extrasynaptic D2-type DA receptor DOP-3 suppress Swip in dat-1 animals, consistent with paralysis as arising from excessive DA signaling. Although animals grown on the vesicular monoamine transporter antagonist reserpine diminish Swip, the drug must be applied chronically, can impact the signaling of multiple biogenic amines, and has been reported to have penetrant, off-target actions. Here, we demonstrate that the antipsychotic drug azaperone potently and rapidly suppresses Swip behavior in either dat-1 mutants, as well as in wildtype animals treated with the DAT-1 antagonist nisoxetine, with genetic experiments consistent with DOP-3 antagonism as the mechanism of Swip suppression. Reversal of Swip in previously paralyzed dat-1 animals by azaperone application demonstrates an otherwise functionally-intact swimming circuit in these mutants. Finally, whereas azaperone suppresses DA-dependent Swip, the drug fails to attenuate the DA-independent paralysis induced by βPEA, aldicarb or genetic disruption of γ-aminobutyric acid (GABA) signaling. We discuss our findings with respect to the use of azaperone as a potent and selective tool in the identification and analysis of presynaptic mechanisms that regulate DA signaling.
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http://dx.doi.org/10.1016/j.neuint.2018.05.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6250597PMC
February 2019

Region-Specific Regulation of Presynaptic Dopamine Homeostasis by D Autoreceptors Shapes the Impact of the Neuropsychiatric Disease-Associated DAT Variant Val559.

J Neurosci 2018 06 8;38(23):5302-5312. Epub 2018 May 8.

Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232,

Disruptions of dopamine (DA) signaling contribute to a broad spectrum of neuropsychiatric disorders, including attention-deficit hyperactivity disorder (ADHD), addiction, bipolar disorder, and schizophrenia. Despite evidence that risk for these disorders derives from heritable variation in DA-linked genes, a better understanding is needed of the molecular and circuit context through which gene variation drives distinct disease traits. Previously, we identified the DA transporter (DAT) variant Val559 in subjects with ADHD and established that the mutation supports anomalous DAT-mediated DA efflux (ADE). Here, we demonstrate that region-specific contributions of D autoreceptors (D2AR) to presynaptic DA homeostasis dictate the consequences of Val559 expression in adolescent male mice. We show that activation of D2ARs in the WT dorsal striatum (DS), but not ventral striatum (VS), increases DAT phosphorylation and surface trafficking. In contrast, the activity of tyrosine hydroxylase (TH) is D2AR-dependent in both regions. In the DS but not VS of Val559 mice, tonic activation of D2ARs drives a positive feedback loop that promotes surface expression of efflux-prone DATs, raising extracellular DA levels and overwhelming DAT-mediated DA clearance capacity. Whereas D2ARs that regulate DAT are tonically activated in the Val559 DS, D2ARs that regulate TH become desensitized, allowing maintenance of cytosolic DA needed to sustain ADE. Together with prior findings, our results argue for distinct D2AR pools that regulate DA synthesis versus DA release and inactivation and offer a clear example of how the penetrance of gene variation can be limited to a subset of expression sites based on differences in intersecting regulatory networks. Altered dopamine (DA) signaling has been linked to multiple neuropsychiatric disorders. In an effort to understand and model disease-associated DAergic disturbances, we previously screened the DA transporter (DAT) in subjects with attention-deficit hyperactivity disorder (ADHD) and identified multiple, functionally impactful, coding variants. One of these variants, Val559, supports anomalous DA efflux (ADE) and in transgenic mice leads to changes in locomotor patterns, psychostimulant sensitivity, and impulsivity. Here, we show that the penetrance of Val559 ADE is dictated by region-specific differences in how presynaptic D-type autoreceptors (D2ARs) constrain DA signaling, biasing phenotypic effects to dorsal striatal projections. The Val559 model illustrates how the impact of genetic variation underlying neuropsychiatric disorders can be shaped by the differential engagement of synaptic regulatory mechanisms.
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http://dx.doi.org/10.1523/JNEUROSCI.0055-18.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5990980PMC
June 2018

Analysis of neuroanatomical differences in mice with genetically modified serotonin transporters assessed by structural magnetic resonance imaging.

