GLRA1 Research Articles

Clinical and genetic analysis of three children with Hyperekplexia

To explore the clinical and genetic characteristics of three children with Hyperekplexia. Three children who were diagnosed with Hyperekplexia at the Third Affiliated Hospital of Zhengzhou University between June 2018 and March 2020 were selected as the study subjects. Clinical data of the three children were collected. All children were subjected to whole exome sequencing. Pathogenicity of candidate variants were verified by Sanger sequencing and bioinformatic analysis. The three children were all males, and had presented exaggerated startle reflexes and generalized stiffness in response to unexpected auditory or tactile stimulation, or had frequent traumatic falls following exaggerated startle. All children had shown positive nose-tapping reflex, though EEG and cranial MRI exams were all negative. Whole exome sequencing revealed that two children had harbored homozygous variants of the GLRB gene, of which the c.1017_c.1018insAG (p.G340Rfs*14) was unreported previously. The third child had harbored compound heterozygous variants of the GLRA1 gene, among which the c.1262T>A (p.IIe421Asn) variant showed an unreported autosomal recessive inheritance. All children had responded well to clonazepam treatment. Patients with Hyperekplexia have typical clinical manifestations. Early clinical identification and genetic analysis can facilitate their diagnosis.

Changes in ethanol effects in knock-in mice expressing ethanol insensitive alpha1 and alpha2 glycine receptor subunits

AimsGlycine receptors (GlyRs) are potentiated by physiologically relevant concentrations of ethanol, and mutations in the intracellular loop of α1 and α2 subunits reduced the effect of the drug. Knock-in (KI) mice having these individual mutations revealed that α1 and α2 subunits played a role in ethanol-induced sedation and ethanol intake. In this study, we wanted to examine if the effects of stacking both mutations in a 2xKI mouse model (α1/α2) generated by a selective breeding strategy further impacted cellular and behavioral responses to ethanol. Main methodsWe used electrophysiological recordings to examine ethanol's effect on GlyRs and evaluated ethanol-induced neuronal activation using c-Fos immunoreactivity and the genetically encoded calcium indicator GCaMP6s in the nucleus accumbens (nAc). We also examined ethanol-induced behavior using open field, loss of the righting response, and drinking in the dark (DID) paradigm. Key findingsEthanol did not potentiate GlyRs nor affect neuronal excitability in the nAc from 2xKI. Moreover, ethanol decreased the Ca2+ signal in WT mice, whereas there were no changes in the signal in 2xKI mice. Interestingly, there was an increase in c-Fos baseline in the 2xKI mice in the absence of ethanol. Behavioral assays showed that 2xKI mice recovered faster from a sedative dose of ethanol and had higher ethanol intake on the first test day of the DID test than WT mice. Interestingly, an open-field assay showed that 2xKI mice displayed less anxiety-like behavior than WT mice. SignificanceThe results indicate that α1 and α2 subunits are biologically relevant targets for regulating sedative effects and ethanol consumption.

Spinal Glycine Receptor Alpha 3 Cells Communicate Sensations of Chemical Itch in Hairy Skin.

Glycinergic neurons regulate nociceptive and pruriceptive signaling in the spinal cord, but the identity and role of the glycine-regulated neurons are not fully known. Herein, we have characterized spinal glycine receptor alpha 3 (Glra3) subunit-expressing neurons in Glra3-Cre female and male mice. Glra3-Cre(+) neurons express Glra3, are located mainly in laminae III-VI, and respond to glycine. Chemogenetic activation of spinal Glra3-Cre(+) neurons induced biting/licking, stomping, and guarding behaviors, indicative of both a nociceptive and pruriceptive role for this population. Chemogenetic inhibition did not affect mechanical or thermal responses but reduced behaviors evoked by compound 48/80 and chloroquine, revealing a pruriceptive role for these neurons. Spinal cells activated by compound 48/80 or chloroquine express Glra3, further supporting the phenotype. Retrograde tracing revealed that spinal Glra3-Cre(+) neurons receive input from afferents associated with pain and itch, and dorsal root stimulation validated the monosynaptic input. In conclusion, these results show that spinal Glra3(+) neurons contribute to acute communication of compound 48/80- and chloroquine-induced itch in hairy skin.

