Glycine and glycine transport control dendritic excitability and spiking.

Perfluorooctane sulfonate (PFOS) poses significant health and environmental risks due to its persistence and widespread use and has been linked to various adverse outcomes, such as liver toxicity. Although the molecular responses and toxicity effects of PFOS exposure have been extensively studied, considerable uncertainty remains regarding the causal mechanisms leading to PFOS-associated adverse effects. To help bridge this gap, we conducted CRISPR screens in HepG2/C3A human liver cells exposed to IC25 (170 μM) of PFOS to identify genes and pathways influencing PFOS-induced cytotoxicity. Using a genome-wide CRISPR knockout library targeting 18,819 genes, we identified 340 candidate genes that modulate PFOS-induced cytotoxicity when genetically disrupted (189 gene disruptions increased sensitivity and 151 gene disruptions increased resistance). From these candidate genes, we individually disrupted two candidate genes, SLC6A9 which encodes the glycine transporter GlyT1, and CPSF2, and confirmed increased resistance to PFOS exposure. Further, molecular docking analysis predicts that PFOS directly binds to GlyT1 and functional inhibition of GlyT1 also increases resistance to PFOS exposure. Gene-Disease outcome association analysis using the Comparative Toxicogenomics Database (CTD) indicated an enrichment of candidate genes associated with cancer-related and liver disease phenotypes. KEGG and STRING enrichment analyses found over representation of several biological pathways including DNA damage response and cell cycle. Lastly, cross-species conservation analysis using the top two validated gene targets found that their pathways were highly conserved in several environmentally relevant species. These findings provide new mechanistic and functional insights into PFOS-induced cytotoxicity, highlight potential molecular targets for toxicity mitigation, and establish a foundation for cross-species toxicogenomic modeling of PFOS health effects.

Background/Objectives: The neurotransmitter glycine is involved in several physiological and pathological conditions in the Central Nervous System. Different biological structures, including glycine receptors and transporters, are under study as targets for potential drugs acting against serious neurological and psychiatric disorders. The regulation of glycine release from nerve terminals is only partially understood. We report here preliminary evidence of the modulation of glycine release through presynaptic metabotropic glutamate receptors 1 (mGlu1) from glycinergic nerve terminals in mouse hippocampi. Methods: Purified mouse hippocampal synaptosomes labeled with [3H]glycine were used to study glycine release under superfusion conditions. Results: The group I metabotropic glutamate receptor agonist 3,5-DHPG potentiated depolarization-evoked [3H]glycine release from hippocampal synaptosomes, an effect strongly counteracted by the selective mGlu1 antagonist LY 367385. 3,5-DHPG failed to increase [3H]glycine release in Grm1crv4/crv4 mice, a mouse model lacking mGlu1. Although further research is needed to clarify these mechanisms, data suggest that glycine-releasing hippocampal nerve terminals are endowed with presynaptic mGlu1 receptors whose activation potentiates glycine release. Conclusions: Considering that in the hippocampus, glycine is relevant as a co-agonist of glutamate at NMDA receptors and that mGlu1 receptor ligands are under study as potential drugs, we propose that the possible effects of these agents on the release of glycine should be considered when studying these compounds.

Bitopertin shows efficacy in patients with erythropoietic protoporphyria: Results from the randomized, double-blind, placebo-controlled AURORA trial.

