Normal Striatal Vesicular Acetylcholine Transporter Expression in Tourette Syndrome.
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ABSTRACT:
SUBMITTER: Albin RL
PROVIDER: S-EPMC5547197 | biostudies-other | 2017 Jul-Aug
REPOSITORIES: biostudies-other
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Project description:ObjectiveTo identify the genetic basis of a recessive congenital neurologic syndrome characterized by severe hypotonia, arthrogryposis, and respiratory failure.MethodsIdentification of the responsible gene by exome sequencing and assessment of the effect of the mutation on protein stability in transfected rat neuronal-like PC12A123.7 cells.ResultsTwo brothers from a nonconsanguineous Yemeni Jewish family manifested at birth with severe hypotonia and arthrogryposis. The older brother died of respiratory failure at 5 days of age. The proband, now 4.5 years old, has been mechanically ventilated since birth with virtually no milestones achievement. Whole exome sequencing revealed homozygosity of SLC18A3 c.1078G>C, p.Gly360Arg in the affected brothers but not in other family members. SLC18A3 p.Gly360Arg is not reported in world populations but is present at a carrier frequency of 1:30 in healthy Yemeni Jews. SLC18A3 encodes the vesicular acetylcholine transporter (VAChT), which loads newly synthesized acetylcholine from the neuronal cytoplasm into synaptic vesicles. Mice that are VAChT-null have been shown to die at birth of respiratory failure. In human VAChT, residue 360 is located in a conserved region and substitution of arginine for glycine is predicted to disrupt proper protein folding and membrane embedding. Stable transfection of wild-type and mutant human VAChT into neuronal-like PC12A123.7 cells revealed similar mRNA levels, but undetectable levels of the mutant protein, suggesting post-translational degradation of mutant VAChT.ConclusionLoss of function of VAChT underlies severe arthrogryposis and respiratory failure. While most congenital myasthenic syndromes are caused by defects in postsynaptic proteins, VAChT deficiency is a presynaptic myasthenic syndrome.
| S-EPMC5384838 | biostudies-literature
Project description:ObjectiveTo describe the clinical and genetic characteristics of presynaptic congenital myasthenic syndrome secondary to biallelic variants in SLC18A3.MethodsIndividuals from 2 families were identified with biallelic variants in SLC18A3, the gene encoding the vesicular acetylcholine transporter (VAChT), through whole-exome sequencing.ResultsThe patients demonstrated features seen in presynaptic congenital myasthenic syndrome, including ptosis, ophthalmoplegia, fatigable weakness, apneic crises, and deterioration of symptoms in cold water for patient 1. Both patients demonstrated moderate clinical improvement on pyridostigmine. Patient 1 had a broader phenotype, including learning difficulties and left ventricular dysfunction. Electrophysiologic studies were typical for a presynaptic defect. Both patients showed profound electrodecrement on low-frequency repetitive stimulation followed by a prolonged period of postactivation exhaustion. In patient 1, this was unmasked only after isometric contraction, a recognized feature of presynaptic disease, emphasizing the importance of activation procedures.ConclusionsVAChT is responsible for uptake of acetylcholine into presynaptic vesicles. The clinical and electrographic characteristics of the patients described are consistent with previously reported mouse models of VAChT deficiency. These findings make it very likely that defects in VAChT due to variants in SLC18A3 are a cause of congenital myasthenic syndrome in humans.
| S-EPMC5075972 | biostudies-literature
Project description:Invariant E309 is in contact with critical and invariant D398 in a three-dimensional homology model of vesicular acetylcholine transporter (VAChT, TC 2.A.1.2.13) [Vardy, E., et al. (2004) Protein Sci. 13, 1832-1840]. In the work reported here, E309 and D398 in human VAChT were mutated singly and together to test their functions, assign pK values to them, and determine whether the residues are close to each other in three-dimensional space. Mutants were stably expressed in the PC12(A123.7) cell line, and transport and binding properties were characterized at different pH values using radiolabeled ligands and filtration assays. Contrary to a prior conclusion, the results demonstrate that most D398 mutants do not bind the allosteric inhibitor vesamicol even weakly. Earlier work showed that most D398 mutants do not transport ACh. D398 therefore probably is the residue that must deprotonate with a pK of 6.5 for binding of vesamicol and with a pK of approximately 5.9 for transport of ACh. Because E309Q has no effect on VAChT functions at physiological pH, E309 has no apparent critical role. However, radical mutations in E309 cause decreases in ACh and vesamicol affinities and total loss of ACh transport. Unlike wild-type VAChT, which exhibits a peak of [(3)H]vesamicol binding centered at pH 7.4, mutants E309Q, E309D, E309A, and E309K all exhibit peaks of binding centered at pH >or=9. The combination of high pH and mutated E309 apparently produces a relaxed (in contrast to tense) conformation of VAChT that binds vesamicol exceptionally tightly. No compensatory interactions between E309 and D398 in double mutants were discovered. Proof of a close spatial relationship between E309 and D398 was not found. Nevertheless, the data are more consistent with the homology model than an alternative hydropathy model of VAChT that likely locates E309 far from D398 and the ACh binding site in three-dimensional space. Also, a probable network of interactions involving E309 and an unknown residue having a pK of 10 has been revealed.
