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Tatsuya Haga - One of the best experts on this subject based on the ideXlab platform.
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Molecular properties of the high-affinity Choline Transporter CHT1
Journal of biochemistry, 2014Co-Authors: Tatsuya HagaAbstract:This article summarizes molecular properties of the high-affinity Choline Transporter (CHT1) with reference to the historical background focusing studies performed in laboratories of the author. CHT1 is present on the presynaptic terminal of Cholinergic neurons, and takes up Choline which is the precursor of acetylCholine. The Na(+)-dependent uptake of Choline by CHT1 is the rate-limiting step for synthesis of acetylCholine. CHT1 is the integral membrane protein with 13 transmembrane segments, belongs to the Na(+)/glucose co-Transporter family (SLC5), and has 20-25% homology with members of this family. A single nucleotide polymorphism (SNP) for human CHT1 has been identified, which has a replacement from isoleucine to valine in the third transmembrane segment and shows the Choline uptake activity of 50-60% as much as that of wild-type CHT1. The proportion of this SNP is high among Asians. Possible importance of Choline diet for those with this SNP was discussed.
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Transmembrane topology and oligomeric structure of the high-affinity Choline Transporter
The Journal of biological chemistry, 2012Co-Authors: Takashi Okuda, Haruhiko Yamada, Chieko Osawa, Kengo Hayashi, Shizue Nishikawa, Tomoko Ushio, Yuji Kubo, Motoyasu Satou, Haruo Ogawa, Tatsuya HagaAbstract:The high-affinity Choline Transporter CHT1 mediates Choline uptake essential for acetylCholine synthesis in Cholinergic nerve terminals. CHT1 belongs to the Na+/glucose coTransporter family (SLC5), which is postulated to have a common 13-transmembrane domain core; however, no direct experimental evidence for CHT1 transmembrane topology has yet been reported. We examined the transmembrane topology of human CHT1 using cysteine-scanning analysis. Single cysteine residues were introduced into the putative extra- and intracellular loops and probed for external accessibility for labeling with a membrane-impermeable, sulfhydryl-specific biotinylating reagent in intact cells expressing these mutants. The results provide experimental evidence for a topological model of a 13-transmembrane domain protein with an extracellular amino terminus and an intracellular carboxyl terminus. We also constructed a three-dimensional homology model of CHT1 based on the crystal structure of the bacterial Na+/galactose coTransporter, which supports our conclusion of CHT1 transmembrane topology. Furthermore, we examined whether CHT1 exists as a monomer or oligomer. Chemical cross-linking induces the formation of a higher molecular weight form of CHT1 on the cell surface in HEK293 cells. Two different epitope-tagged CHT1 proteins expressed in the same cells can be co-immunoprecipitated. Moreover, co-expression of an inactive mutant I89A with the wild type induces a dominant-negative effect on the overall Choline uptake activity. These results indicate that CHT1 forms a homo-oligomer on the cell surface in cultured cells.
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The high-affinity Choline Transporter CHT1 is regulated by the ubiquitin ligase Nedd4-2.
Biomedical research (Tokyo Japan), 2012Co-Authors: Haruhiko Yamada, Shinobu Imajoh-ohmi, Tatsuya HagaAbstract:The high-affinity Choline Transporter (CHT1), which is specifically expressed in Cholinergic neurons, constitutes a rate-limiting step for acetylCholine synthesis. We have found that the exogenous ubiquitin ligase Nedd4-2 interacts with CHT1 expressed in HEK293 cells decreasing the amount of cell surface CHT1 by approximately 40%, and that small interfering RNA for endogenous Nedd4-2 enhances the Choline uptake activity by CHT1 in HEK293 cells. These results indicate that Nedd4-2-mediated ubiquitination regulates the cell surface expression of CHT1 in cultured cells and suggest a possibility that treatments or drugs which inhibit the interaction between CHT1 and Nedd4-2 might be useful for diseases involving decrease in acetylCholine level such as Alzheimer's disease.
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Substrate-Induced Internalization of the High-Affinity Choline Transporter
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2011Co-Authors: Takashi Okuda, Hidemi Misawa, Asami Konishi, Tatsuya HagaAbstract:Cholinergic neurons are endowed with a high-affinity Choline uptake system for efficient synthesis of acetylCholine at the presynaptic terminals. The high-affinity Choline Transporter CHT1 is responsible for Choline uptake, the rate-limiting step in acetylCholine synthesis. However, endogenous physiological factors that affect CHT1 expression or function and consequently regulate the acetylCholine synthesis rate are essentially unknown. Here we demonstrate that extracellular substrate decreases the cell-surface expression of CHT1 in rat brain synaptosomes, primary cultures from the basal forebrain, and mammalian cell lines transfected with CHT1. Extracellular Choline rapidly decreases cell-surface CHT1 expression by accelerating its internalization, a process that is mediated by a dynamin-dependent endocytosis pathway in HEK293 cells. Specific inhibitor hemicholinium-3 decreases the constitutive internalization rate and thereby increases cell-surface CHT1 expression. We also demonstrate that the constitutive internalization of CHT1 depends on extracellular pH in cultured cells. Our results collectively suggest that the internalization of CHT1 is induced by extracellular substrate, providing a novel feedback mechanism for the regulation of acetylCholine synthesis at the Cholinergic presynaptic terminals.