Dopamine-mediated striatal activity and function is enhanced in GlyRα2 knockout animals

The glycine receptor alpha 2 (GlyRα2) is a ligand-gated ion channel which upon activation induces a chloride conductance. Here, we investigated the role of GlyRα2 in dopamine-stimulated striatal cell activity and behavior. We show that depletion of GlyRα2 enhances dopamine-induced increases in the activity of putative dopamine D1 receptor-expressing striatal projection neurons, but does not alter midbrain dopamine neuron activity. We next show that the locomotor response to d-amphetamine is enhanced in GlyRα2 knockout animals, and that this increase correlates with c-fos expression in the dorsal striatum. 3-D modeling revealed an increase in the neuronal ensemble size in the striatum in response to D-amphetamine in GlyRα2 KO mice. Finally, we show enhanced appetitive conditioning in GlyRα2 KO animals that is likely due to increased motivation, but not changes in associative learning or hedonic response. Taken together, we show that GlyRα2 is an important regulator of dopamine-stimulated striatal activity and function.

The glycine receptor alpha 3 subunit mRNA expression shows sex-dependent differences in the adult mouse brain

BackgroundThe glycinergic system plays an important inhibitory role in the mouse central nervous system, where glycine controls the excitability of spinal itch- and pain-mediating neurons. Impairments of the glycine receptors can cause motor and sensory deficits. Glycine exerts inhibition through interaction with ligand-gated ion channels composed of alpha and beta subunits. We have investigated the mRNA expression of the glycine receptor alpha 3 (Glra3) subunit in the nervous system as well as in several peripheral organs of female and male mice.ResultsSingle-cell RNA sequencing (scRNA-seq) data analysis on the Zeisel et al. (2018) dataset indicated widespread but low expression of Glra3 in vesicular glutamate transporter 2 (Vglut2, Slc17a6) positive and vesicular inhibitory amino acid transporter (Viaat, Slc32a1)positive neurons of the mouse central nervous system. Highest occurrence of Glra3 expression was identified in the cortex, amygdala, and striatal regions, as well as in the hypothalamus, brainstem and spinal cord. Bulk quantitative real-time-PCR (qRT-PCR) analysis demonstrated Glra3 expression in cortex, amygdala, striatum, hypothalamus, thalamus, pituitary gland, hippocampus, cerebellum, brainstem, and spinal cord. Additionally, male mice expressed higher levels of Glra3 in all investigated brain areas compared with female mice. Lastly, RNAscope spatially validated Glra3 expression in the areas indicated by the single-cell and bulk analyses. Moreover, RNAscope analysis confirmed co-localization of Glra3 with Slc17a6 or Slc32a1 in the central nervous system areas suggested from the single-cell data.ConclusionsGlra3 expression is low but widespread in the mouse central nervous system. Clear sex-dependent differences have been identified, indicating higher levels of Glra3 in several telencephalic and diencephalic areas, as well as in cerebellum and brainstem, in male mice compared with female mice.

A loss-of-function variant in canine GLRA1 associates with a neurological disorder resembling human hyperekplexia

Hereditary hyperekplexia is a rare neuronal disorder characterized by an exaggerated startle response to sudden tactile or acoustic stimuli. In this study, we present a Miniature Australian Shepherd family showing clinical signs, which have genetic and phenotypic similarities with human hereditary hyperekplexia: episodes of muscle stiffness that could occasionally be triggered by acoustic stimuli. Whole genome sequence data analysis of two affected dogs revealed a 36-bp deletion spanning the exon–intron boundary in the glycine receptor alpha 1 (GLRA1) gene. Further validation in pedigree samples and an additional cohort of 127 Miniature Australian Shepherds, 45 Miniature American Shepherds and 74 Australian Shepherds demonstrated complete segregation of the variant with the disease, according to an autosomal recessive inheritance pattern. The protein encoded by GLRA1 is a subunit of the glycine receptor, which mediates postsynaptic inhibition in the brain stem and spinal cord. The canine GLRA1 deletion is located in the signal peptide and is predicted to cause exon skipping and subsequent premature stop codon resulting in a significant defect in glycine signaling. Variants in GLRA1 are known to cause hereditary hyperekplexia in humans; however, this is the first study to associate a variant in canine GLRA1 with the disorder, establishing a spontaneous large animal disease model for the human condition.

