GLYT1 encephalopathy: Further delineation of disease phenotype and discussion of pathophysiological mechanisms.

GLYT1 encephalopathy is a form of glycine encephalopathy caused by disturbance of glycine transport. The phenotypic spectrum of the disease has not yet been completely described, as only four unrelated families with the disorder have been reported to date. Common features of affected patients include neonatal hypotonia, respiratory failure, encephalopathy, myoclonic jerks, dysmorphic features, and musculoeskeletal anomalies. All reported affected patients harbor biallelic genetic variants in SLC6A9. SNP array together with Sanger sequencing were performed in a newborn with arthrogryposis and severe neurological impairment. The novel genetic variant c.997delC in SLC6A9 was detected in homozygous state in the patient. At protein level, the predicted change is p.(Arg333Alafs*3), which most probably results in a loss of protein function. The variant cosegregated with the disease in the family. A subsequent pregnancy with ultrasound anomalies was also affected. The proband presented the core phenotypic features of GLYT1 encephalopathy, but also a burst suppression pattern on the electroencephalogram, a clinical feature not previously associated with the disorder. Our results suggest that the appearance of this pattern correlates with higher cerebrospinal fluid glycine levels and cerebrospinal fluid/plasma glycine ratios. A detailed discussion on the possible pathophysiological mechanisms of the disorder is also provided.

Loss of Glycine Transporter 1 Causes a Subtype of Glycine Encephalopathy with Arthrogryposis and Mildly Elevated Cerebrospinal Fluid Glycine.

Glycine is a major neurotransmitter that activates inhibitory glycine receptors and is a co-agonist for excitatory glutamatergic N-methyl-D-aspartate (NMDA) receptors. Two transporters, GLYT1 and GLYT2, regulate extracellular glycine concentrations within the CNS. Dysregulation of the extracellular glycine has been associated with hyperekplexia and nonketotic hyperglycinemia. Here, we report four individuals from two families who presented at birth with facial dysmorphism, encephalopathy, arthrogryposis, hypotonia progressing to hypertonicity with startle-like clonus, and respiratory failure. Only one individual survived the respiratory failure and was weaned off ventilation but has significant global developmental delay. Mildly elevated cerebrospinal fluid (CSF) glycine and normal serum glycine were observed in two individuals. In both families, we identified truncating mutations in SLC6A9, encoding GLYT1. We demonstrate that pharmacologic or genetic abolishment of GlyT1 activity in mice leads to mildly elevated glycine in the CSF but not in blood. Additionally, previously reported slc6a9-null mice and zebrafish mutants also display phenotypes consistent with the affected individuals we examined. Our data suggest that truncating SLC6A9 mutations lead to a distinct human neurological syndrome hallmarked by mildly elevated CSF glycine and normal serum glycine.

Forebrain glycine transporter 1 deletion enhances sensitivity to CS-US discontiguity in classical conditioning.

The deletion of glycine transporter 1 (GlyT1) in forebrain neurons can apparently strengthen Pavlovian aversive conditioning, but this phenotype is not expressed if conditioning followed non-reinforced pre-exposures of the to-be-conditioned stimulus (CS). To examine whether GlyT1 disruption may only enhance aversive associative learning under conditions that most favour the formation of CS-US excitatory link, we evaluated the impact of GlyT1 disruption on the trace conditioning procedure whereby a trace interval between a tone CS and a shock US was introduced during conditioning. CS and US occurrences were thus rendered discontiguous, which was expected to impede conditioning compared with contiguous CS-US pairing. Conditioned freezing to the CS was measured in a retention test conducted 48 h after conditioning. The genetic disruption significantly modified the temporal dynamics of the freezing response over the course of the 8-min presentation of the CS, although the immediate conditioned response to the CS was unaffected. The separation between "trace" and "no-trace" conditions was augmented in the mutant mice, but this only became apparent in mid-session; and the augmentation can be attributed to the combined effects of (i) weaker conditioned freezing in the mutant relative to control subjects in the "trace" condition, and (ii) stronger conditioned freezing in mutants relative controls in the "no-trace" condition. The demonstrated increased sensitivity to the effect of CS-US temporal discontiguity further highlights the importance of GlyT1-dependent mechanisms in the regulation of associative learning.