Mol Autism 2018 10;9:24. Epub 2018 Apr 10.

1Mouse Imaging Centre (MICe), Hospital for Sick Children, 25 Orde Street, Toronto, Ontario M5T 3H7 Canada.

Background: The serotonin (5-HT) system has long been implicated in autism spectrum disorder (ASD) as indicated by elevated whole blood and platelet 5-HT, altered platelet and brain receptor and transporter binding, and genetic linkage and association findings. Based upon work in genetically modified mice, 5-HT is known to influence several aspects of brain development, but systematic neuroimaging studies have not previously been reported. In particular, the 5-HT transporter (serotonin transporter, SERT; 5-HTT) gene, , has been extensively studied.

Methods: Using a 7-T MRI and deformation-based morphometry, we assessed neuroanatomical differences in an knockout mouse on a C57BL/6 genetic background, along with an Ala56 knockin mouse on two different genetic backgrounds (129S and C57BL/6).

Results: Individually (same sex, same background, same genotype), the only differences found were in the female knockout mouse; all the others had no significant differences. However, an analysis of variance across the whole study sample revealed a significant effect of on the amygdala, thalamus, dorsal raphe nucleus, and lateral and frontal cortices.

Conclusions: This work shows that an increase or decrease in SERT function has a significant effect on the neuroanatomy in 5-HT relevant regions, particularly the raphe nuclei. Notably, the Ala56 knockin alone appears to have an insignificant, but suggestive, effect compared to the KO, which is consistent with function. Despite the small number of 5-HT neurons and their localization to the brainstem, it is clear that 5-HT plays an important role in neuroanatomical organization.
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http://dx.doi.org/10.1186/s13229-018-0210-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5894125PMC
October 2018

Glial loss of the metallo β-lactamase domain containing protein, SWIP-10, induces age- and glutamate-signaling dependent, dopamine neuron degeneration.

PLoS Genet 2018 03 28;14(3):e1007269. Epub 2018 Mar 28.

Department of Pharmacology, Vanderbilt University, Nashville, TN, United States of America.

Across phylogeny, glutamate (Glu) signaling plays a critical role in regulating neural excitability, thus supporting many complex behaviors. Perturbed synaptic and extrasynaptic Glu homeostasis in the human brain has been implicated in multiple neuropsychiatric and neurodegenerative disorders including Parkinson's disease, where theories suggest that excitotoxic insults may accelerate a naturally occurring process of dopamine (DA) neuron degeneration. In C. elegans, mutation of the glial expressed gene, swip-10, results in Glu-dependent DA neuron hyperexcitation that leads to elevated DA release, triggering DA signaling-dependent motor paralysis. Here, we demonstrate that swip-10 mutations induce premature and progressive DA neuron degeneration, with light and electron microscopy studies demonstrating the presence of dystrophic dendritic processes, as well as shrunken and/or missing cell soma. As with paralysis, DA neuron degeneration in swip-10 mutants is rescued by glial-specific, but not DA neuron-specific expression of wildtype swip-10, consistent with a cell non-autonomous mechanism. Genetic studies implicate the vesicular Glu transporter VGLU-3 and the cystine/Glu exchanger homolog AAT-1 as potential sources of Glu signaling supporting DA neuron degeneration. Degeneration can be significantly suppressed by mutations in the Ca2+ permeable Glu receptors, nmr-2 and glr-1, in genes that support intracellular Ca2+ signaling and Ca2+-dependent proteolysis, as well as genes involved in apoptotic cell death. Our studies suggest that Glu stimulation of nematode DA neurons in early larval stages, without the protective actions of SWIP-10, contributes to insults that ultimately drive DA neuron degeneration. The swip-10 model may provide an efficient platform for the identification of molecular mechanisms that enhance risk for Parkinson's disease and/or the identification of agents that can limit neurodegenerative disease progression.
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http://dx.doi.org/10.1371/journal.pgen.1007269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5891035PMC
March 2018

Truncating mutations underlie a spectrum of dominant hereditary motor neuropathies.

Neurol Genet 2018 Apr 23;4(2):e222. Epub 2018 Mar 23.