Comprehensive behavioral analyses of mice with a glycine receptor alpha 4 deficiency

Glycine receptors (GlyRs) are ligand-gated chloride channels comprising alpha (α1–4) and β subunits. The GlyR subunits play major roles in the mammalian central nervous system, ranging from regulating simple sensory information to modulating higher-order brain function. Unlike the other GlyR subunits, GlyR α4 receives relatively little attention because the human ortholog lacks a transmembrane domain and is thus considered a pseudogene. A recent genetic study reported that the GLRA4 pseudogene locus on the X chromosome is potentially involved in cognitive impairment, motor delay and craniofacial anomalies in humans. The physiologic roles of GlyR α4 in mammal behavior and its involvement in disease, however, are not known. Here we examined the temporal and spatial expression profile of GlyR α4 in the mouse brain and subjected Glra4 mutant mice to a comprehensive behavioral analysis to elucidate the role of GlyR α4 in behavior. The GlyR α4 subunit was mainly enriched in the hindbrain and midbrain, and had relatively lower expression in the thalamus, cerebellum, hypothalamus, and olfactory bulb. In addition, expression of the GlyR α4 subunit gradually increased during brain development. Glra4 mutant mice exhibited a decreased amplitude and delayed onset of the startle response compared with wild-type littermates, and increased social interaction in the home cage during the dark period. Glra4 mutants also had a low percentage of entries into open arms in the elevated plus-maze test. Although mice with GlyR α4 deficiency did not show motor and learning abnormalities reported to be associated in human genomics studies, they exhibited behavioral changes in startle response and social and anxiety-like behavior. Our data clarify the spatiotemporal expression pattern of the GlyR α4 subunit and suggest that glycinergic signaling modulates social, startle, and anxiety-like behaviors in mice.

Structural basis for cannabinoid-induced potentiation of alpha1-glycine receptors in lipid nanodiscs.

Nociception and motor coordination are critically governed by glycine receptor (GlyR) function at inhibitory synapses. Consequentially, GlyRs are attractive targets in the management of chronic pain and in the treatment of several neurological disorders. High-resolution mechanistic details of GlyR function and its modulation are just emerging. While it has been known that cannabinoids such as Δ9-tetrahydrocannabinol (THC), the principal psychoactive constituent in marijuana potentiate GlyR in the therapeutically relevant concentration range, the molecular mechanism underlying this effect is still not understood. Here, we present Cryo-EM structures of full-length GlyR reconstituted into lipid nanodisc in complex with THC under varying concentrations of glycine. The GlyR-THC complexes are captured in multiple conformational states that reveal the basis for THC-mediated potentiation, manifested as different extents of opening at the level of the channel pore. Taken together, these structural findings, combined with molecular dynamics simulations and functional analysis, provide new insights into the potential THC binding site and the allosteric coupling to the channel pore.

Four Turkish families with hyperekplexia: A missense mutation and the exon 1–7 deletion in the GLRA1 gene

BackgroundHyperekplexia is a disease that progresses with excessive startle attacks and is included in the differential diagnosis of epilepsy and many movement disorders. MethodsThe WES results were validated in available family members by Sanger sequencing, or in the case of deletion, PCR followed by agarose gel electrophoresis was performed. ResultsWES analysis revealed the previously reported homozygous c.277C>T p.Arg93Trp variant in the GLRA1 gene (ENST00000455880.2) in Family 1. In all other three families, the previously reported homozygous deletion of exons 1–7 of the GLRA1 gene was identified using CNV analysis based on the WES data. ConclusionsThe homozygous exon1-7 deletion has been described several times in different populations and may be a founder mutation in the Kurdish people in Turkey. The family with Arg93Trp variant stems from the Black Sea region of Turkey where close consanguinity is common. These analyses are important to provide genetic counseling to families and for a better understanding of the pathophysiology of the disease.