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Detection of the high-affinity Choline Transporter in the MOLT-3 human leukemic T-cell line.
Life sciences, 2003Co-Authors: Takeshi Fujii, Takashi Okuda, Tatsuya Haga, Koichiro KawashimaAbstract:We previously showed that lymphocytes possess the necessary components to constitute an independent, non-neuronal Cholinergic system; these include acetylCholine (ACh) itself, Choline acetyltransferase (the ACh-synthesizing enzyme), and both muscarinic and nicotinic ACh receptors (AChRs). In addition, we showed that stimulation of AChRs with their respective agonists elicits a variety of biochemical and functional effects, suggesting that lymphocytic Cholinergic system is involved in the regulation of immune function. In nerve terminals, Choline taken up via the high-affinity Choline Transporter (CHT1) is exclusively utilized for ACh synthesis. In the present study, therefore, we investigated the expression of CHT1 in T-lymphocytes. Reverse transcription-polymerase chain reaction analysis revealed that MOLT-3 cells, a human leukemic T-cell line used as a T-lymphocyte model, expressed CHT1 mRNA, but that the CEM and Jurkat T-cell lines did not. Consistent with that finding, specific binding of [3H]hemicholinium-3 (HC-3), an inhibitor of CHT1, and HC-3-sensitive [3H]Choline uptake were also detected in MOLT-3 cells. These results suggest that CHT1 plays a role in mediating Choline uptake in T-lymphocytes and provides further evidence for the presence of an independent lymphocytic Cholinergic system.
Randy D Blakely - One of the best experts on this subject based on the ideXlab platform.
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Cholinergic genetics of visual attention: Human and mouse Choline Transporter capacity variants influence distractibility.
Journal of Physiology-paris, 2016Co-Authors: Martin Sarter, Randy D Blakely, Cindy Lustig, Ajeesh Koshy CherianAbstract:The basal forebrain Cholinergic projection system to the cortex mediates essential aspects of visual attention performance, including the detection of cues and the response to performance challenges (top-down control of attention). Higher levels of top-down control are mediated via elevated levels of Cholinergic neuromodulation. The neuronal Choline Transporter (CHT) strongly influences the synthesis and release of acetylCholine (ACh). As the capacity of the CHT to import Choline into the neuron is a major, presynaptic determinant of Cholinergic neuromodulation, we hypothesize that genetically-imposed CHT capacity variation impacts the balance of bottom-up versus top-down control of visual attention. Following a brief review of the cognitive concepts relevant for this hypothesis, we describe the key results from our research in mice and humans that possess genetically-imposed changes in Choline uptake capacity. CHT subcapacity is associated with poor top-down attentional control and attenuated (Cholinergic) activation of right frontal regions. Conversely, mice overexpressing the CHT, and humans expressing a CHT variant hypothesized to enhance Choline Transporter function, are relatively resistant to challenges of visual attention performance. Genetic or environmental modulation of CHT expression and function may be associated with vulnerabilities for cognitive disorders.
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Choline on the Move: Perspectives on the Molecular Physiology and Pharmacology of the Presynaptic Choline Transporter.
Advances in pharmacology (San Diego Calif.), 2016Co-Authors: Elizabeth A. Ennis, Randy D BlakelyAbstract:Genetic, biochemical, physiological, and pharmacological approaches have advanced our understanding of Cholinergic biology for over 100 years. High-affinity Choline uptake (HACU) was one of the last features of Cholinergic signaling to be defined at a molecular level, achieved through the cloning of the Choline Transporter (CHT, SLC5A7). In retrospect, the molecular era of CHT studies initiated with the identification of hemicholinium-3 (HC-3), a potent, competitive CHT antagonist, though it would take another 30 years before HC-3, in radiolabeled form, was used by Joseph Coyle's laboratory to identify and monitor the dynamics of CHT proteins. Though HC-3 studies provided important insights into CHT distribution and regulation, another 15 years would pass before the structure of CHT genes and proteins were identified, a full decade after the cloning of most other neurotransmitter-associated Transporters. The availability of CHT gene and protein probes propelled the development of cell and animal models as well as efforts to gain insights into how human CHT gene variation affects the risk for brain and neuromuscular disorders. Most recently, our group has pursued a broadening of CHT pharmacology, elucidating novel chemical structures that may serve to advance Cholinergic diagnostics and medication development. Here we provide a short review of the transformation that has occurred in HACU research and how such advances may promote the development of novel therapeutics.