RILD Wellcome Wolfson Centre (C.G.S., H.H., K.E.S.B., M.A.R., B.A.C., J.K.C., E.L.B., A.H.C.), Royal Devon & Exeter NHS Foundation Trust, Exeter; Wessex Clinical Genetics Service (C.G.S.), Princess Anne Hospital, Southampton, United Kingdom; Neurogenetics Group (D.B., I.M., P.D.J., T.D., J.B.), Center for Molecular Neurology, VIB; Laboratory of Neuromuscular Pathology (D.B., I.M., P.D.J., T.D., J.B.), Institute Born-Bunge, University of Antwerp; Department of Neurology (M.B., D.W.), University of Minnesota, Minneapolis, MN; Department of Neurology (P.D.J., J.B.), Neuromuscular Reference Centre, Antwerp University Hospital, Antwerpen, Belgium; Clinical Genetics (M.M.M.), St. George's University of London, London, United Kingdom; Biomedical Science (R.D.B.), Florida Atlantic University, Jupiter Campus, FL; and Department of Neurology (J.D.B.), University Hospital Ghent, Ghent, Belgium; Peninsula Clinical Genetics Service (E.L.B.), Royal Devon and Exeter Hospital, Exeter, United Kingdom.

Objective: To identify the genetic cause of disease in 2 previously unreported families with forms of distal hereditary motor neuropathies (dHMNs).

Methods: The first family comprises individuals affected by dHMN type V, which lacks the cardinal clinical feature of vocal cord paralysis characteristic of dHMN-VII observed in the second family. Next-generation sequencing was performed on the proband of each family. Variants were annotated and filtered, initially focusing on genes associated with neuropathy. Candidate variants were further investigated and confirmed by dideoxy sequence analysis and cosegregation studies. Thorough patient phenotyping was completed, comprising clinical history, examination, and neurologic investigation.

Results: dHMNs are a heterogeneous group of peripheral motor neuron disorders characterized by length-dependent neuropathy and progressive distal limb muscle weakness and wasting. We previously reported a dominant-negative frameshift mutation located in the concluding exon of the gene encoding the choline transporter (CHT), leading to protein truncation, as the likely cause of dominantly-inherited dHMN-VII in an extended UK family. In this study, our genetic studies identified distinct heterozygous frameshift mutations located in the last coding exon of , predicted to result in the truncation of the CHT C-terminus, as the likely cause of the condition in each family.

Conclusions: This study corroborates C-terminal CHT truncation as a cause of autosomal dominant dHMN, confirming upper limb predominating over lower limb involvement, and broadening the clinical spectrum arising from CHT malfunction.
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http://dx.doi.org/10.1212/NXG.0000000000000222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866402PMC
April 2018

Interrogating the Spatiotemporal Landscape of Neuromodulatory GPCR Signaling by Real-Time Imaging of cAMP in Intact Neurons and Circuits.

Cell Rep 2018 01;22(1):255-268

Department of Neuroscience, The Scripps Research Institute, Jupiter, FL 33458 USA. Electronic address:

Modulation of neuronal circuits is key to information processing in the brain. The majority of neuromodulators exert their effects by activating G-protein-coupled receptors (GPCRs) that control the production of second messengers directly impacting cellular physiology. How numerous GPCRs integrate neuromodulatory inputs while accommodating diversity of incoming signals is poorly understood. In this study, we develop an in vivo tool and analytical suite for analyzing GPCR responses by monitoring the dynamics of a key second messenger, cyclic AMP (cAMP), with excellent quantitative and spatiotemporal resolution in various neurons. Using this imaging approach in combination with CRISPR/Cas9 editing and optogenetics, we interrogate neuromodulatory mechanisms of defined populations of neurons in an intact mesolimbic reward circuit and describe how individual inputs generate discrete second-messenger signatures in a cell- and receptor-specific fashion. This offers a resource for studying native neuronal GPCR signaling in real time.
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http://dx.doi.org/10.1016/j.celrep.2017.12.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761078PMC
January 2018

Is dopamine transporter-mediated dopaminergic signaling in the retina a noninvasive biomarker for attention-deficit/ hyperactivity disorder? A study in a novel dopamine transporter variant Val559 transgenic mouse model.