Structural basis for cannabinoid-induced potentiation of alpha1-glycine receptors in lipid nanodiscs

Nociception and motor coordination are critically governed by glycine receptor (GlyR) function at inhibitory synapses. Consequentially, GlyRs are attractive targets in the management of chronic pain and in the treatment of several neurological disorders. High-resolution mechanistic details of GlyR function and its modulation are just emerging. While it has been known that cannabinoids such as Δ9-tetrahydrocannabinol (THC), the principal psychoactive constituent in marijuana, potentiate GlyR in the therapeutically relevant concentration range, the molecular mechanism underlying this effect is still not understood. Here, we present Cryo-EM structures of full-length GlyR reconstituted into lipid nanodisc in complex with THC under varying concentrations of glycine. The GlyR-THC complexes are captured in multiple conformational states that reveal the basis for THC-mediated potentiation, manifested as different extents of opening at the level of the channel pore. Taken together, these structural findings, combined with molecular dynamics simulations and functional analysis, provide insights into the potential THC binding site and the allosteric coupling to the channel pore.

Modulation of Ligand-Gated Glycine Receptors Via Functional Monoclonal Antibodies.

Ion channels are targets of considerable therapeutic interest to address a wide variety of neurologic indications, including pain perception. Current pharmacological strategies have focused mostly on small molecule approaches that can be limited by selectivity requirements within members of a channel family or superfamily. Therapeutic antibodies have been proposed, designed, and characterized to alleviate this selectivity limitation; however, there are no Food and Drug Administration-approved therapeutic antibody-based drugs targeting ion channels on the market to date. Here, in an effort to identify novel classes of engineered ion channel modulators for potential neurologic therapeutic applications, we report the generation and characterization of six (EC50 < 25nM) Cys-loop receptor family monoclonal antibodies with modulatory function against rat and human glycine receptor alpha 1 (GlyRα1) and/or GlyRα3. These antibodies have activating (i.e., positive modulator) or inhibiting (i.e., negative modulator) profiles. Moreover, GlyRα3 selectivity was successfully achieved for two of the three positive modulators identified. When dosed intravenously, the antibodies achieved sufficient brain exposure to cover their calculated in vitro EC50 values. When compared head-to-head at identical exposures, the GlyRα3-selective antibody showed a more desirable safety profile over the nonselective antibody, thus demonstrating, for the first time, an advantage for GlyRα3-selectivity. Our data show that ligand-gated ion channels of the glycine receptor family within the central nervous system can be functionally modulated by engineered biologics in a dose-dependent manner and that, despite high protein homology between the alpha subunits, selectivity can be achieved within this receptor family, resulting in future therapeutic candidates with more desirable drug safety profiles. SIGNIFICANCE STATEMENT: This study presents immunization and multiplatform screening approaches to generate a diverse library of functional antibodies (agonist, potentiator, or inhibitory) raised against human glycine receptors (GlyRs). This study also demonstrates the feasibility of acquiring alpha subunit selectivity, a desirable therapeutic profile. When tested in vivo, these tool molecules demonstrated an increased safety profile in favor of GlyRα3-selectivity. These are the first reported functional GlyR antibodies that may open new avenues to treating central nervous system diseases with subunit selective biologics.

Novel GLRA1 Variants Causing Neonatal Arthrogryposis and Developmental Delay in Three Unrelated Cases. Early Signs of Autosomal Dominant Hyperekplexia

Hereditary hyperekplexia (HPX) is an inherited neuronal disorder characterized by exaggerated startle responses and stiffness to unexpected stimuli. In the newborn period, the phenotype may present as decreased range of motion of all joints, neonatal arthrogryposis or generalized neonatal hypertonia [1]. HPX is associated with three main genes: GLRA1, GLRB, and SLC6A5 [2]. We report three unrelated families with variants in the GLRA1 gene that expand the understanding of the clinical presentation of affected individuals with GLRA1 variants. No variants observed have been previously reported, two of them are familial and one a de-novo change. One of the families presents three generations of affected individuals with neonatal joint contractures. All three probands in this report present with significant developmental delay, speech delay, feeding difficulties, and motor delay. Hearing loss was seen in one of the patients. All patients appear to present with central hypotonia and two of them present with central hypotonia and peripheral hypertonia with hyperreflexia. Currently it has been suggested that GLRA1 dysfunction results in failure of inhibition of potent neurotransmitters in the central nervous system [3]. This may explain the intrauterine signs and perinatal presentation of neuromuscular dysfunction in the reported patients. We believe that the information presented here helps to understand the natural history of hyperekplexia. Early recognition of this rare disorder is important due to the high risk of sudden infant death in children due to apnea and the potential to implement rescue treatments. Based on these cases, patients with genetic variants in the GLRA1 gene may present with congenital arthrogryposis that improves with time and results in a milder form of distal joint contractures in adulthood. Patients with hyperekplexia may have a better prognosis than other forms of arthrogryposis and we suggest that GLRA1 gene testing should be included in neonatal arthrogryposis panels.

