Role of palmitoylation on the neuronal glycine transporter GlyT2.

The neuronal glycine transporter GlyT2 removes glycine from the synaptic cleft through active Na+, Cl-, and glycine cotransport contributing to the termination of the glycinergic signal as well as supplying substrate to the presynaptic terminal for the maintenance of the neurotransmitter content in synaptic vesicles. Patients with mutations in the human GlyT2 gene (SLC6A5), develop hyperekplexia or startle disease (OMIM 149400), characterized by hypertonia and exaggerated startle responses to trivial stimuli that may have lethal consequences in the neonates as a result of apnea episodes. Post-translational modifications in cysteine residues of GlyT2 are an aspect of structural interest we analyzed. Our study is compatible with a reversible and short-lived S-acylation in spinal cord membranes, detectable by biochemical and proteomics methods (acyl-Rac binding and IP-ABE) confirmed with positive and negative controls (palmitoylated and non-palmitoylated proteins). According to a short-lived modification, direct labeling using click chemistry was faint but mostly consistent. We have analyzed the physiological properties of a GlyT2 mutant lacking the cysteines with high prediction of palmitoylation and the mutant is less prone to be included in lipid rafts, an effect also observed upon treatment with the palmitoylation inhibitor 2-bromopalmitate. This work demonstrates there are determinants of lipid raft inclusion associated with the GlyT2 mutated cysteines, which are presumably modified by palmitoylation.

Dual Role of Dysfunctional Asc-1 Transporter in Distinct Human Pathologies, Human Startle Disease, and Developmental Delay.

Human startle disease is associated with mutations in distinct genes encoding glycine receptors, transporters or interacting proteins at glycinergic synapses in spinal cord and brainstem. However, a significant number of diagnosed patients does not carry a mutation in the common genes GLRA1, GLRB, and SLC6A5 Recently, studies on solute carrier 7 subfamily 10 (SLC7A10; Asc-1, alanine-serine-cysteine transporter) knock-out (KO) mice displaying a startle disease-like phenotype hypothesized that this transporter might represent a novel candidate for human startle disease. Here, we screened 51 patients from our patient cohort negative for the common genes and found three exonic (one missense, two synonymous), seven intronic, and single nucleotide changes in the 5' and 3' untranslated regions (UTRs) in Asc-1. The identified missense mutation Asc-1G307R from a patient with startle disease and developmental delay was investigated in functional studies. At the molecular level, the mutation Asc-1G307R did not interfere with cell-surface expression, but disrupted glycine uptake. Substitution of glycine at position 307 to other amino acids, e.g., to alanine or tryptophan did not affect trafficking or glycine transport. By contrast, G307K disrupted glycine transport similar to the G307R mutation found in the patient. Structurally, the disrupted function in variants carrying positively charged residues can be explained by local structural rearrangements because of the large positively charged side chain. Thus, our data suggest that SLC7A10 may represent a rare but novel gene associated with human startle disease and developmental delay.

Botulinum toxin type A counteracts neuropathic pain by countering the increase of GlyT2 expression in the spinal cord of CCI rats.

Botulinum toxin type A (BoNT/A) is a potent toxin, acts by cleaving synaptosome-associated-protein-25 (SNAP-25) to regulate the release of the neural transmitter and shows analgesic effect in neuropathic pain. However, the mechanisms of BoNT/A actions involved in nociceptions remain unclear. Glycine transporter 2 (GlyT2) is an isoform of glycine transporters, which plays an important role in the regulation of glycinergic neurotransmission. Inhibition of GlyTs could decrease pain sensation in neuropathic pain, the role of GlyT2 in the analgesic effect of BoNT/A has not been studied yet. In our present study, we demonstrated that the protein levels of GlyT2 and SNAP-25 were upregulated in the spinal cord after the development of chronic constriction injury (CCI)-induced neuropathic pain. Intraplantar application of BoNT/A (20 U/kg) attenuated mechanical allodynia induced by CCI and downregulated GlyT2 expression in the spinal cord. The application of BoNT/A s also decreased the expression of GlyT2 in pheochromocytoma (PC12) cells. Moreover, intrathecal application of lentivirus-mediated GlyT2 reversed the antinociceptive effect of BoNT/A in CCI rats. These findings indicate that GlyT2 contributes to the antinociceptive effect of BoNT/A and suggest a novel mechanism underlying BoNT/A's antinociception action.