| S-EPMC2875949 | biostudies-literature
Project description:ObjectiveTourette syndrome (TS) is a common neurodevelopmental disorder marked by tics and behavioral comorbidities. Clinical pharmacology suggests that dopaminergic signaling abnormalities are part of the pathophysiology of TS. Prior molecular imaging studies of nigrostriatal dopaminergic terminal markers report conflicting results. Our goal was to characterize the distribution of nigrostriatal dopaminergic terminals in subjects with TS.MethodsThirty-three adult subjects with TS were studied with PET using [11C]dihydrotetrabenazine (DTBZ), a ligand for the type 2 vesicular monoamine transporter, and with [11C] methylphenidate (MP), a ligand for the plasmalemmal dopamine transporter. Subjects were characterized with standard rating instruments for tic severity, obsessive-compulsive behaviors, and attentional deficits.ResultsWe found no differences between subjects with TS and control subjects in DTBZ and MP binding in any striatal region. There was no correlation between binding measures and clinical variables. Ventral striatal DTBZ and MP binding distributions in subjects with TS were normal.ConclusionsWe found no evidence of increased striatal dopaminergic innervation in Tourette syndrome (TS). Discrepancy between our present results and those of other studies may be explained by heterogeneity of TS.
| S-EPMC2677509 | biostudies-literature
Project description:Classical neurotransmitters such as acetylcholine (ACh) require transport into synaptic vesicles for regulated exocytotic release. The Caenorhabditis elegans gene unc-17 encodes a protein with homology to mammalian transporters that concentrate monoamine neurotransmitters into synaptic vesicles. Mutations in unc-17 protect against organophosphorus toxicity, indicating a role in cholinergic neurotransmission. Using the relationship of unc-17 to the vesicular amine transporters, we first isolated a related sequence from the electric ray Torpedo californica [Torpedo vesicular ACh transporter (TorVAChT)] that is expressed by the electric lobe but not by peripheral tissues. Using the relationship of the Torpedo sequence to unc-17, we then isolated the cDNA for a rat homologue (rVAChT). Northern blot analysis shows expression of these sequences in the basal forebrain, basal ganglia, and spinal cord but not cerebellum or peripheral tissues. In situ hybridization shows expression of rVAChT mRNA in all cholinergic cell groups, including those in the basal forebrain, brainstem, and spinal cord that previously have been shown to express choline acetyltransferase mRNA. The human VAChT gene also localizes to chromosome 10 near the gene for choline acetyltransferase. Taken together, these observations support a role for rVAChT in vesicular ACh transport and indicate its potential as a novel marker for cholinergic neurons.
| S-EPMC45073 | biostudies-other
Project description:Vesicular acetylcholine transporter (VAChT) function is essential for organismal survival, mediating the packaging of acetylcholine (ACh) for exocytotic release. However, its expression pattern in the Drosophila brain has not been fully elucidated. To investigate the localization of VAChT, we developed an antibody against the C terminal region of the protein and we show that this antibody recognizes a 65KDa protein corresponding to VAChT on an immunoblot in both Drosophila head homogenates and in Schneider 2 cells. Further, we report for the first time the expression of VAChT in the antennal lobe and ventral nerve cord of Drosophila larva; and we independently confirm the expression of the protein in mushroom bodies and optic lobes of adult Drosophila. Importantly, we show that VAChT co-localizes with a synaptic vesicle marker in vivo, confirming previous reports of the localization of VAChT to synaptic terminals. Together, these findings help establish the vesicular localization of VAChT in cholinergic neurons in Drosophila and present an important molecular tool with which to dissect the function of the transporter in vivo.