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The Presynaptic Choline Transporter Imposes Limits on Sustained Cortical AcetylCholine Release and Attention
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2013Co-Authors: Vinay Parikh, Randy D Blakely, Megan St. Peters, Martin SarterAbstract:Functional variation in the gene encoding the presynaptic Choline Transporter (CHT) has been linked to attention-deficit/hyperactivity disorder. Here, we report that a heterozygous deletion in the CHT gene in mice (CHT+/−) limits the capacity of Cholinergic neurons to sustain acetylCholine (ACh) release and attentional performance. Cortical microdialysis and amperometric methods revealed that, whereas wild-type and CHT+/− animals support equivalent basal ACh release and Choline clearance, CHT+/− animals exhibit a significant inability to elevate extracellular ACh following basal forebrain stimulation, in parallel with a diminished Choline clearance capacity following cessation of stimulation. Consistent with these findings, the density of CHTs in cortical synaptosomal plasma membrane-enriched fractions from unstimulated CHT+/− animals matched those observed in wild-type animals despite reductions in CHT levels in total extracts, achieved via a redistribution of CHT from vesicle pools. As a consequence, in CHT+/− animals, basal forebrain stimulation was unable to mobilize wild-type quantities of CHT to the plasma membrane. In behavioral studies, CHT+/− mice were impaired in performing a sustained attention task known to depend on cortical Cholinergic activity. In wild-type mice, but not CHT+/− mice, attentional performance increased the density of CHTs in the synaptosomal membrane in the right frontal cortex. Basal CHT levels in vesicle-enriched membranes predicted the degree of CHT mobilization as well as individual variations in performance on the sustained attention task. Our findings demonstrate biochemical and physiological alterations that underlie cognitive impairments associated with genetically imposed reductions in Choline uptake capacity.
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Choline Transporter gene variation is associated with attention-deficit hyperactivity disorder
Journal of Neurodevelopmental Disorders, 2009Co-Authors: Brett A. English, Maureen K. Hahn, Ian R. Gizer, Michelle Mazei-robison, Angela Steele, Daniel M. Kurnik, Mark A. Stein, Irwin D. Waldman, Randy D BlakelyAbstract:The neurotransmitter acetylCholine (ACh) plays a critical role in brain circuits mediating motor control, attention, learning and memory. Cholinergic dysfunction is associated with multiple brain disorders including Alzheimer’s Disease, addiction, schizophrenia and Attention-Deficit Hyperactivity Disorder (ADHD). The presynaptic Choline Transporter (CHT, SLC5A7 ) is the major, rate-limiting determinant of ACh production in the brain and periphery and is consequently upregulated during tasks that require sustained attention. Given the contribution of central Cholinergic circuits to the control of movement and attention, we hypothesized that functional CHT gene variants might impact risk for ADHD. We performed a case-control study, followed by family-based association tests on a separate cohort, of two purportedly functional CHT polymorphisms (coding variant Ile89Val (rs1013940) and a genomic SNP 3’ of the CHT gene (rs333229), affording both a replication sample and opportunities to reduce potential population stratification biases. Initial genotyping of pediatric ADHD subjects for two purportedly functional CHT alleles revealed a 2–3 fold elevation of the Val89 allele ( n = 100; P = 0.02) relative to healthy controls, as well as a significant decrease of the 3’SNP minor allele in Caucasian male subjects ( n = 60; P = 0.004). In family based association tests, we found significant overtransmission of the Val89 variant to children with a Combined subtype diagnosis (OR = 3.16; P = 0.01), with an increased Odds Ratio for a haplotype comprising both minor alleles. These studies show evidence of Cholinergic deficits in ADHD, particularly for subjects with the Combined subtype, and, if replicated, may encourage further consideration of Cholinergic agonist therapy in the disorder.
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Aberrant trafficking of the high-affinity Choline Transporter in AP-3-deficient mice.