Synergistic Control of Transmitter Turnover at Glycinergic Synapses by GlyT1, GlyT2, and ASC-1.

In addition to being involved in protein biosynthesis and metabolism, the amino acid glycine is the most important inhibitory neurotransmitter in caudal regions of the brain. These functions require a tight regulation of glycine concentration not only in the synaptic cleft, but also in various intracellular and extracellular compartments. This is achieved not only by confining the synthesis and degradation of glycine predominantly to the mitochondria, but also by the action of high-affinity large-capacity glycine transporters that mediate the transport of glycine across the membranes of presynaptic terminals or glial cells surrounding the synapses. Although most cells at glycine-dependent synapses express more than one transporter with high affinity for glycine, their synergistic functional interaction is only poorly understood. In this review, we summarize our current knowledge of the two high-affinity transporters for glycine, the sodium-dependent glycine transporters 1 (GlyT1; SLC6A9) and 2 (GlyT2; SLC6A5) and the alanine-serine-cysteine-1 transporter (Asc-1; SLC7A10).

Bupivacaine reduces GlyT1 expression by potentiating the p-AMPKα/BDNF signalling pathway in spinal astrocytes of rats.

Bupivacaine, a local anaesthetic, is widely applied in the epidural or subarachnoid space to clinically manage acute and chronic pain. However, the underlying mechanisms are complex and unclear. Glycine transporter 1 (GlyT1) in the spinal cord plays a critical role in various pathologic pain conditions. Therefore, we sought to determine whether bupivacaine exerts its analgesic effect by regulating GlyT1 expression and to determine the underlying mechanisms of regulation. Primary astrocytes prepared from the spinal cord of rats were treated with bupivacaine. The protein levels of GlyT1, brain-derived neurotrophic factor (BDNF) and phosphorylated adenosine 5'-monophosphate (AMP)-activated protein kinase α (p-AMPKα) were measured by western blotting or immunofluorescence. In addition, 7,8-dihydroxyflavone (7,8-DHF, BDNF receptor agonist) and AMPK shRNA were applied to verify the relationship between the regulation of GlyT1 by bupivacaine and the p-AMPKα/BDNF signalling pathway. After treatment with bupivacaine, GlyT1 expression was diminished in a concentration-dependent manner, while the expression of BDNF and p-AMPK was increased. Moreover, 7,8-DHF decreased GlyT1 expression, and AMPK knockdown suppressed the upregulation of BDNF expression by bupivacaine. Finally, we concluded that bupivacaine reduced GlyT1 expression in spinal astrocytes by activating the p-AMPKα/BDNF signalling pathway. These results provide a new mechanism for the analgesic effect of intrathecal bupivacaine in the treatment of acute and chronic pain.

Glucose metabolic profiles evaluated by PET associated with molecular characteristic landscape of gastric cancer.

BACKGROUND: Although FDG-PET is widely used in cancer, its role in gastric cancer (GC) is still controversial due to variable [18F]fluorodeoxyglucose ([18F]FDG) uptake. Here, we sought to develop a genetic signature to predict high FDG-avid GC to plan individualized PET and investigate the molecular landscape of GC and its association with glucose metabolic profiles noninvasively evaluated by [18F]FDG-PET. METHODS: Based on a genetic signature, PETscore, representing [18F]FDG avidity, was developed by imaging data acquired from thirty patient-derived xenografts (PDX). The PETscore was validated by [18F]FDG-PET data and gene expression data of human GC. The PETscore was associated with genomic and transcriptomic profiles of GC using The Cancer Genome Atlas. RESULTS: Five genes, PLS1, PYY, HBQ1, SLC6A5, and NAT16, were identified for the predictive model for [18F]FDG uptake of GC. The PETscore was validated in independent PET data of human GC with qRT-PCR and RNA-sequencing. By applying PETscore on TCGA, a significant association between glucose uptake and tumor mutational burden as well as genomic alterations were identified. CONCLUSION: Our findings suggest that molecular characteristics are underlying the diverse metabolic profiles of GC. Diverse glucose metabolic profiles may apply to precise diagnostic and therapeutic approaches for GC.

Assessment of the Anti-Allodynic and Anti-Hyperalgesic Efficacy of a Glycine Transporter 2 Inhibitor Relative to Pregabalin, Duloxetine and Indomethacin in a Rat Model of Cisplatin-Induced Peripheral Neuropathy.