Identify the conformational states of glycine receptor alpha3 through umbrella sampling

Ligand-gated ion channels allows cells to respond rapidly to changes in their external environment. The structure change from one state to other states is the key to understand how ion channels function. In many cases due to the limitation in experiment, only some state structures, a closed, open state, or desensitized state, are available. A reliable method is needed to derive the structures of the missing state structures. Even in cases structures of all states are available, it is desired to understand how conformation changes during state transition.

Structural Investigation into Gating and Modulation of Alpha1 Glycine Receptor in Lipid Nanodiscs

Glycine receptors (GlyR) mediate fast chemical transmission of synaptic signals in the central and peripheral nervous system. GlyR are anionic members of pentameric ligand gated ion channels and are central players in motor coordination and pain perception. Potentiation of GlyR activity results in dampening pain signals in the spinal cord making these receptors an attractive target for pain therapy. Here, we present Cryo-EM structures of the full-length alpha1 GlyR reconstituted into lipid nanodiscs that are captured in multiple functional states that include, the unliganded state (closed) and glycine-bound conformations (open and desensitized). in addition, we present conformations of GlyR in various ligand-bound forms that provide insights into the mechanisms underlying allosteric modulation. The functional state assignments were validated by molecular dynamics simulations. A comparison of these states reveals global conformational changes underlying GlyR channel gating and modulation.

Sex and Menopause Modify the Effect of Single Nucleotide Polymorphism Genotypes on Fibrosis in NAFLD.

The development of fibrosis in nonalcoholic fatty liver disease (NAFLD) is influenced by genetics, sex, and menopausal status, but whether genetic susceptibility to fibrosis is influenced by sex and reproductive status is unclear. Our aim was to identify metabolism‐related single nucleotide polymorphisms (SNPs), whose effect on NAFLD fibrosis is significantly modified by sex and menopausal status. We performed a cross‐sectional, proof‐of‐concept study of 616 patients in the Duke NAFLD Clinical Database and Biorepository. The primary outcome was nonalcoholic steatohepatitis–Clinical Research Network (NASH–CRN) fibrosis stage. Menopause status was self‐reported; age 51 years was used as a surrogate for menopause in patients with missing menopause data. The Metabochip was used to obtain 98,359 SNP genotypes in known metabolic pathway genes for each patient. We used additive genetic models to characterize sex and menopause‐specific effects of SNP genotypes on NAFLD fibrosis stage. In the main effects analysis, none of the SNPs were associated with fibrosis at P < 0.05 after correcting for multiple comparisons. Twenty‐five SNPs significantly interacted with sex/menopause to affect fibrosis stage (interaction P < 0.0001). After removal of loci in linkage disequilibrium, 10 independent loci were identified. Six were in the following genes: KCNIP4 (potassium voltage‐gated channel interacting protein 4), PSORS1C1 (psoriasis susceptibility 1 candidate 1), KLHL8 (Kelch‐like family member 8), GLRA1 (glycine receptor alpha 1), NOTCH2 (notch receptor 2), and PRKCH (protein kinase C eta), and four SNPs were intergenic. In stratified models, four SNPs were significant in premenopausal and postmenopausal women, three only in postmenopausal women, two in men and postmenopausal women, and one only in premenopausal women. Conclusion: We identified 10 loci with a significant sex/menopause interaction with respect to fibrosis. None of these SNPs were significant in all sex/menopause groups, suggesting modulation of genetic susceptibility to fibrosis by sex and menopause status. Future studies of genetic predictors of NAFLD progression should account for sex and menopause.

Transcriptomic analysis of the liver in aged laying hens with different intensity of brown eggshell color.

