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1.  The Glycinergic System in Human Startle Disease: A Genetic Screening Approach 
Human startle disease, also known as hyperekplexia (OMIM 149400), is a paroxysmal neurological disorder caused by defects in glycinergic neurotransmission. Hyperekplexia is characterised by an exaggerated startle reflex in response to tactile or acoustic stimuli which first presents as neonatal hypertonia, followed in some with episodes of life-threatening infantile apnoea. Genetic screening studies have demonstrated that hyperekplexia is genetically heterogeneous with several missense and nonsense mutations in the postsynaptic glycine receptor (GlyR) α1 subunit gene (GLRA1) as the primary cause. More recently, missense, nonsense and frameshift mutations have also been identified in the glycine transporter GlyT2 gene, SLC6A5, demonstrating a presynaptic component to this disease. Further mutations, albeit rare, have been identified in the genes encoding the GlyR β subunit (GLRB), collybistin (ARHGEF9) and gephyrin (GPHN) – all of which are postsynaptic proteins involved in orchestrating glycinergic neurotransmission. In this review, we describe the clinical ascertainment aspects, phenotypic considerations and the downstream molecular genetic tools utilised to analyse both presynaptic and postsynaptic components of this heterogeneous human neurological disorder. Moreover, we will describe how the ancient startle response is the preserve of glycinergic neurotransmission and how animal models and human hyperekplexia patients have provided synergistic evidence that implicates this inhibitory system in the control of startle reflexes.
doi:10.3389/fnmol.2010.00008
PMCID: PMC2854534  PMID: 20407582
glycine; hyperekplexia; receptor; transporter; mutation
2.  A Critical Role for Glycine Transporters in Hyperexcitability Disorders 
Defects in mammalian glycinergic neurotransmission result in a complex motor disorder characterized by neonatal hypertonia and an exaggerated startle reflex, known as hyperekplexia (OMIM 149400). This affects newborn children and is characterized by noise or touch-induced seizures that result in muscle stiffness and breath-holding episodes. Although rare, this disorder can have serious consequences, including brain damage and/or sudden infant death. The primary cause of hyperekplexia is missense and non-sense mutations in the glycine receptor (GlyR) α1 subunit gene (GLRA1) on chromosome 5q33.1, although we have also discovered rare mutations in the genes encoding the GlyR β subunit (GLRB) and the GlyR clustering proteins gephyrin (GPNH) and collybistin (ARHGEF9). Recent studies of the Na+/Cl−-dependent glycine transporters GlyT1 and GlyT2 using mouse knockout models and human genetics have revealed that mutations in GlyT2 are a second major cause of hyperekplexia, while the phenotype of the GlyT1 knockout mouse resembles a devastating neurological disorder known as glycine encephalopathy (OMIM 605899). These findings highlight the importance of these transporters in regulating the levels of synaptic glycine.
doi:10.3389/neuro.02.001.2008
PMCID: PMC2526004  PMID: 18946534
glycine transporters; GlyT1; GlyT2; VIAAT; hyperekplexia; startle disease; glycine encephalopathy
3.  Mutations in the human GlyT2 gene define a presynaptic component of human startle disease 
Nature genetics  2006;38(7):801-806.
Hyperekplexia is a human neurological disorder characterized by an excessive startle response and is typically caused by missense and nonsense mutations in the gene encoding the inhibitory glycine receptor (GlyR) α1 subunit (GLRA1)1-3. Genetic heterogeneity has been confirmed in isolated sporadic cases with mutations in other postsynaptic glycinergic proteins including the GlyR β subunit (GLRB)4, gephyrin (GPHN)5 and RhoGEF collybistin (ARHGEF9)6. However, many sporadic patients diagnosed with hyperekplexia do not carry mutations in these genes2-7. Here we reveal that missense, nonsense and frameshift mutations in the presynaptic glycine transporter 2 (GlyT2) gene (SLC6A5)8 also cause hyperekplexia. Patients harbouring mutations in SLC6A5 presented with hypertonia, an exaggerated startle response to tactile or acoustic stimuli, and life-threatening neonatal apnoea episodes. GlyT2 mutations result in defective subcellular localisation and/or decreased glycine uptake, with selected mutations affecting predicted glycine and Na+ binding sites. Our results demonstrate that SLC6A5 is a major gene for hyperekplexia and define the first neurological disorder linked to mutations in a Na+/Cl−-dependent transporter for a classical fast neurotransmitter. By analogy, we suggest that in other human disorders where defects in postsynaptic receptors have been identified, similar symptoms could result from defects in the cognate presynaptic neurotransmitter transporter.