Cisplatin, which is a chemotherapy drug listed on the World Health Organisation's List of Essential Medicines, commonly induces dose-limiting side effects including chemotherapy-induced peripheral neuropathy (CIPN) that has a major negative impact on quality of life in cancer survivors. Although adjuvant drugs including anticonvulsants and antidepressants are used for the relief of CIPN, analgesia is often unsatisfactory. Herein, we used a rat model of CIPN (cisplatin) to assess the effect of a glycine transporter 2 (GlyT2) inhibitor, relative to pregabalin, duloxetine, indomethacin and vehicle. Male Sprague-Dawley rats with cisplatin-induced mechanical allodynia and mechanical hyperalgesia in the bilateral hindpaws received oral bolus doses of the GlyT2 inhibitor (3-30 mg/kg), pregabalin (3-100 mg/kg), duloxetine (3-100 mg/kg), indomethacin (1-10 mg/kg) or vehicle. The GlyT2 inhibitor alleviated both mechanical allodynia and hyperalgesia in the bilateral hindpaws at a dose of 10 mg/kg, but not at higher or lower doses. Pregabalin and indomethacin induced dose-dependent relief of mechanical allodynia but duloxetine lacked efficacy. Pregabalin and duloxetine alleviated mechanical hyperalgesia in the bilateral hindpaws while indomethacin lacked efficacy. The mechanism underpinning pain relief induced by the GlyT2 inhibitor at 10 mg/kg is likely due to increased glycinergic inhibition in the lumbar spinal cord, although the bell-shaped dose-response curve warrants further translational considerations.

Repurposing of glycine transport inhibitors for the treatment of erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is a rare disease in which patients experience severe light sensitivity. It is caused by a deficiency of ferrochelatase (FECH), the last enzyme in heme biosynthesis (HBS). The lack of FECH causes accumulation of its photoreactive substrate protoporphyrin IX (PPIX) in patients' erythrocytes. Here, we explored an approach for the treatment of EPP by decreasing PPIX synthesis using small-molecule inhibitors directed to factors in the HBS pathway. We generated a FECH-knockout clone from K562 erythroleukemia cells, which accumulates PPIX and undergoes oxidative stress upon light exposure. We used these matched cell lines to screen a set of publicly available inhibitors of factors in the HBS pathway. Inhibitors of the glycine transporters GlyT1 and GlyT2 lowered levels of PPIX and markers of oxidative stress selectively in K56211B4 cells, and in primary erythroid cultures from an EPP patient. Our findings open the door to investigation of glycine transport inhibitors for HBS disorders.

Chloride-dependent conformational changes in the GlyT1 glycine transporter.

The human GlyT1 glycine transporter requires chloride for its function. However, the mechanism by which Cl- exerts its influence is unknown. To examine the role that Cl- plays in the transport cycle, we measured the effect of Cl- on both glycine binding and conformational changes. The ability of glycine to displace the high-affinity radioligand [3H]CHIBA-3007 required Na+ and was potentiated over 1,000-fold by Cl- We generated GlyT1b mutants containing reactive cysteine residues in either the extracellular or cytoplasmic permeation pathways and measured changes in the reactivity of those cysteine residues as indicators of conformational changes in response to ions and substrate. Na+ increased accessibility in the extracellular pathway and decreased it in the cytoplasmic pathway, consistent with stabilizing an outward-open conformation as observed in other members of this transporter family. In the presence of Na+, both glycine and Cl- independently shifted the conformation of GlyT1b toward an outward-closed conformation. Together, Na+, glycine, and Cl- stabilized an inward-open conformation of GlyT1b. We then examined whether Cl- acts by interacting with a conserved glutamine to allow formation of an ion pair that stabilizes the closed state of the extracellular pathway. Molecular dynamics simulations of a GlyT1 homolog indicated that this ion pair is formed more frequently as that pathway closes. Mutation of the glutamine blocked the effect of Cl-, and substituting it with glutamate or lysine resulted in outward- or inward-facing transporter conformations, respectively. These results provide an unexpected insight into the role of Cl- in this family of transporters.

Phosphorylation of Serine 157 Protects the Rat Glycine Transporter GlyT2 from Calpain Cleavage.