The European journal of neuroscience, 2008Co-Authors: Hidemi Misawa, Randy D Blakely, Kazuko Nakata, Takashi Okuda, Hirofumi Fujigaya, Takashi Nishimura, Yasuhiro Moriwaki, Koichiro Kawashima, Alicia M. Ruggiero, Fubito NakatsuAbstract:The high-affinity Choline Transporter (CHT) is expressed in Cholinergic neurons and efficiently transported to axon terminals where it controls the rate-limiting step in acetylCholine synthesis. Recent studies have shown that the majority of CHT is unexpectedly localized on synaptic vesicles (SV) rather than the presynaptic plasma membrane, establishing vesicular CHT trafficking as a basis for activity-dependent CHT regulation. Here, we analyse the intracellular distribution of CHT in the adaptor protein-3 (AP-3)-deficient mouse model mocha. In the mocha mouse, granular structures in cell bodies are intensely labelled with CHT antibody, indicating possible deficits in CHT trafficking from the cell body to the axon terminal. Western blot analyses reveal that CHT on SV in mocha mice is decreased by 30% compared with wild-type mice. However, no significant difference in synaptosomal Choline uptake activity is detected, consistent with the existence of a large reservoir pool for CHT. To further characterize CHT trafficking, we established a PC12D-CHT cell line. In this line, CHT is found associated with a subpopulation of synaptophysin-positive synaptic-like microvesicles (SLMV). The amounts of CHT detected on SLMV are greatly reduced by treating the cell with agents that halt AP-dependent membrane trafficking. These results demonstrate that APs have important functions for CHT trafficking in neuronal cells.
Takashi Okuda - One of the best experts on this subject based on the ideXlab platform.
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Competitive inhibition of the high-affinity Choline Transporter by tetrahydropyrimidine anthelmintics.
European journal of pharmacology, 2021Co-Authors: Takashi Okuda, Yuki Nomura, Asami Konishi, Hidemi MisawaAbstract:Abstract The high-affinity Choline Transporter CHT 1 mediates Choline uptake, the rate-limiting and regulatory step in acetylCholine synthesis at Cholinergic presynaptic terminals. CHT1-medated Choline uptake is specifically inhibited by hemicholinium-3, which is a type of Choline analog that acts as a competitive inhibitor. Although the substrate Choline and the inhibitor hemicholinium-3 are well-established ligands of CHT 1, few potent ligands other than Choline analogs have been reported. Here we show that tetrahydropyrimidine anthelmintics, known as nicotinic acetylCholine receptor agonists, act as competitive inhibitors of CHT 1. A ligand-dependent trafficking assay in cell lines expressing human CHT 1 was designed to search for CHT 1 ligands from a collection of biologically active compounds. We found that morantel as well as other tetrahydropyrimidines, pyrantel and oxantel, potently inhibits the high-affinity Choline uptake activity of CHT 1 in a competitive manner similar to the inhibitor hemicholinium-3. They also inhibit the high-affinity Choline Transporter from the nematode Caenorhabditis elegans. Finally, tetrahydropyrimidines potently inhibit the high-affinity Choline uptake in rat brain synaptosomes at a low micromolar level, resulting in the inhibition of acetylCholine synthesis. The rank order of potency in synaptosomes is as follows: morantel > pyarantel > oxantel (Ki = 1.3, 5.7, and 8.3 μM, respectively). Our results reveal that tetrahydropyrimidine anthelmintics are novel CHT 1 ligands that inhibit the high-affinity Choline uptake for acetylCholine synthesis in Cholinergic neurons.
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Transmembrane topology and oligomeric structure of the high-affinity Choline Transporter
The Journal of biological chemistry, 2012Co-Authors: Takashi Okuda, Haruhiko Yamada, Chieko Osawa, Kengo Hayashi, Shizue Nishikawa, Tomoko Ushio, Yuji Kubo, Motoyasu Satou, Haruo Ogawa, Tatsuya HagaAbstract:The high-affinity Choline Transporter CHT1 mediates Choline uptake essential for acetylCholine synthesis in Cholinergic nerve terminals. CHT1 belongs to the Na+/glucose coTransporter family (SLC5), which is postulated to have a common 13-transmembrane domain core; however, no direct experimental evidence for CHT1 transmembrane topology has yet been reported. We examined the transmembrane topology of human CHT1 using cysteine-scanning analysis. Single cysteine residues were introduced into the putative extra- and intracellular loops and probed for external accessibility for labeling with a membrane-impermeable, sulfhydryl-specific biotinylating reagent in intact cells expressing these mutants. The results provide experimental evidence for a topological model of a 13-transmembrane domain protein with an extracellular amino terminus and an intracellular carboxyl terminus. We also constructed a three-dimensional homology model of CHT1 based on the crystal structure of the bacterial Na+/galactose coTransporter, which supports our conclusion of CHT1 transmembrane topology. Furthermore, we examined whether CHT1 exists as a monomer or oligomer. Chemical cross-linking induces the formation of a higher molecular weight form of CHT1 on the cell surface in HEK293 cells. Two different epitope-tagged CHT1 proteins expressed in the same cells can be co-immunoprecipitated. Moreover, co-expression of an inactive mutant I89A with the wild type induces a dominant-negative effect on the overall Choline uptake activity. These results indicate that CHT1 forms a homo-oligomer on the cell surface in cultured cells.