Aging results in decreased collagen synthesis and the disruption of extracellular matrix integrity, primarily due to increased oxidative stress. This study evaluated whether atelocollagen can restore collagen synthesis in aged skin by modulating glycine transporter 1 (GlyT1)-mediated glycine uptake, regulating oxidative stress, and influencing extracellular matrix remodeling factors in senescent human cells and the skin of older mice. Human dermal fibroblasts (HDFs) and aged mouse skin were treated with atelocollagen, with analyses of GlyT1 expression, glutathione and intracellular glycine concentrations, oxidative stress markers, nuclear factor-kappa-B (NF-κB) activity, matrix metalloproteinases (MMPs), SMAD proteins (SMADs) signaling, and collagen I/III. Treatment with GlyT1 inhibitors and glycine was tested. Atelocollagen significantly increased GlyT1 expression and intracellular glycine concentration, promoted glutathione synthesis, and reduced oxidative stress. These effects led to decreased NF-κB activity and MMP1/3/9 expression, increased SMAD2/3 phosphorylation, and upregulated type I/III collagen synthesis in senescent HDFs and aged mouse skin. All beneficial effects were blocked by GlyT1 inhibition and were generally superior to glycine alone. Histology showed increased collagen density and improved skin elasticity in atelocollagen-treated mice. In summary, atelocollagen enhances collagen synthesis and reduces oxidative stress in aged skin through GlyT1-dependent glycine transport, providing a potential strategy for skin rejuvenation.

In primates, the OFF midget retinal ganglion cells (OFF mRGCs) have a high spatial density and small dendritic arbors. Their axons provide input to the parvocellular pathway mediating both colour vision and the highest-acuity spatial vision. This study aimed to understand the basis for their light responses by identifying the presynaptic amacrine and bipolar cells. Retinal tissue from an adult macaque was processed for serial block-face scanning electron microscopy, and a volume of images of the inner retina located 2mm temporal to the centre of the fovea was analysed. Ten OFF mRGCs and many of their presynaptic cells were reconstructed. Both midget and diffuse types of bipolar cells provided excitatory, glutamatergic input. Axons and long dendrites of wide-field amacrine cells made synapses, and we propose that these mediate tonic, GABAergic inhibition. Narrow-field amacrine cells also made synapses onto the OFF mRGCs, and we propose that most of them are glycinergic, inhibitory synapses. One presynaptic narrow-field amacrine cell was the knotty bistratified type 1 (KB1), which contains immunoreactive glycine and vesicular glutamate transporter 3. We propose that they enlarge the receptive field centers of OFF mRGCs via direct, excitatory synapses. The KB1 cell studied most extensively was presynaptic to some of the same types of amacrine cells that made inhibitory synapses onto OFF mRGCs. We propose that the knotty bistratifed type 1cells release glycine at those synapses and disinhibit responses of OFF mRGCs. KEY POINTS: In primates, OFF midget ganglion cells have the highest spatial density of any projection neurons, and they mediate high acuity vision. Ten of these cells and the neurons providing their inputs were reconstructed from a volume of serial ultrathin sections taken 2mm temporal to the centre of the macaque fovea. They received the majority of their inputs from amacrine cells, local circuit neurons that are typically inhibitory. One of the presynaptic amacrine cells resembled those containing vesicular glutamate transporter 3, and we propose that they provide excitatory input that enlarges the receptive field centers of OFF midget ganglion cells. They also receive excitatory input from both midget and diffuse bipolar cells. The results provide an explanation for some apparent contradictions between anatomical and physiological studies and are potentially important for understanding the etiology of retinal diseases.

Efficacy and safety of iclepertin for cognitive impairment associated with schizophrenia (CONNEX programme): results from three phase 3 randomised controlled trials.

Glycine transporter 2 (GlyT2) regulates inhibitory glycinergic neurotransmission, and its inhibition potentiates glycinergic signaling, which is a promising strategy for managing neuropathic pain. This study presents high-resolution structures of GlyT2 in its apo state and in complexes with the substrate glycine, analgesic inhibitors, captured in three functional states: outward-facing, occluded, and inward-facing. The glycine-bound structure reveals the binding mode of the substrate, Na+ and Cl-. Specifically, we identified the Na3 binding site, offering fundamental insights into Na+/Cl- coupled substrate binding and conformational changes. Moreover, we clearly elucidate a previously unseen allosteric binding pocket for the lipid-based oleoyl-D-lysine, which acts as a wedge to stabilize GlyT2 in the outward-facing conformation and prevents its transition. Furthermore, the complex structures with small compounds ALX1393, opiranserin, and ORG25543 reveal their competitive and allosteric inhibition mechanisms. Overall, our study provides a solid foundation for understanding glycine reuptake mechanisms and developing effective and safer analgesic agents.