The N-terminal region of the rat glycine transporter 2 (rGlyT2, SLC6A5) is cleaved by calpain protease in vitro, which raises the question of its protection against calpain in vivo. Here, we used a phosphomimetic and orthogonal phosphoserine translation approach to investigate the possible role of phosphorylation in the protection of two calpain cleavage sites, M156/S157 and G164/T165, previously identified in the N-terminus region of the rat GlyT2. Replacement of serine 157 with phosphomimetic aspartate or with orthogonal phosphoserine blocked both calpain cleavage sites and caused an electrophoretic mobility shift of rGlyT2N fusion proteins. Both effects can be reversed by dephosphorylation, suggesting that phosphorylation might induce structural changes in the rGlyT2 N-terminus, preventing the accessibility of the M156/S157 and G164/T165 cleavage sites to calpain in vivo. In comparison with the wild type, the phosphomimetic mutation S157D increased the total immunoreactivity of the transporter expressed in neuroblastoma cells, suggesting that serine 157 phosphorylation or phosphorylation-regulated calpain cleavage might contribute to the turnover of the glycine transporter GlyT2.

The PERK-EIF2α-ATF4 signaling branch regulates osteoblast differentiation and proliferation by PTH.

Parathyroid hormone (PTH) and its related peptide (PTH-related peptide 1-34) are two of the Food and Drug Administration-approved bone-promoting drugs for age-related osteoporosis. Treatment with PTH stimulates bone formation. However, the molecular mechanisms of PTH-mediated osteoblast differentiation and cell proliferation are still not completely understood. In this study, we showed that PTH induced endoplasmic reticulum (ER) stress in osteoblasts through the PKR-like endoplasmic reticulum kinase (PERK)-eukaryotic initiation factor 2α (EIF2α)-activating transcription factor 4 (ATF4)-signaling pathway. After separately blocking PERK-EIF2α-ATF4 signaling with two different inhibitors [AMG'44 and integrated stress response inhibitor (ISRIB)] or specific small interfering RNA for PERK and ATF4, the following targets were all downregulated: expression of osteoblast differentiation markers [runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alp), type I collagen (Col1a1), and osteocalcin (Ocn)], cell proliferation markers (CyclinE, CyclinD, and CDC2), amino acid import (Glyt1), and metabolism-related genes (Asns). Additionally, Alp-positive staining cells, Alp activity, matrix mineralization, Ocn secretion, and cell proliferation indexes were inhibited. Interestingly, we found that salubrinal enhanced PTH-induced osteoblast differentiation and proliferation by maintenance of phosphorylation of EIF2α. Furthermore, we observed that PTH increased the association between heat shock protein 90 (HSP90) and PERK and maintained PERK protein stabilization in the early stages of PTH-induced ER stress. Treatment of MC3T3-E1 cells with geldanamycin, an HSP90 inhibitor, decreased PERK protein expression and inhibited osteoblast differentiation and cell proliferation upon PTH treatment. Taken together, our data demonstrate that PTH regulates osteoblast differentiation and cell proliferation, partly by activating the HSP90-dependent PERK-EIF2α-ATF4 signaling pathway.

Vascular endothelial growth factor signaling requires glycine to promote angiogenesis.

Peripheral vascular occlusive disease (PVOD) is a common manifestation of atherosclerosis, and it has a high rate of morbidity. Therapeutic angiogenesis would re-establish blood perfusion and rescue ischemic tissue. Vascular endothelial growth factor (VEGF) induces angiogenesis and can potentially be used to treat ischemic diseases, yet in clinical trials VEGF has not fulfilled its full potential with side effects. Whether amino acids promote angiogenesis and the molecular mechanisms are largely unknown. Here we showed that (1) Glycine significantly promoted angiogenesis both in vitro and in vivo and effectively protected mitochondrial function. (2) Activation of glycine transporter 1(GlyT1) induced by VEGF led to an increase in intracellular glycine. (3) Glycine directly bounded to voltage dependent anion channel 1 (VDAC1) on the mitochondrial outer membrane and inhibited its opening. These original results highlight glycine as a necessary mediator in VEGF signalling via the GlyT1-glycine-mTOR-VDAC1 axis pathway. Therefore, the findings in this study are of significance providing new mechanistic insights into angiogenesis and providing better understanding of glycine function in angiogenesis, which may provide valuable information for development of novel therapeutic targets for the treatment of angiogenic vascular disorders.

P2X receptors up-regulate the cell-surface expression of the neuronal glycine transporter GlyT2.