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Substrate-Induced Internalization of the High-Affinity Choline Transporter
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2011Co-Authors: Takashi Okuda, Hidemi Misawa, Asami Konishi, Tatsuya HagaAbstract:Cholinergic neurons are endowed with a high-affinity Choline uptake system for efficient synthesis of acetylCholine at the presynaptic terminals. The high-affinity Choline Transporter CHT1 is responsible for Choline uptake, the rate-limiting step in acetylCholine synthesis. However, endogenous physiological factors that affect CHT1 expression or function and consequently regulate the acetylCholine synthesis rate are essentially unknown. Here we demonstrate that extracellular substrate decreases the cell-surface expression of CHT1 in rat brain synaptosomes, primary cultures from the basal forebrain, and mammalian cell lines transfected with CHT1. Extracellular Choline rapidly decreases cell-surface CHT1 expression by accelerating its internalization, a process that is mediated by a dynamin-dependent endocytosis pathway in HEK293 cells. Specific inhibitor hemicholinium-3 decreases the constitutive internalization rate and thereby increases cell-surface CHT1 expression. We also demonstrate that the constitutive internalization of CHT1 depends on extracellular pH in cultured cells. Our results collectively suggest that the internalization of CHT1 is induced by extracellular substrate, providing a novel feedback mechanism for the regulation of acetylCholine synthesis at the Cholinergic presynaptic terminals.
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Aberrant trafficking of the high-affinity Choline Transporter in AP-3-deficient mice.
The European journal of neuroscience, 2008Co-Authors: Hidemi Misawa, Randy D Blakely, Kazuko Nakata, Takashi Okuda, Hirofumi Fujigaya, Takashi Nishimura, Yasuhiro Moriwaki, Koichiro Kawashima, Alicia M. Ruggiero, Fubito NakatsuAbstract:The high-affinity Choline Transporter (CHT) is expressed in Cholinergic neurons and efficiently transported to axon terminals where it controls the rate-limiting step in acetylCholine synthesis. Recent studies have shown that the majority of CHT is unexpectedly localized on synaptic vesicles (SV) rather than the presynaptic plasma membrane, establishing vesicular CHT trafficking as a basis for activity-dependent CHT regulation. Here, we analyse the intracellular distribution of CHT in the adaptor protein-3 (AP-3)-deficient mouse model mocha. In the mocha mouse, granular structures in cell bodies are intensely labelled with CHT antibody, indicating possible deficits in CHT trafficking from the cell body to the axon terminal. Western blot analyses reveal that CHT on SV in mocha mice is decreased by 30% compared with wild-type mice. However, no significant difference in synaptosomal Choline uptake activity is detected, consistent with the existence of a large reservoir pool for CHT. To further characterize CHT trafficking, we established a PC12D-CHT cell line. In this line, CHT is found associated with a subpopulation of synaptophysin-positive synaptic-like microvesicles (SLMV). The amounts of CHT detected on SLMV are greatly reduced by treating the cell with agents that halt AP-dependent membrane trafficking. These results demonstrate that APs have important functions for CHT trafficking in neuronal cells.
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Ultrastructural localization of high-affinity Choline Transporter in the rat neuromuscular junction: Enrichment on synaptic vesicles
Synapse (New York N.Y.), 2004Co-Authors: Kazuko Nakata, Takashi Okuda, Hidemi MisawaAbstract:In Cholinergic neurons, Na+- and Cl−-dependent, hemicholinium-3-sensitive, high-affinity Choline uptake system is thought to be the rate-limiting step in acetylCholine (ACh) synthesis. The system is highly regulated by neuronal activity; the Choline uptake is increased by a condition in which ACh release is favored. Here we analyzed the ultrastructural localization of the high-affinity Choline Transporter (CHT) in the rat neuromuscular junctions with two separate antibodies. The majority (>90%) of immunogold labeling of CHT was observed on synaptic vesicles rather than the presynaptic plasma membrane. Less than 5% of the gold-silver particles were associated with the plasma membrane, and more than 70% of such particles were localized within or in close vicinity to presynaptic active zones. Our morphological data support the recent hypothesis that trafficking of CHT from synaptic vesicles to the plasma membrane couples neuronal activity and Choline uptake. Synapse 53:53–56, 2004. © 2004 Wiley-Liss, Inc.
R.j. Rylett - One of the best experts on this subject based on the ideXlab platform.
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Modulation of sodium-coupled Choline Transporter CHT function in health and disease.