Chronic neuropathic pain, caused by nerve damage or disease, is increasing in prevalence, but current treatments are ineffective and over-reliant on opioids. The neuronal glycine transporter, GlyT2, regulates inhibitory glycinergic neurotransmission and represents a promising target for new analgesics. However, most GlyT2 inhibitors cause significant side effects, in part due to irreversible inhibition at analgesic doses. Here we develop a reversible inhibitor of GlyT2, RPI-GLYT2–82, and identify its binding site by determining cryo-EM structures of human GlyT2. We capture three fundamental conformational states of GlyT2 in the substrate-free state, and bound to either glycine, RPI-GLYT2–82 or the pseudo-irreversible inhibitor ORG25543. We demonstrate that RPI-GLYT2–82 dissociates from GlyT2 faster than ORG25543, providing analgesia in mouse neuropathic pain models without on-target side-effects or addiction liability. Our data provide a mechanistic understanding of allosteric inhibition of glycine transport, enabling structure-based design of non-opioid analgesics.

The proline transporter (PROT, SLC6A7) and the neutral and cationic amino acid transporter (ATB0,+, SLC6A14) belong to the glycine transporter subfamily, exhibiting distinct substrate specificities and physiological functions. PROT modulates neurotransmission through proline transport in the brain, while ATB0,+ facilitates nutrient uptake, especially in the gastrointestinal tract. Impaired function of PROT has been associated with neurological disorders, while ATB0,+ overexpression has been linked to cancers. Despite their biological relevance, the pool of known ligands for these transporters is limited, and their exact 3D structures remain unknown. Therefore, we conducted an in silico analysis of PROT and ATB0,+ and compared the obtained results with available literature data on the glycine transporter GlyT1, from the same subfamily. Using homology modelling, docking studies, and molecular dynamics simulations, we investigated the structural properties of PROT and ATB0,+ and described protein–ligand interactions. We pointed crucial residues responsible for ligand binding, including Tyr133, Tyr297, Phe303, and Phe403 in PROT and Trp327, Val128, and Tyr321 in ATB0,+. This work provides new insights into the molecular features of PROT and ATB0,+ transporters, which could support the development of novel transporter inhibitors.

GlyT1 inhibition promotes post-ischemic neuroprotection in the MCAO model.

Glycine is a key inhibitory neurotransmitter in the central nervous system. Its synaptic concentration is regulated by glycine transporters (GlyT1,2), which belong to the solute carrier family 6 (SLC6). GlyT1 is predominantly expressed in astrocytes and glutamatergic neurons, whereas GlyT2 is localized to presynaptic glycinergic terminals. Both transporters mediate glycine reuptake coupled with Na⁺ and Cl⁻ ions. The dual role of GlyT1 in modulating inhibitory neurotransmission and excitatory NMDA receptor signaling makes its investigation crucial for understanding the molecular mechanisms of synaptic activity and developing therapies for schizophrenia, neuropathic pain, and other neurological disorders. In this study, we employed molecular dynamics (MD) simulations to examine the coordination of ionic and substrate interactions within GlyT1. The initial protein coordinates were obtained from the outward-open GlyT1 structure (PDB ID: 8WFL). The transporter was embedded in a phosphatidylcholine bilayer and solvated with 150 mM NaCl. Classical MD simulations were performed for 2 µs. To capture rare substrate-binding events and quantify glycine affinity, we also employed enhanced sampling simulations using nonequilibrium MD methods. Analysis of MD trajectories revealed the sequence of substrate binding and identified key amino acid residues involved in ligand coordination. Free energy calculations derived from enhanced sampling simulations allowed us to estimate glycine affinity under varying scenarios of ion occupancy at binding sites. These findings provide critical insights into the molecular mechanism of GlyT1 and other SLC6 sodium-dependent neurotransmitter transporters. Furthermore, the results can be used to explore potential pharmacological compounds capable of modulating glycinergic and glutamatergic systems functioning.