Glycinergic inhibitory neurons of the spinal dorsal horn exert critical control over the conduction of nociceptive signals to higher brain areas. The neuronal glycine transporter 2 (GlyT2) is involved in the recycling of synaptic glycine from the inhibitory synaptic cleft and its activity modulates intra and extracellular glycine concentrations. In this report we show that the stimulation of P2X purinergic receptors with ßγ-methylene adenosine 5'-triphosphate induces the up-regulation of GlyT2 transport activity by increasing total and plasma membrane expression and reducing transporter ubiquitination. We identified the receptor subtypes involved by combining pharmacological approaches, siRNA-mediated protein knockdown, and dorsal root ganglion cell enrichment in brainstem and spinal cord primary cultures. Up-regulation of GlyT2 required the combined stimulation of homomeric P2X3 and P2X2 receptors or heteromeric P2X2/3 receptors. We measured the spontaneous glycinergic currents, glycine release and GlyT2 uptake concurrently in response to P2X receptor agonists, and showed that the impact of P2X3 receptor activation on glycinergic neurotransmission involves the modulation of GlyT2 expression or activity. The recognized pro-nociceptive action of P2X3 receptors suggests that the fine-tuning of GlyT2 activity may have consequences in nociceptive signal conduction.

Regional differences in glutathione accumulation pathways in the rat cornea: Mapping of amino acid transporters involved in glutathione synthesis.

In this study we have sought to complete the identification and localisation of uptake pathways involved in accumulating precursor amino acids involved in GSH synthesis in the rat cornea. To do this, we performed reverse transcription PCR (RT-PCR) to identify the Excitatory Amino Acid Transporters (EAAT 1-5) responsible for glutamate uptake, and glycine transporters (GLYT 1-2) at the transcript level. Western blotting was used to verify protein expression, while immunolabelling of sagittal sections was used to localise transporters to the different layers of the cornea. Immunolabelling of en face sections was used to examine the subcellular distribution of proteins in the corneal endothelium. Our findings revealed EAAT 1-5 and GLYT 1-2 to be expressed at the transcript and protein level in the rat cornea. Immunohistochemistry revealed all amino acid transporters to be localised to the epithelium. In the majority of cases, labelling was restricted to the epithelium, and labelling absent from the stroma or endothelium. However, EAAT 4 and GLYT 2 labelling was detected in the stroma with EAAT 4 labelling also present in the endothelium. Overall, the identification of amino acid transporters strongly supports the existence of an intracellular GSH synthesis pathway in the rat corneal epithelium. This suggests that regional differences in GSH accumulation pathways exist, with direct uptake of GSH and intracellular synthesis of GSH restricted to the endothelial and epithelial cell layers, respectively. This information is important in the design of targeted strategies to enhance GSH levels in specific layers of the cornea to prevent against oxidative damage, corneal swelling and loss of corneal transparency.

Neurochemistry of neurons in the ventrolateral medulla activated by hypotension: Are the same neurons activated by glucoprivation?

Previous studies have demonstrated that a range of stimuli activate neurons, including catecholaminergic neurons, in the ventrolateral medulla. Not all catecholaminergic neurons are activated and other neurochemical content is largely unknown hence whether stimulus specific populations exist is unclear. Here we determine the neurochemistry (using in situ hybridization) of catecholaminergic and noncatecholaminergic neurons which express c-Fos immunoreactivity throughout the rostrocaudal extent of the ventrolateral medulla, in Sprague Dawley rats treated with hydralazine or saline. Distinct neuronal populations containing PPCART, PPPACAP, and PPNPY mRNAs, which were largely catecholaminergic, were activated by hydralazine but not saline. Both catecholaminergic and noncatecholaminergic neurons containing preprotachykinin and prepro-enkephalin (PPE) mRNAs were also activated, with the noncatecholaminergic population located in the rostral C1 region. Few GlyT2 neurons were activated. A subset of these data was then used to compare the neuronal populations activated by 2-deoxyglucose evoked glucoprivation (Brain Structure and Function (2015) 220:117). Hydralazine activated more neurons than 2-deoxyglucose but similar numbers of catecholaminergic neurons. Commonly activated populations expressing PPNPY and PPE mRNAs were defined. These likely include PPNPY expressing catecholaminergic neurons projecting to vasopressinergic and corticotrophin releasing factor neurons in the paraventricular nucleus, which when activated result in elevated plasma vasopressin and corticosterone. Stimulus specific neurons included noncatecholaminergic neurons and a few PPE positive catecholaminergic neuron but neurochemical codes were largely unidentified. Reasons for the lack of identification of stimulus specific neurons, readily detectable using electrophysiology in anaesthetized preparations and for which neural circuits can be defined, are discussed.