Dynamic role of GlyT1 as glycine sink or source: Pharmacological implications for the gain control of NMDA receptors.

Glycine transporter 1 (GlyT1) mediates the termination of inhibitory glycinergic receptor signaling in the spinal cord and brainstem, and is also present diffusely in the forebrain. Here, it regulates the ambient glycine concentration and influences the 'glycine' site occupancy of N-methyl-d-aspartate receptors (NMDARs). GlyT1 is a reversible transporter with a substantial, but not excessive, sodium-motive force for uphill transport. This study investigates its role as a potential source of glycine supply, either by reverse uptake or heteroexchange. Indeed, glutamate alone does not induce NMDAR current in "naive" oocytes co-expressing GluN1/GluN2A and GlyT1, a previously characterized cellular model. However, after substantial intracellular glycine accumulation, GlyT1 reverses its transport mode, and begins to release glycine into the external compartment, allowing NMDAR activation by glutamate alone. These uptake-dependent glutamate currents were blocked by ALX-5407 and potentiated by sarcosine, a specific inhibitor and substrate of GlyT1, respectively, suggesting a higher occupancy of the co-agonist site when GlyT1 functions as a glycine source either by reversed-uptake or by heteroexchange. These two glycine release mechanisms can be distinguished by their voltage dependence, as the reversed-uptake cycle decreases at hyperpolarized potentials, whereas heteroexchange electroneutrality preserves glycine efflux and NMDAR activation at these potentials. These results establish GlyT1-mediated efflux as a positive regulator of NMDAR coagonist site occupancy, and demonstrate the efficacy of sarcosine heteroexchange in enhancing coagonist site occupancy. Because NMDAR facilitation by GlyT1-inhibitors and sarcosine relies on different transport mechanisms, their actions may be a source of variability in reversing NMDAR hypofunction in schizophrenia.

A lethal and rare cause of arthrogryposis: Glyt1 encephalopathy.

Glycine encephalopathy with normal serum glycine (MIM #617301), also known as GLYT1 encephalopathy, is an extremely rare disorder caused by biallelic variants in SLC6A9 and characterised by facial dysmorphic features, skeletal findings including contractures, knee hyperextension, and joint dislocations and seizures. To date, only ten patients from five families have been reported and only two of them could survive until childhood. In this study, we report on a consanguineous Turkish couple with a history of six pregnancies with three habitual abortions and three postpartum exitus. While in three pregnancies the babies were born prematurely at 32nd gestational week by emergency ceserean section due to hydrops and fetal distress, the other pregnancy was medically terminated at 16th gestational week due to absent fetal heart activity. The product of all these three pregnancies exhibited similar phenotype including short neck, thoracic kyphosis, hypertrichosis, joint contractures and dislocations, hypertonia, knee hyperextension and facial dysmorphic features. Trio exome sequencing was performed prenatally during the last pregnancy and a novel VUS variant in SLC6A9 and a likely pathogenic variant in MTOR gene were detected. DNA isolation was performed from frozen muscle and adrenal tissue of previously autopsied fetuses with similar clinical features, and the same variants were confirmed in both of them. Our data suggest that SLC6A9 and MTOR variants may be responsible for this extremely lethal phenotype in this family.

Glycine encephalopathy.

Inherited neurotransmitter diseases are a subset of rare neurometabolic disorders characterized by hereditary deficiencies in neurotransmitter metabolism or transport. Non-ketotic hyperglycinaemia (NKH), called glycine encephalopathy, is an autosomal recessive glycine metabolism disorder characterized by an abnormal accumulation of glycine in all bodily tissues, including the CNS. The SLC6A9 gene, which codes for the GLYT1 protein, a biochemical abnormality in the GCS, and dihydrolipoamide dehydrogenase enzymes, which function as a GCS component, are responsible for the neonatal form's symptoms, which include progressive encephalopathy, hypotonia, seizures, and occasionally mortality in the first few days of life. By changing the MAPK signalling pathways, glycine deprivation in the brain damages neurons by increasing NMDA receptor activation, increasing intracellular Ca levels, and leading to DNA breakage and cell death in the neuron region. In addition to the previously mentioned clinical diagnosis, NKH or GE would be determined by MLPA and 13C glycine breath tests. Pediatricians, surgeons, neurologists, and geneticists treat NKH and GE at the newborn period; there is no cure for either condition.