Glycine plays a dual role in the central nervous system as a fast inhibitory neurotransmitter and a co-agonist at N-methyl-D-aspartate receptors (NMDARs). Its extracellular levels are tightly regulated by two glycine transporters (GlyTs): GlyT1, which modulates glycine near excitatory synapses to influence glutamatergic transmission, and GlyT2, which sustains presynaptic glycine for inhibitory signalling. Dysregulation of GlyT function has been linked to numerous neurological and psychiatric disorders, including schizophrenia, mood and anxiety disorders, neurodegeneration, epilepsy, stroke, addiction, and pain. This review examines recent preclinical and clinical progress in targeting GlyTs, with an emphasis on GlyT1 inhibition to enhance NMDAR function. Among GlyT1 inhibitors, sarcosine shows consistent promise, particularly for schizophrenia and depressive symptoms. However, the limited clinical success of other compounds underscores challenges in translating preclinical efficacy. Addressing issues such as selectivity, patient stratification, and novel regulatory mechanisms will be key. Future research leveraging imaging biomarkers and next-generation pharmacological tools may help unlock the full therapeutic value of GlyTs.

Iclepertin is a selective inhibitor of glycine transporter 1 recently investigated as a novel treatment for cognitive impairment associated with schizophrenia. Iclepertin is a potential mild inducer of liver cytochrome P450 3A4, which metabolises ethinylestradiol and levonorgestrel, which are used in combined oral contraceptives (OCs). This trial investigated the potential drug interaction effect of steady-state iclepertin on the steady-state pharmacokinetics of combined OCs. This phase I, non-randomised, open-label, two-period, fixed-sequence trial was conducted in healthy pre-menopausal female volunteers aged 18-35 years. In period 1, participants received a combined OC (ethinylestradiol 30 µg/levonorgestrel 150 µg once daily; reference treatment). In period 2, participants received a combined OC and iclepertin 10 mg once daily (test treatment). Primary pharmacokinetic endpoints of ethinylestradiol or levonorgestrel in plasma at steady state over a uniform dosing interval τ were area under the concentration-time curve (AUCτ,ss) and maximum and minimum measured concentration (Cmax,ss and Cmin,ss); drug interaction potential was estimated by geometric mean ratios (test treatment/reference treatment) with two-sided 90% confidence intervals (CIs) using analysis of variance. Safety assessments included monitoring adverse events (AEs). In total, 19 participants entered the trial; 17 were treated (periods 1 and 2). Steady-state pharmacokinetics of ethinylestradiol and levonorgestrel were similar with and without iclepertin; geometric mean ratios for AUCτ,ss, Cmax,ss, and Cmin,ss were slightly above 100%, and 90% CIs were within standard bioequivalence boundaries (80-125%). The number of on-treatment AEs was similar in period 1 (n=13) and period 2 (n=15); AEs were of mild-to-moderate severity. Iclepertin 10 mg had no meaningful effect on the pharmacokinetics of ethinylestradiol and levonorgestrel, suggesting that these drugs can be administered concomitantly. ClinicalTrials.gov (NCT05613777; registered on 18 October 2023).