Neurochemistry international, 2020Co-Authors: O.a. Ojiakor, R.j. RylettAbstract:Abstract The sodium-coupled high-affinity Choline Transporter CHT plays a critical role in acetylCholine (ACh) synthesis by taking up the substrate Choline from the synaptic cleft after neurotransmitter release; this conservation mechanism is the rate-limiting step for production of ACh, thereby facilitating communication by subsequent action potentials. Mice carrying a null mutation for CHT die within an hour of birth due to respiratory failure, indicating the essential role of CHT proteins for sustaining Cholinergic transmission. Choline uptake activity is regulated dynamically by CHT proteins undergoing rapid trafficking between subcellular compartments and the plasma membrane where they are functionally active. CHT proteins internalize from the cell surface into the endolysosomal pathway by a clathrin-mediated mechanism, but can undergo ubiquitination and proteosomal degradation under conditions such as cellular oxidative stress. Over the years, functionally-relevant CHT polymorphisms have been linked to a range of neurological and psychiatric disorders, including ADHD and depression; the impact of these mutations and the extent to which they alter Cholinergic signaling have not been addressed fully. Recent studies have identified compounds that can either promote or diminish Cholinergic neurotransmission by modulating CHT function, thus having the potential to serve as pharmacological tools or therapeutic prototypes. Here, we review regulation of CHT activity, trafficking and subcellular disposition of CHT proteins, alteration of Transporter function in genetic, neurological and psychiatric diseases, and investigations of compounds that modulate activity of the Transporter.
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Differential regulation of the high‐affinity Choline Transporter by wild‐type and Swedish mutant amyloid precursor protein
Journal of neurochemistry, 2015Co-Authors: Leah Katherine Cuddy, Claudia Seah, Stephen H. Pasternak, R.j. RylettAbstract:The high-affinity Choline Transporter (CHT) is responsible for Choline uptake into Cholinergic neurons, with this being the rate-limiting step for acetylCholine production. Altering CHT protein disposition directly impacts Choline uptake activity and Cholinergic neurotransmission. Amyloid precursor protein (APP) interacts with CHT proteins and increases their endocytosis from the cell surface. The goal of this study was to examine regulation of CHT trafficking and activity by wild-type APP (APPwt) and determine if this differs with Swedish mutant APP (APPSwe) in SH-SY5Y human neuroblastoma cells. APPSwe differs from APPwt in its trafficking from the cell surface through endosomes. We report for the first time that CHT interacts significantly less with APPSwe than with APPwt. Surprisingly, however, CHT cell surface levels and Choline uptake activity are decreased to the same extent and CHT co-localization to early endosomes increased similarly in cells expressing either APPwt or APPSwe. A critical observation is that CHT co-immunoprecipitates with βCTF from APPSwe-expressing cells. We propose that decreased CHT function is mediated differently by APPwt and APPSwe; APPwt interaction with CHT facilitates its endocytosis from the cell surface, whereas the effect of APPSwe on CHT is mediated indirectly potentially by binding to the βCTF fragment or by Aβ released from cells. High-affinity Choline Transporter (CHT) takes Choline into Cholinergic neurons for acetylCholine synthesis. Amyloid precursor protein (APP) interacts with CHT proteins, but this is decreased for Swedish mutant APP (APPSwe). CHT cell surface levels and localization to early endosomes, and Choline uptake activity are changed similarly by APPwt or APPSwe. APPSwe mediates effects indirectly potentially by βCTF or Aβ.
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Regulation of the high-affinity Choline Transporter activity and trafficking by its association with cholesterol-rich lipid rafts.
Journal of neurochemistry, 2013Co-Authors: Leah Katherine Cuddy, Warren Winick-ng, R.j. RylettAbstract:The sodium-coupled, hemicholinium-3-sensitive, high-affinity Choline Transporter (CHT) is responsible for transport of Choline into Cholinergic nerve terminals from the synaptic cleft following acetylCholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts in both SH-SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH-SY5Y cells expressing rat CHT with filipin, methyl-β-cyclodextrin (MβC) or cholesterol oxidase significantly decreased Choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol-saturated MβC. Kinetic analysis of binding of [3H]hemicholinium-3 to CHT revealed that reducing membrane cholesterol with MβC decreased both the apparent binding affinity (KD) and maximum number of binding sites (Bmax); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol-rich rafts is critical for Transporter function and localization. Alterations in plasma membrane cholesterol Cholinergic nerve terminals could diminish Cholinergic transmission by reducing Choline availability for acetylCholine synthesis. The sodium-coupled Choline Transporter CHT moves Choline into Cholinergic nerve terminals to serve as substrate for acetylCholine synthesis. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts, and decreasing membrane cholesterol significantly reduces both Choline uptake activity and cell surface CHT protein levels. CHT association with cholesterol-rich rafts is critical for its function, and alterations in plasma membrane cholesterol could diminish Cholinergic transmission by reducing Choline availability for acetylCholine synthesis.