Identifying Conformation-Selective Heavy-Chain-Only Antibodies Against Membrane Proteins by a Thermal-Shift Scintillation Proximity Assay.

Over the last decades, the use of heavy-chain-only antibodies has received growing attention in academia and industry as research and diagnostic tools as well as therapeutics. Their generation has improved with the help of innovative new methods such as the sybody technology; however, identifying conformation-selective compounds against membrane proteins remains a major challenge. In this chapter, we apply a thermal shift scintillation proximity assay (SPA-TS) to identify sybodies from an in vitro display campaign with the ability to selectively stabilize the inhibitor-bound conformation of the human solute carrier (SLC) family transporter SC6A9 (GlyT1). Using detergent-purified GlyT1 protein and a tritium-labeled glycine uptake inhibitor small molecule, we find sybody candidates that increase the apparent melting temperature in SPA-TS by several degrees. The thermal shift stabilizes the GlyT1-inhibitor complex and qualifies the sybodies for structural studies and inhibitor-selective small molecule screening assays. The SPA-TS assay in its current form is adaptable to any antibody discovery campaign for membrane proteins and permits the generation of highly valuable tools in most stages of drug discovery and development.

Glycine Transporter 1 Encephalopathy From Biochemical Pathway to Clinical Disease: Review.

Glycine transporter 1 encephalopathy (OMIM# 617301; glycine encephalopathy with normal serum glycine, GLYT1 transporter dysfunction, and nonketotic hyperglycinemia) is caused by mutations in the SLC6A9 gene. To date, 6 cases have been reported in the literature, characterized as having neonatal onset, respiratory failure that required mechanical ventilation, severe hypotonia at birth that progressed to limb hypertonicity, and startle-like responses provoked by sudden loud noises and tactile stimulation. Additional characteristics included dysmorphic features, musculoskeletal abnormalities, and abnormal antenatal findings. Initial diagnosis include elevated levels of glycine in cerebrospinal fluid and an elevated cerebrospinal fluid to plasma glycine ratio. Abnormal magnetic resonance imaging findings included white matter abnormalities, thin corpus callosum, dilatation of the lateral and third ventricles, caudate atrophy, and tiny cysts. Patients reported so far showed normal electroencephalogram results. Treatment was supportive and appeared severe as 50% of the patients died between 2 days and 7 months of age, while surviving children had global developmental delay. In this report, we reviewed the published cases having glycine transporter 1 encephalopathy and retrospectively characterizing the disease phenotypes, affected biochemical pathways, neuroradiological abnormalities, diagnosis, genetic issues, and treatment; additionally, key discussion points are also presented.

Pharmacogenetic Analysis of Functional Glutamate System Gene Variants and Clinical Response to Clozapine.

Altered glutamate neurotransmission is implicated in the etiology of schizophrenia (SCZ) and the pharmacogenetics of response to clozapine (CLZ), which is the drug of choice for treatment-resistant SCZ. Response to antipsychotic therapy is highly variable, although twin studies suggest a genetic component. We investigated the association of 10 glutamate system gene variants with CLZ response using standard genotyping procedures. GRM2 (rs4067 and rs2518461), SLC1A2 (rs4354668, rs4534557, and rs2901534), SLC6A9 (rs12037805, rs1978195, and rs16831558), GRIA1 (rs2195450), and GAD1 (rs3749034) were typed in 163 European SCZ/schizoaffective disorder patients deemed resistant or intolerant to previous pharmacotherapy. Response was assessed following 6 months of CLZ monotherapy using change in Brief Psychiatric Rating Scale (BPRS) scores. Categorical and continuous response variables were analyzed using χ2 tests and analysis of covariance, respectively. We report no significant associations following correction for multiple testing. Prior to correction, nominally significant associations were observed for SLC6A9, SLC1A2, GRM2, and GRIA1. Most notably, CC homozygotes of rs16831558 located in the glycine transporter 1 gene (SLC6A9) exhibited an allele dose-dependent improvement in positive symptoms compared to T allele carriers (puncorrected = 0.008, pcorrected = 0.08). To clarify the role of SLC6A9 in clinical response to antipsychotic medication, and CLZ in particular, this finding warrants further investigation in larger well-characterized samples.