Some individuals exposed to traumatic stressors develop psychiatric disorders while others remain resilient. The Brief Resilience Scale (BRS) assesses the ability to "bounce back" from stress and is a widely used measure of trait resilience. We performed the first genome-wide association study of BRS in the UK Biobank (UKB). Beginning 2022, a subset of UKB participants completed an on-line mental-health questionnaire that included the six-item BRS. BRS data and genome-wide typing and imputation were available for 124,774 participants of European ancestry. Genome-wide linear tests of BRS were performed, followed by SNP-based heritability and cross-trait genetic correlation analyses. Nominally significant loci (P < 5 × 10-6) were followed up for candidate gene mapping. SNP-based heritability of BRS was 7.3% and strong genetic correlations (rg) were observed with neuroticism (rg, - 0.70 to - 0.44), depression (rg, - 0.63 to - 0.37) and anxiety (rg, - 0.81 to - 0.46). Three loci met genome-wide significance (P < 5 × 10-8, near VRK2, TNKS/MSRA and RAB36) and 29 loci met nominal significance (P < 5 × 10-6). None of these were replicated in prior GWAS using different measures of resilience. The strongest candidate genes prioritized on the basis of both functional and biological evidence include VRK2 (2p16.1) (previously associated with neuropsychiatric disorders) and MSRA (8p23.1) (reduces methionine sulfoxide to methionine). Others at nominally significant loci include SLC6A9 (1p34.1) (encodes a glycine transporter), NPY (7p15.2) (involved in stress response), CADPS2 (7q31.32) (involved in synaptic vesicle exocytosis), and PCDH9 (13q21.32) (involved in neural tissue cell adhesion). CONCLUSIONS: Our findings provide further support for a genetic basis to trait resilience and one shared with psychiatric disorders and personality traits including depression, anxiety, and neuroticism. Our promising loci warrant replication but offer new biological insight to resilience.

ObjectiveTransplant rejection driven by Th1 cell-mediated immune responses remains a critical challenge. This study aimed to investigate the role of glycine transporter 1 (GlyT1/SLC6A9) in Th1 differentiation and evaluate the therapeutic potential of its inhibitor, ALX-5407, in attenuating allograft rejection.MethodsRNA sequencing, flow cytometry, and qRT-PCR were employed to analyze GlyT1 expression in Th1-polarized CD4+T cells. ALX-5407 (0.5–500 nM) was tested in vitro under Th1-polarizing conditions. A murine skin allograft model (BALB/c to C57BL/6) was established to assess graft survival and immune responses. Combination therapy with rapamycin and ALX-5407 was evaluated through histopathology, immunofluorescence, and splenocyte profiling. Mechanistic insights were derived from RNA-seq, KEGG/GO enrichment, and Western blotting.ResultsGlyT1 expression was significantly upregulated in Th1 cells and rejection cohorts. ALX-5407 suppressed Th1 differentiation, reducing IFN-γ+CD4+T cells proportions (p < 0.05) and activation markers (CD25, CD69), while inducing apoptosis via caspase-3 activation and BCL-2 downregulation. Although ALX-5407 monotherapy failed to prolong graft survival, combination with rapamycin synergistically enhanced efficacy (p = 0.018), reduced inflammatory infiltration, and attenuated splenic Th1 polarization. Mechanistically, ALX-5407 inhibited MAPK signaling but activated the PI3K-AKT-mTOR pathway, which rapamycin counteracted to amplify suppression.ConclusionsGlyT1 serves as a metabolic checkpoint in Th1 differentiation, and its inhibition by ALX-5407 attenuates allograft rejection through dual suppression of Th1 function and apoptosis induction. Synergy with rapamycin highlights a novel combinatorial strategy to mitigate rejection with reduced toxicity. These findings position GlyT1 targeting as a promising approach for clinical translation in transplantation immunotherapy.

Differential synaptic inhibition and serotonin 5-HT7 receptor-mediated modulation in identified dorsal horn neurons.