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Choline Transporter cht regulation and function in Cholinergic neurons
Central nervous system agents in medicinal chemistry, 2012Co-Authors: S A Black, R.j. RylettAbstract:Choline uptake into Cholinergic nerve terminals by the sodium-dependent high-affinity Choline Transporter CHT is essential for providing Choline as substrate for synthesis of acetylCholine (ACh); ACh is used by Cholinergic neurons to communicate information to a wide range of tissues in central and peripheral nervous systems. CHT is expressed almost exclusively in Cholinergic neurons, and is subject to transcriptional and post-translational control by factors that promote or diminish Cholinergic neurotransmission. The distribution of CHT proteins within Cholinergic presynaptic terminals is dynamically regulated. Thus, Choline uptake activity is determined largely by the plasma membrane CHT level, and this is finely controlled by a balance between internalization and recycling of CHT proteins in endosomal compartments. CHT proteins are also in synaptic vesicle membranes, thereby allowing cell surface CHT levels to increase rapidly in conjunction with exocytotic transmitter release to provide enhanced Choline for ACh re-synthesis. Little is known about post-translational modification of CHT, although data is emerging that CHT activity and subcellular trafficking is modulated by kinase-mediated phosphorylation. Recent studies have also identified proteins with which CHT interacts, but this requires further investigation to reveal the role of other proteins in regulating CHT function and activity. Polymorphisms in CHT protein and modifications in its expression are linked to neurological and psychiatric disorders, and can alter function of peripheral systems that are regulated by Cholinergic innervation, such as the cardiovascular system. The critical role of CHT in maintaining Cholinergic transmission indicates that it could be a target for therapeutic intervention to promote ACh synthesis, but mechanisms by which this can be accomplished have not been adequately addressed.
R. Jane Rylett - One of the best experts on this subject based on the ideXlab platform.
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Insulin Regulates the Activity of the High-Affinity Choline Transporter CHT.
PloS one, 2015Co-Authors: Katherine J. Fishwick, R. Jane RylettAbstract:Studies in humans and animal models show that neuronal insulin resistance increases the risk of developing Alzheimer’s Disease (AD), and that insulin treatment may promote memory function. Cholinergic neurons play a critical role in cognitive and attentional processing and their dysfunction early in AD pathology may promote the progression of AD pathology. Synthesis and release of the neurotransmitter acetylCholine (ACh) is closely linked to the activity of the high-affinity Choline Transporter protein (CHT), but the impact of insulin receptor signaling and neuronal insulin resistance on these aspects of Cholinergic function are unknown. In this study, we used differentiated SH-SY5Y cells stably-expressing CHT proteins to study the effect of insulin signaling on CHT activity and function. We find that Choline uptake activity measured after acute addition of 20 nM insulin is significantly lower in cells that were grown for 24 h in media containing insulin compared to cells grown in the absence of insulin. This coincides with loss of ability to increase phospho-Protein Kinase B (PKB)/Akt levels in response to acute insulin stimulation in the chronic insulin-treated cells. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3-kinase) in cells significantly lowers phospho-PKB/Akt levels and decreases Choline uptake activity. We show total internal reflection microscopy (TIRF) imaging of the dynamic movement of CHT proteins in live cells in response to depolarization and drug treatments. These data show that acute exposure of depolarized cells to insulin is coupled to transiently increased levels of CHT proteins at the cell surface, and that this is attenuated by chronic insulin exposure. Moreover, prolonged inhibition of PI3-kinase results in enhanced levels of CHT proteins at the cell surface by decreasing their rate of internalization.
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Regulated recycling and plasma membrane recruitment of the high-affinity Choline Transporter.
The European journal of neuroscience, 2007Co-Authors: Fabiola M. Ribeiro, Stefanie A. G. Black, Vania F. Prado, R. Jane Rylett, Marco A. M. Prado, Metta Pinthong, Alexis C. Gordon, Stephen S. G. FergusonAbstract:The high-affinity Choline Transporter (CHT1) is responsible for uptake of Choline from the synaptic cleft and supplying Choline for acetylCholine synthesis. CHT1 internalization by clathrin-coated vesicles is proposed to represent a mechanism by which high-affinity Choline uptake can be modulated. We show here that internalized CHT1 is rapidly recycled back to the cell surface in both human embryonic kidney cells (HEK 293 cells) and SH-SY5Y neuroblastoma cells. This rapidly recycling pool of CHT1 comprises about 10% of total CHT1 protein. In the SH-SY5Y neuroblastoma cell line K + -depolarization promotes Ca 2+ -dependent increase in the rate of CHT1 recycling to the plasma membrane without affecting the rate of CHT1 internalization. K + -depolarization also increases the size of the pool of CHT1 protein that can be mobilized to the plasma membrane. Thus, the activity-dependent increase in plasma membrane CHT1 localization appears to be regulated by two mechanisms: (i) an increase in the rate of externalization of the intracellular CHT1 pool; and (ii) the recruitment of additional intracellular Transporters to the recycling pool.