ObjectiveEggshell color is an important indicator of egg quality for consumers, especially for brown eggs. Various factors related to laying hens and their environment affect brown eggshell coloration. However, there have been no studies investigating hepatic functions of laying hens with variable intensity of brown eggshell color. Therefore, this study was aimed to identify potential factors affecting brown eggshell coloration in aged laying hens at the hepatic transcriptomic level.MethodsFive hundred 92-wk-old Hy-line Brown laying hens were screened to select laying hens with different intensity of brown eggshell color based on eggshell color fans. Based on eggshell color scores, hens with dark brown eggshells (DBE; eggshell color fan score = 14.8) and hens with light brown eggshells (LBE; eggshell color fan score = 9.7) were finally selected for the liver sampling. We performed RNA-seq analysis using the liver samples through the paired-end sequencing libraries. Differentially expressed genes (DEGs) profiling was carried out to identify their biological meaning by bioinformatics.ResultsA total of 290 DEGs were identified with 196 being up-regulated and 94 being down-regulated in DBE groups as compared to LBE groups. The Kyoto encyclopedia of genes and genomes (KEGG) analysis revealed that these DEGs belong to several biological pathways including herpes simplex infection (toll-like receptor 3 [TLR3], cyclin-dependent kinase 1, etc.) and influenza A (TLR3, radical S-adenosyl methionine domain containing 2, myxovirus [influenza virus] resistance 1, etc.). Genes related to stress response (ceremide kinase like) and nutrient metabolism (phosphoenolpyruvate carboxy-kinase 1, methylmalonic aciduria [cobalamin deficiency] cblB type, glycine receptor alpha 2, solute carrier family 7 member 11, etc.) were also identified to be differentially expressed.ConclusionThe current results provide new insights regarding hepatic molecular functions related to different intensity of brown eggshell color in aged laying hens. These insights will contribute to future studies aiming to optimize brown eggshell coloration in aged laying hens.

Anxiety and Startle Phenotypes in Glrb Spastic and Glra1 Spasmodic Mouse Mutants.

A GWAS study recently demonstrated single nucleotide polymorphisms (SNPs) in the human GLRB gene of individuals with a prevalence for agoraphobia. GLRB encodes the glycine receptor (GlyRs) β subunit. The identified SNPs are localized within the gene flanking regions (3′ and 5′ UTRs) and intronic regions. It was suggested that these nucleotide polymorphisms modify GlyRs expression and phenotypic behavior in humans contributing to an anxiety phenotype as a mild form of hyperekplexia. Hyperekplexia is a human neuromotor disorder with massive startle phenotypes due to mutations in genes encoding GlyRs subunits. GLRA1 mutations have been more commonly observed than GLRB mutations. If an anxiety phenotype contributes to the hyperekplexia disease pattern has not been investigated yet. Here, we compared two mouse models harboring either a mutation in the murine Glra1 or Glrb gene with regard to anxiety and startle phenotypes. Homozygous spasmodic animals carrying a Glra1 point mutation (alanine 52 to serine) displayed abnormally enhanced startle responses. Moreover, spasmodic mice exhibited significant changes in fear-related behaviors (freezing, rearing and time spent on back) analyzed during the startle paradigm, even in a neutral context. Spastic mice exhibit reduced expression levels of the full-length GlyRs β subunit due to aberrant splicing of the Glrb gene. Heterozygous animals appear normal without an obvious behavioral phenotype and thus might reflect the human situation analyzed in the GWAS study on agoraphobia and startle. In contrast to spasmodic mice, heterozygous spastic animals revealed no startle phenotype in a neutral as well as a conditioning context. Other mechanisms such as a modulatory function of the GlyRs β subunit within glycinergic circuits in neuronal networks important for fear and fear-related behavior may exist. Possibly, in human additional changes in fear and fear-related circuits either due to gene-gene interactions e.g., with GLRA1 genes or epigenetic factors are necessary to create the agoraphobia and in particular the startle phenotype.

Inhibition of the Glycine Receptor alpha 3 Function by Colchicine.

Colchicine is a plant alkaloid that is widely used as a therapeutic agent. It is widely accepted that colchicine reduces the production of inflammatory mediators mainly by altering cytoskeleton dynamics due to its microtubule polymerization inhibitory activity. However, other lines of evidence have shown that colchicine exerts direct actions on the function of ion channels, which are independent of cytoskeleton alterations. Colchicine is able to modify the function of several pentameric ligand-gated ion channels, including glycine receptors (GlyRs). Previous electrophysiological studies have shown that colchicine act as an antagonist of GlyRs composed by the α 1 subunit. In addition, it was recently demonstrated that colchicine directly bind to the α 3 subunit of GlyRs. Interestingly, other studies have shown a main role of α 3GlyRs on chronic inflammatory pain. Nevertheless, the functional effects of colchicine on the α 3GlyR function are still unknown. Here, by using electrophysiological techniques and bioinformatics, we show that colchicine inhibited the function of the α 3GlyRs. Colchicine elicited concentration-dependent inhibitory effects on α 3GlyRs at micromolar range and decreased the apparent affinity for glycine. Single-channel recordings show that the colchicine inhibition is associated with a decrease in the open probability of the ion channel. Molecular docking assays suggest that colchicine preferentially bind to the orthosteric site in the closed state of the ion channel. Altogether, our results suggest that colchicine is a competitive antagonist of the α 3GlyRs.