LncRNA-SLC6A9-5:2: A potent sensitizer in 131I-resistant papillary thyroid carcinoma with PARP-1 induction.

Recent studies have indicated that long non-coding RNAs play crucial roles in numerous cancers, including thyroid cancer, while their function in the mechanism of thyroid cancer 131I resistance has not been elucidated to date. The present study identified a functional long non-coding RNA, SLC6A9-5:2, which was involved in the radioactive therapy resistance of thyroid cancer. We demonstrated that SLC6A9-5:2 was remarkably downregulated in 131I-resistant thyroid cancer cell lines and 131I-insensitive patients and was positively correlated with Poly (ADP-ribose) polymerase 1 (PARP-1) expression and its activation. After downregulating SLC6A9 or blocking PARP-1 artificially, the sensitive thyroid cancer cells mostly displayed a tolerant phenotype under 131I exposure. Furthermore, SLC6A9-5:2 overexpression was positively correlated with PARP-1 mRNA and protein levels, which restored the sensitivity of resistant thyroid cancer cells. The present study further revealed that cancer cell death was primarily caused by ATP exhaustion in excessive DNA repair with high PARP-1 activity. In patients with thyroid cancer, a positive correlation between SLC6A9-5:2 and PARP-1 was identified, and low SLC6A9-5:2 expression was associated with a worse prognosis of papillary thyroid carcinoma. Hence, our data provide a new lncRNA-mediated regulatory mechanism implying that SLC6A9-5:2 can be used as a novel therapeutic target for 131I-resistant thyroid cancer.

Effects of the glycine reuptake inhibitors bitopertin and RG7118 on glycine in cerebrospinal fluid: results of two proofs of mechanism studies in healthy volunteers.

RATIONALE: Hypofunction of NMDA receptors has been implicated in neuropsychiatric disorders including schizophrenia. NMDA receptor neurotransmission can be enhanced through inhibition of glycine reuptake by the glycine transporter type 1 (GlyT1). OBJECTIVES: The primary objective of these studies was to explore the relationship between plasma exposure and glycine cerebrospinal fluid (CSF) concentrations following administration of bitopertin and RG7118 in healthy volunteers. METHODS: The bitopertin study comprised four dose levels (3, 10, 30 and 60 mg) administered once daily for 10 days. In the RG7118 study, placebo, 15 or 30 mg RG7118 was administered once daily for 28 days. CSF samples were taken on day -2 and day 10, and day -1 and day 26 for bitopertin and RG7118, respectively. RESULTS: Twenty-two and 24 subjects participated in the bitopertin and RG7118 study, respectively. In the bitopertin study, CSF glycine concentrations showed a dose-dependent increase from baseline to day 10. The geometric mean ratios (coefficient of variation) of AUC0-12 h on day 10 over baseline were 1.3 (17 %), 1.3 (49 %), 1.7 (18 %) and 2.3 (14 %) after 3, 10, 30 and 60 mg, respectively. In the RG7118 study, the geometric mean ratio of glycine concentration (CV) on day 26 at 6 h post-dose over time-matched baseline was approx. 1.9 (24 and 15 %) for 15 and 30 mg. CONCLUSIONS: The mechanism of action of bitopertin and RG7118, i.e. inhibition of glycine reuptake in the brain, was confirmed. The maximal increase observed in healthy volunteers was similar to the one observed in animals showing the good translatability of this biomarker.