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Activity and subcellular trafficking of the sodium-coupled Choline Transporter CHT is regulated acutely by peroxynitrite.
Molecular pharmacology, 2007Co-Authors: Metta Pinthong, Fabiola M. Ribeiro, Stefanie A. G. Black, Stephen S. G. Ferguson, Chumpol Pholpramool, R. Jane RylettAbstract:Excess formation of nitric oxide and superoxide by-products (peroxynitrite, reactive oxygen, and reactive nitrogen species) attenuates Cholinergic transmission potentially having a role in Alzheimer disease pathogenesis. In this study, we investigated mechanisms by which acute exposure to peroxynitrite impairs function of the sodium-dependent hemicholinium-3 (HC-3)sensitive Choline Transporter (CHT) that provides substrate for acetylCholine synthesis. The peroxynitrite generator 3-morpholinosydnonimine (SIN-1) acutely inhibited Choline uptake in cells stably expressing FLAG-tagged rat CHT in a dose- and timedependent manner, with an IC50 0.9 0.14 mM and t1/2 4 min. SIN-1 significantly reduced Vmax of Choline uptake without altering the Km. This correlated with a SIN-1-induced decrease in cell surface CHT protein, observed as lowered levels of HC-3 binding and biotinylated CHT at the plasma membrane. It is noteworthy that short-term exposure of cells to SIN-1 accelerated the rate of internalization of CHT from the plasma membrane, but it did not alter return of CHT back to the cell surface. SIN-1 did not disrupt cell membrane integrity or cause cell death. Thus, the inhibitory effect of SIN-1 on Choline uptake activity and HC-3 binding was related to enhanced internalization of CHT proteins from the plasma membrane to subcellular organelles.
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The “ins” and “outs” of the high‐affinity Choline Transporter CHT1
Journal of neurochemistry, 2006Co-Authors: Fabiola M. Ribeiro, Stefanie A. G. Black, Vania F. Prado, R. Jane Rylett, Stephen S. G. Ferguson, Marco A. M. PradoAbstract:Maintenance of acetylCholine (ACh) synthesis depends on the activity of the high-affinity Choline Transporter (CHT1), which is responsible for the reuptake of Choline from the synaptic cleft into presynaptic neurons. In this review, we discuss the current understanding of mechanisms involved in the cellular trafficking of CHT1. CHT1 protein is mainly found in intracellular organelles, such as endosomal compartments and synaptic vesicles. The presence of CHT1 at the plasma membrane is limited by rapid endocytosis of the Transporter in clathrin-coated pits in a mechanism dependent on a dileucine-like motif present in the carboxyl-terminal region of the Transporter. The intracellular pool of CHT1 appears to constitute a reserve pool of Transporters, important for maintenance of Cholinergic neurotransmission. However, the physiological basis of the presence of CHT1 in intracellular organelles is not fully understood. Current knowledge about CHT1 indicates that stimulated and constitutive exocytosis, in addition to endocytosis, will have major consequences for regulating Choline uptake. Future investigations of CHT1 trafficking should elucidate such regulatory mechanisms, which may aid in understanding the pathophysiology of diseases that affect Cholinergic neurons, such as Alzheimer's disease.
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Constitutive high-affinity Choline Transporter endocytosis is determined by a carboxyl-terminal tail dileucine motif.
Journal of neurochemistry, 2005Co-Authors: Fabiola M. Ribeiro, Stefanie A. G. Black, Vania F. Prado, R. Jane Rylett, Marco A. M. Prado, Sean P. Cregan, Stephen S. G. FergusonAbstract:Maintenance of acetylCholine synthesis depends on the effective functioning of a high-affinity sodium-dependent Choline Transporter (CHT1). Recent studies have shown that this Transporter is predominantly localized inside the cell, unlike other neurotransmitter Transporters, suggesting that the trafficking of CHT1 to and from the plasma membrane may play a crucial role in regulating Choline uptake. Here we found that CHT1 is rapidly and constitutively internalized in clathrin-coated vesicles to Rab5-positive early endosomes. CHT1 internalization is controlled by an atypical carboxyl-terminal dileucine-like motif (L531, V532) which, upon replacement by alanine residues, blocks CHT1 internalization in both human embryonic kidney 293 cells and primary cortical neurons and results in both increased CHT1 cell surface expression and Choline transport activity. Perturbation of clathrin-mediated endocytosis with dynamin-I K44A increases cell surface expression and transport activity to a similar extent as mutating the dileucine motif, suggesting that we have identified the motif responsible for constitutive CHT1 internalization. Based on the observation that the localization of CHT1 to the plasma membrane is transient, we propose that acetylCholine synthesis may be influenced by processes that lead to the attenuation of constitutive CHT1 endocytosis.