| S-EPMC5382984 | biostudies-literature
Project description:BackgroundAcetylcholine-mediated transmission plays a central role in the impairment of corticostriatal synaptic activity and plasticity in multiple DYT1 mouse models. However, the nature of such alteration remains unclear.ObjectiveThe aim of the present work was to characterize the mechanistic basis of cholinergic dysfunction in DYT1 dystonia to identify potential targets for pharmacological intervention.MethodsWe utilized electrophysiology recordings, immunohistochemistry, enzymatic activity assays, and Western blotting techniques to analyze in detail the cholinergic machinery in the dorsal striatum of the Tor1a+/- mouse model of DYT1 dystonia.ResultsWe found a significant increase in the vesicular acetylcholine transporter (VAChT) protein level, the protein responsible for loading acetylcholine (ACh) from the cytosol into synaptic vesicles, which indicates an altered cholinergic tone. Accordingly, in Tor1a+/- mice we measured a robust elevation in basal ACh content coupled to a compensatory enhancement of acetylcholinesterase (AChE) enzymatic activity. Moreover, pharmacological activation of dopamine D2 receptors, which is expected to reduce ACh levels, caused an abnormal elevation in its content, as compared to controls. Patch-clamp recordings revealed a reduced effect of AChE inhibitors on cholinergic interneuron excitability, whereas muscarinic autoreceptor function was preserved. Finally, we tested the hypothesis that blockade of VAChT could restore corticostriatal long-term synaptic plasticity deficits. Vesamicol, a selective VAChT inhibitor, rescued a normal expression of synaptic plasticity.ConclusionsOverall, our findings indicate that VAChT is a key player in the alterations of striatal plasticity and a novel target to normalize cholinergic dysfunction observed in DYT1 dystonia. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
| S-EPMC9291835 | biostudies-literature
Project description:Rett syndrome (RTT) is a neurodevelopmental disability characterized by mutations in the X-linked methyl-CpG-binding protein 2 located at the Xq28 region. The severity is modified in part by X chromosomal inactivation resulting in wide clinical variability. We hypothesized that the ability to perform the activities of daily living (ADL) is correlated with the density of vesicular acetylcholine transporters in the striata of women with RTT. The density of the vesicular acetylcholine transporters in the living human brain can be estimated by single-photon emission-computed tomography (SPECT) after the administration of (-)-5-[¹²³I]iodobenzovesamicol ([¹²³I]IBVM). Twenty-four hours following the intravenous injection of ?333 MBq (9 mCi) [¹²³ I]IBVM, four women with RTT and nine healthy adult volunteer control participants underwent SPECT brain scans for 60 min. The Vesicular Acetylcholine Transporter Binding Site Index (Kuhl et al., 1994), a measurement of the density of vesicular acetylcholine transporters, was estimated in the striatum and the reference structure, the cerebellum. The women with RTT were assessed for certain ADL. Although the striatal Vesicular Acetylcholine Transporter Binding Site Index was not significantly lower in RTT (5.2 ± 0.9) than in healthy adults (5.7 ± 1.6), RTT striatal Vesicular Acetylcholine Transporter Binding Site Indices and ADL scores were linearly associated (ADL = 0.89*(Vesicular Acetylcholine Transporter Binding Site Index) + 4.5; R² = 0.93; P < 0.01), suggesting a correlation between the ability to perform ADL and the density of vesicular acetylcholine transporters in the striata of women with RTT. [¹²³I]IBVM is a promising tool to characterize the pathophysiological mechanisms of RTT and other neurodevelopmental disabilities.
| S-EPMC3480211 | biostudies-literature
Project description:Gilles de la Tourette syndrome (GTS) is a complex neurodevelopmental disorder characterized by motor and vocal tics. Most of the GTS individuals have comorbid diagnoses, of which obsessive-compulsive disorder (OCD) and attention deficit-hyperactivity disorder (ADHD) are the most common. Several neurotransmitter systems have been implicated in disease pathogenesis, and amongst these, the dopaminergic and the serotonergic pathways are the most widely studied. In this study, we aimed to investigate whether the serotonin transporter (SERT) gene (SLC6A4) was differentially expressed among GTS individuals compared to healthy controls, and whether DNA variants (the SERT-linked polymorphic region 5-HTTLPR, together with the associated rs25531 and rs25532 variants, and the rare Ile425Val variant) or promoter methylation of SLC6A4 were associated with gene expression levels or with the presence of OCD as comorbidity. We observed that SLC6A4 expression is upregulated in GTS individuals compared to controls. Although no specific genotype, allele or haplotype was overrepresented in GTS individuals compared to controls, we observed that the LAC/LAC genotype of the 5-HTTLPR/rs25531/rs25532 three-locus haplotype was associated with higher SLC6A4 mRNA expression levels in GTS individuals, but not in the control group.
| S-EPMC7827645 | biostudies-literature
Project description:To identify suitable lipophilic compounds having high potency and selectivity for vesicular acetylcholine transporter (VAChT), a heteroaromatic ring or a phenyl group was introduced into the carbonyl-containing scaffold for VAChT ligands. Twenty new compounds with ALogD values between 0.53 and 3.2 were synthesized, and their in vitro binding affinities were assayed. Six of them (19a, 19e, 19g, 19k, and 24a-b) displayed high affinity for VAChT (Ki = 0.93-18 nM for racemates) and moderate to high selectivity for VAChT over ?1 and ?2 receptors (Ki = 44-4400-fold). These compounds have a methyl or a fluoro substitution that provides the position for incorporating PET radioisotopes C-11 or F-18. Compound (-)-[(11)C]24b (Ki = 0.78 nM for VAChT, 1200-fold over ? receptors) was successfully synthesized and evaluated in vivo in rats and nonhuman primates. The data revealed that (-)-[(11)C]24b has highest binding in striatum and has favorable pharmacokinetics in the brain.
| S-EPMC3804129 | biostudies-literature