Astrocytic transporters in Alzheimer's disease.

Astrocytes play a fundamental role in maintaining the health and function of the central nervous system. Increasing evidence indicates that astrocytes undergo both cellular and molecular changes at an early stage in neurological diseases, including Alzheimer's disease (AD). These changes may reflect a change from a neuroprotective to a neurotoxic phenotype. Given the lack of current disease-modifying therapies for AD, astrocytes have become an interesting and viable target for therapeutic intervention. The astrocyte transport system covers a diverse array of proteins involved in metabolic support, neurotransmission and synaptic architecture. Therefore, specific targeting of individual transporter families has the potential to suppress neurodegeneration, a characteristic hallmark of AD. A small number of the 400 transporter superfamilies are expressed in astrocytes, with evidence highlighting a fraction of these are implicated in AD. Here, we review the current evidence for six astrocytic transporter subfamilies involved in AD, as reported in both animal and human studies. This review confirms that astrocytes are indeed a viable target, highlights the complexities of studying astrocytes and provides future directives to exploit the potential of astrocytes in tackling AD.

Genetic inactivation of glutamate neurons in the rat sublaterodorsal tegmental nucleus recapitulates REM sleep behaviour disorder.

SEE SCHENCK AND MAHOWALD DOI101093/AWW329 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Idiopathic REM sleep behaviour disorder is characterized by the enactment of violent dreams during paradoxical (REM) sleep in the absence of normal muscle atonia. Accumulating clinical and experimental data suggest that REM sleep behaviour disorder might be due to the neurodegeneration of glutamate neurons involved in paradoxical sleep and located within the pontine sublaterodorsal tegmental nucleus. The purpose of the present work was thus to functionally determine first, the role of glutamate sublaterodorsal tegmental nucleus neurons in paradoxical sleep and second, whether their genetic inactivation is sufficient for recapitulating REM sleep behaviour disorder in rats. For this goal, we first injected two retrograde tracers in the intralaminar thalamus and ventral medulla to disentangle neuronal circuits in which sublaterodorsal tegmental nucleus is involved; second we infused bilaterally in sublaterodorsal tegmental nucleus adeno-associated viruses carrying short hairpin RNAs targeting Slc17a6 mRNA [which encodes vesicular glutamate transporter 2 (vGluT2)] to chronically impair glutamate synaptic transmission in sublaterodorsal tegmental nucleus neurons. At the neuroanatomical level, sublaterodorsal tegmental nucleus neurons specifically activated during paradoxical sleep hypersomnia send descending efferents to glycine/GABA neurons within the ventral medulla, but not ascending projections to the intralaminar thalamus. These data suggest a crucial role of sublaterodorsal tegmental nucleus neurons rather in muscle atonia than in paradoxical sleep generation. In line with this hypothesis, 30 days after adeno-associated virus injections into sublaterodorsal tegmental nucleus rats display a decrease of 30% of paradoxical sleep daily quantities, and a significant increase of muscle tone during paradoxical sleep concomitant to a tremendous increase of abnormal motor dream-enacting behaviours. These animals display symptoms and behaviours during paradoxical sleep that closely mimic human REM sleep behaviour disorder. Altogether, our data demonstrate that glutamate sublaterodorsal tegmental nucleus neurons generate muscle atonia during paradoxical sleep likely through descending projections to glycine/GABA premotor neurons in the ventral medulla. Although playing a role in paradoxical sleep regulation, they are, however, not necessary for inducing the state itself. The present work further validates a potent new preclinical REM sleep behaviour disorder model that opens avenues for studying and treating this disabling sleep disorder, and advances potential regions implicated in prodromal stages of synucleinopathies such as Parkinson's disease.

GlyT1 Inhibitor NFPS Exerts Neuroprotection via GlyR Alpha1 Subunit in the Rat Model of Transient Focal Cerebral Ischaemia and Reperfusion.