Erythropoietic protoporphyria (EPP) is a genetic disorder typically resulting from decreased ferrochelatase (FECH) activity, the last enzyme in heme biosynthesis. Patients with X-linked protoporphyria (XLPP) have an overlapping phenotype caused by increased activity of 5-aminolevulinic acid synthase 2 (ALAS2), the first enzyme in erythroid heme synthesis. In both cases, protoporphyrin IX (PPIX) accumulates in erythrocytes and secondarily in plasma and tissues. Patients develop acute phototoxicity reactions upon brief exposure to sunlight. Some also experience chronic liver disease, and a small fraction develop acute cholestatic liver failure. Therapeutic options are limited, and none, save hematopoietic stem cell transplantation, directly targets erythroid PPIX accumulation. Bitopertin is an investigational orally available small-molecule inhibitor of the erythroid cell-surface glycine transporter GLYT1. We established the bitopertin PPIX inhibitory half-maximal effective concentration in a human erythroblast EPP model and confirmed a marked reduction of PPIX in erythroblasts derived from patients with EPP. We demonstrate that bitopertin also reduced erythrocyte and plasma PPIX accumulation in vivo in both EPP and XLPP mouse models. Finally, the reduction in erythroid PPIX ameliorated liver disease in the EPP mouse model. Altogether, these data support the development of bitopertin to treat patients with EPP or XLPP.

Hyperekplexia (OMIM 149400), a sensorimotor syndrome of perinatal clinical relevance, causes newborns to display an energic startle reflex in response to certain trivial stimuli. This condition can be lethal due to apnea episodes. The disease is caused by a blockade of glycinergic neurotransmission. Glycinergic interneurons preserve their identity by the activity of the surface glycine transporter GlyT2, which supplies glycine to presynaptic terminals to maintain glycine content in synaptic vesicles. Loss-of-function mutations in the GlyT2 gene (SLC6A5) cause a presynaptic form of human hyperekplexia. Here, we describe a new GlyT2 variant found in an infantile patient diagnosed with hyperekplexia. A missense mutation in the open reading frame of the GlyT2 gene inherited in homozygosity caused the substitution G449E in a residue highly conserved across the phylogenetic scale. The sequences of the glycine receptor genes GLRA1 and GLRB did not show abnormalities. We expressed the recombinant GlyT2 variant in heterologous cells and analyzed its pathogenic mechanism. The transporter was totally inactive, behaving as a bona fide loss-of-function mutant. Furthermore, the mutation promoted the abnormal insertion of the protein into the membrane, leading to its large incorporation into lipid rafts. However, there was no apparent alteration of wild-type trafficking upon mutant coexpression, as the mutant was prematurely degraded from the endoplasmic reticulum. Rescue with chemical chaperones was not possible for this mutant. Proteomics demonstrated that the expression of the mutant induced the unfolded protein response and interfered with raft-dependent processes. Therefore, the new variant causes a loss of function regarding GlyT2 activity but a gain of function as a cell proteostasis disturber.

Histotroph is regulated by P4 and paracrine factors and contains amino acids essential for porcine conceptus development. We hypothesized P4 contributes to amino acid transport during pregnancy in pigs. Ovariectomized gilts received daily P4 or corn oil (CO) injections from Days 12 through 39 post-estrus. High-performance liquid chromatography (HPLC) determined increased abundances of amino acids including glycine, in uterine flushings from P4-treated gilts. SLCs transport amino acids across membranes. rtPCR analyses revealed endometrial expression of SLC6A9 mRNA increased during the peri-implantation period of pregnancy. SLC7A8 and SLC38A1 mRNA expression also increased during that period but decreased later in pregnancy. SLC1A4, SLC1A5, and SLC7A10 mRNA expression was greatest later in pregnancy. Immunofluorescence analyses localized SLC6A9, which transports glycine exclusively, to the endometrial luminal epithelium from Day 12 through 25, uterine glandular epithelium throughout gestation, conceptus trophectoderm and endoderm between Days 15 and 20, and chorionic epithelia (CE) of interareolar and areolar CE between Days 40 and 60. Expression in interareolar regions decreased by Day 60 but remained high in the areolae. Expression of SLC6A9 was greater for endometrium from P4-treated than CO-treated pigs. These results illustrate the complexity of, and stage-dependent changes in, expression of amino acid transporters in endometria from pregnant pigs. The results suggest that transport of glycine into the uterine lumen and across the chorioallantois by SLC6A9 may be critical for delivery of this amino acid that is essential for conceptus development.