J Neurodev Disord 2017 12 28;9(1):38. Epub 2017 Dec 28.

Department of Biological Sciences, Vanderbilt University, Box 35-1634 Station B, Nashville, TN, 37235-1634, USA.

Background: Dopamine (DA) is a critical neuromodulator in the retina. Disruption of retinal DA synthesis and signaling significantly attenuates light-adapted, electroretinogram (ERG) responses, as well as contrast sensitivity and acuity. As these measures can be detected noninvasively, they may provide opportunities to detect disease processes linked to perturbed DA signaling. Recently, we identified a rare, functional DA transporter (DAT, SLC6A3) coding substitution, Ala559Val, in subjects with attention-deficit/hyperactivity disorder (ADHD), demonstrating that DAT Val559 imparts anomalous DA efflux (ADE) with attendant physiological, pharmacological, and behavioral phenotypes. To understand the broader impact of ADE on ADHD, noninvasive measures sensitive to DAT reversal are needed.

Methods: Here, we explored this question through ERG-based analysis of retinal light responses, as well as HPLC measurements of retinal DA in DAT Val559 mice.

Results: Male mice homozygous (HOM) for the DAT Val559 variant demonstrated increased, light-adapted ERG b-wave amplitudes compared to wild type (WT) and heterozygous (HET) mice, whereas dark-adapted responses were indistinguishable across genotypes. The elevated amplitude of the photopic light responses in HOM mice could be mimicked in WT mice by applying D and D DA receptor agonists and suppressed in HOM mice by introducing D antagonist, supporting elevated retinal DA signaling arising from ADE. Following the challenge with amphetamine, WT exhibited an increase in light-adapted response amplitudes, while HOM did not. Total retinal DA content was similar across genotypes. Interestingly, female DAT Val559 HOM animals revealed no significant difference in photopic ERG responses when compared with WT and HET littermates.

Conclusions: These data reveal that noninvasive, in vivo evaluation of retinal responses to light can reveal physiological signatures of ADE, suggesting a possible approach to the segregation of neurobehavioral disorders based on the DAT-dependent control of DA signaling.
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http://dx.doi.org/10.1186/s11689-017-9215-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5745861PMC
December 2017

Choline transporter mutations in severe congenital myasthenic syndrome disrupt transporter localization.

Brain 2017 Nov;140(11):2838-2850

RILD Wellcome Wolfson Centre, Royal Devon and Exeter NHS Foundation Trust, Barrack Road, Exeter, EX2 5DW, UK.

The presynaptic, high-affinity choline transporter is a critical determinant of signalling by the neurotransmitter acetylcholine at both central and peripheral cholinergic synapses, including the neuromuscular junction. Here we describe an autosomal recessive presynaptic congenital myasthenic syndrome presenting with a broad clinical phenotype due to homozygous choline transporter missense mutations. The clinical phenotype ranges from the classical presentation of a congenital myasthenic syndrome in one patient (p.Pro210Leu), to severe neurodevelopmental delay with brain atrophy (p.Ser94Arg) and extend the clinical outcomes to a more severe spectrum with infantile lethality (p.Val112Glu). Cells transfected with mutant transporter construct revealed a virtually complete loss of transport activity that was paralleled by a reduction in transporter cell surface expression. Consistent with these findings, studies to determine the impact of gene mutations on the trafficking of the Caenorhabditis elegans choline transporter orthologue revealed deficits in transporter export to axons and nerve terminals. These findings contrast with our previous findings in autosomal dominant distal hereditary motor neuropathy of a dominant-negative frameshift mutation at the C-terminus of choline transporter that was associated with significantly reduced, but not completely abrogated choline transporter function. Together our findings define divergent neuropathological outcomes arising from different classes of choline transporter mutation with distinct disease processes and modes of inheritance. These findings underscore the essential role played by the choline transporter in sustaining acetylcholine neurotransmission at both central and neuromuscular synapses, with important implications for treatment and drug selection.
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http://dx.doi.org/10.1093/brain/awx249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5844214PMC
November 2017