BACKGROUND/AIMS: Glycine is a strychnine-sensitive inhibitory neurotransmitter in the central nervous system (CNS), especially in the spinal cord, brainstem, and retina. The objective of the present study was to investigate the potential neuroprotective effects of GlyT1 inhibitor N [3-(4'-fluorophenyl)-3-(4'-phenylphenoxy) propyl] sarcosine (NFPS) in the rat model of experimental stroke. METHODS: In vivo ischaemia was induced by transient middle cerebral artery occlusion (tMCAO). The methods of Western Blotting, Nissl Staining and Morris water maze methods were applied to analyze the anti-ischaemia mechanism. RESULTS: The results showed that high dose of NFPS (H-NFPS) significantly reduced infarct volume, neuronal injury and the expression of cleaved caspase-3, enhanced Bcl-2/Bax, and improved spatial learning deficits which were administered three hours after transient middle cerebral artery occlusion (tMCAO) induction in rats, while, low dose of NFPS (L-NFPS) exacerbated the injury of ischaemia. These findings suggested that low and high dose of NFPS produced opposite effects. Importantly, it was demonstrated that H-NFPS-dependent neuronal protection was inverted by salicylate (Sal), a specific GlyR x0251;1 antagonist. Such effects could probably be attributed to the enhanced glycine level in both synaptic and extrasynaptic clefts and the subsequently altered extrasynaptic GlyRs and their subtypes. CONCLUSIONS: These data imply that GlyT1 inhibitor NFPS may be a novel target for clinical treatment of transient focal cerebral ischaemia and reperfusion which are associated with altered GlyR alpha 1 subunits.

Spinal glycine transporter-1 inhibition influences the micturition reflex in urethane-anesthetized rats.

PURPOSE: Glycine is an inhibitory neurotransmitter in the central nervous system. So far, two types of glycine transporters (GlyTs), GlyT-1 and GlyT-2, have been cloned. The aim of this study is to investigate the effects of a selective GlyT-1 inhibitor that can increase endogenous glycine concentration on the micturition reflex in urethane-anesthetized rats. METHODS: Continuous cystometrograms (0.04 ml/min) were performed in female Sprague-Dawley rats (232-265 g) under urethane anesthesia. After stable micturition cycles were established, ALX5407, a selective GlyT-1 inhibitor, was administered intrathecally or intracerebroventricularly to evaluate changes in bladder activity. Cystometric parameters were recorded and compared before and after drug administration. RESULTS: Intrathecal administration of ALX5407 (1, 3, 10 and 30 µg) increased intercontraction intervals at doses of 3 µg or higher in a dose-dependent fashion. Intrathecal administration of ALX5407 (1, 3, 10 and 30 µg) also increased pressure threshold at doses of 3 µg or higher in a dose-dependent fashion. However, when ALX5407 (1, 3, 10 and 30 µg) was administered intracerebroventricularly, there were no significant changes in intercontraction intervals, pressure threshold, maximum voiding pressure or baseline pressure or post-void residual urine volume at any doses tested. CONCLUSION: The results of our study indicate that GlyT-1 plays an important role in the modulation of micturition. Furthermore, these findings indicate that in urethane-anesthetized rats suppression of GlyT-1 can inhibit the micturition reflex at the spinal cord level. Thus, GlyT-1 could be a potential target for the treatment of bladder dysfunction such as overactive bladder.

Structural insights into the benzophenanthridines binding to human glycine transporter GlyT1.

We previously identified cysteine 475 as a key residue for the inhibitory action of sanguinarine on the human glycine transporter GlyT1c. To define potential benzophenanthridine binding pocket more closely, we created a structural homology model of GlyT1 and also mutated several amino acids in the vicinity of cysteine 475. Even though this area contains the molecular determinants of the glycine and sodium permeation pathways, and several mutations resulted in an inactive transporter, we found that the mutation of a polar aromatic tyrosine 370 to purely aromatic phenylalanine, but not to an aliphatic leucine, significantly increased the sensitivity of GlyT1 to both sanguinarine and chelerythrine. The reduction of sanguinarine to dihydrosanguinarine completely eliminated the alkaloid's inhibitory potency. Both these results suggest that aromaticity is important in the interaction of benzophenanthridines with GlyT1. Even though tyrosine 370 is part of the conformationaly highly flexible glycine binding site, and is accesible during the transport process from both intra and extracellular sites, the cytoplasmic location of the second alkaloid sensitive residue, cysteine 475, suggests that the benzophenanthridines might attack the area of the GlyT1 intracellular gates.