Excessive Startle with Novel GLRA1 Mutations in 4 Chinese Patients and a Literature Review of GLRA1-Related Hyperekplexia.

Background and PurposeHyperekplexia (HPX), a rare neurogenetic disorder, is classically characterized by neonatal hypertonia, exaggerated startle response provoked by the sudden external stimuli and followed by a shortly general stiffness. Glycine receptor alpha 1 (GLRA1) is the major pathogenic gene of the disease. We described the clinical manifestations of genetically confirmed HPX patients and made a literature review of GLRA1-related HPX to improve the early recognition and prompt the management of the disorder.MethodsExtensive clinical evaluations were analyzed in 4 Chinese HPX patients from two unrelated families. Next generation sequencing was conducted in the probands. Sanger sequence and segregation analysis were applied to confirm the findings.ResultsAll four patients including 3 males and 1 female presented with excessive startle reflex, a cautious gait and recurrent falls. Moreover, startle episodes were dramatically improved with the treatment of clonazepam in all cases. Exome sequencing revealed 2 homozygous GLRA1 mutations in the patients. The mutation c.1286T>A p.I429N has been previously reported, while c.754delC p.L252* is novel.ConclusionsHPX is a treatable disease, and clonazepam is the drug of choice. By studying and reviewing the disorder, we summarized the phenotype, expanded the genotype spectrum, and discussed the possible pathogenic mechanisms to enhance the understanding and recognition of the disease. Early awareness of the disease is crucial to the prompt and proper administration, as well as the genetic counseling.

Individual knock out of glycine receptor alpha subunits identifies a specific requirement of glra1 for motor function in zebrafish.

Glycine receptors (GlyRs) are ligand-gated chloride channels mediating inhibitory neurotransmission in the brain stem and spinal cord. They function as pentamers composed of alpha and beta subunits for which 5 genes have been identified in human (GLRA1, GLRA2, GLRA3, GLRA4, GLRB). Several in vitro studies showed that the pentameric subtype composition as well as its stoichiometry influence the distribution and the molecular function of the receptor. Moreover, mutations in some of these genes are involved in different human conditions ranging from tinnitus to epilepsy and hyperekplexia, suggesting distinct functions of the different subunits. Although the beta subunit is essential for synaptic clustering of the receptor, the specific role of each alpha subtype is still puzzling in vivo. The zebrafish genome encodes for five glycine receptor alpha subunits (glra1, glra2, glra3, glra4a, glra4b) thus offering a model of choice to investigate the respective role of each subtype on general motor behaviour. After establishing a phylogeny of GlyR subunit evolution between human and zebrafish, we checked the temporal expression pattern of these transcripts during embryo development. Interestingly, we found that glra1 is the only maternally transmitted alpha subunit. We also showed that the expression of the different GlyR subunits starts at different time points during development. Lastly, in order to decipher the role of each alpha subunit on the general motor behaviour of the fish, we knocked out individually each alpha subunit by CRISPR/Cas9-targeted mutagenesis. Surprisingly, we found that knocking out any of the alpha2, 3, a4a or a4b subunit did not lead to any obvious developmental or motor phenotype. However, glra1-/- (hitch) embryos depicted a strong motor dysfunction from 3 days, making them incapable to swim and thus leading to their premature death. Our results infer a strong functional redundancy between alpha subunits and confirm the central role played by glra1 for proper inhibitory neurotransmission controlling locomotion. The genetic tools we developed here will be of general interest for further studies aiming at dissecting the role of GlyRs in glycinergic transmission in vivo and the hitch mutant (hic) is of specific relevance as a new model of hyperekplexia.