doi:10.1038/ng1814
PMCID: PMC3204411  PMID: 16751771
4.  Constitutive Endocytosis and Turnover of the Neuronal Glycine Transporter GlyT2 Is Dependent on Ubiquitination of a C-Terminal Lysine Cluster 
PLoS ONE  2013;8(3):e58863.
Inhibitory glycinergic neurotransmission is terminated by sodium and chloride-dependent plasma membrane glycine transporters (GlyTs). The mainly glial glycine transporter GlyT1 is primarily responsible for the completion of inhibitory neurotransmission and the neuronal glycine transporter GlyT2 mediates the reuptake of the neurotransmitter that is used to refill synaptic vesicles in the terminal, a fundamental role in the physiology and pathology of glycinergic neurotransmission. Indeed, inhibitory glycinergic neurotransmission is modulated by the exocytosis and endocytosis of GlyT2. We previously reported that constitutive and Protein Kinase C (PKC)-regulated endocytosis of GlyT2 is mediated by clathrin and that PKC accelerates GlyT2 endocytosis by increasing its ubiquitination. However, the role of ubiquitination in the constitutive endocytosis and turnover of this protein remains unexplored. Here, we show that ubiquitination of a C-terminus four lysine cluster of GlyT2 is required for constitutive endocytosis, sorting into the slow recycling pathway and turnover of the transporter. Ubiquitination negatively modulates the turnover of GlyT2, such that increased ubiquitination driven by PKC activation accelerates transporter degradation rate shortening its half-life while decreased ubiquitination increases transporter stability. Finally, ubiquitination of GlyT2 in neurons is highly responsive to the free pool of ubiquitin, suggesting that the deubiquitinating enzyme (DUB) ubiquitin C-terminal hydrolase-L1 (UCHL1), as the major regulator of neuronal ubiquitin homeostasis, indirectly modulates the turnover of GlyT2. Our results contribute to the elucidation of the mechanisms underlying the dynamic trafficking of this important neuronal protein which has pathological relevance since mutations in the GlyT2 gene (SLC6A5) are the second most common cause of human hyperekplexia.
doi:10.1371/journal.pone.0058863
PMCID: PMC3590132  PMID: 23484054
5.  Defective Glycinergic Synaptic Transmission in Zebrafish Motility Mutants 
Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Recently, in vivo analysis of glycinergic synaptic transmission has been pursued in zebrafish using molecular genetics. An ENU mutagenesis screen identified two behavioral mutants that are defective in glycinergic synaptic transmission. Zebrafish bandoneon (beo) mutants have a defect in glrbb, one of the duplicated glycine receptor (GlyR) β subunit genes. These mutants exhibit a loss of glycinergic synaptic transmission due to a lack of synaptic aggregation of GlyRs. Due to the consequent loss of reciprocal inhibition of motor circuits between the two sides of the spinal cord, motor neurons activate simultaneously on both sides resulting in bilateral contraction of axial muscles of beo mutants, eliciting the so-called ‘accordion’ phenotype. Similar defects in GlyR subunit genes have been observed in several mammals and are the basis for human hyperekplexia/startle disease. By contrast, zebrafish shocked (sho) mutants have a defect in slc6a9, encoding GlyT1, a glycine transporter that is expressed by astroglial cells surrounding the glycinergic synapse in the hindbrain and spinal cord. GlyT1 mediates rapid uptake of glycine from the synaptic cleft, terminating synaptic transmission. In zebrafish sho mutants, there appears to be elevated extracellular glycine resulting in persistent inhibition of postsynaptic neurons and subsequent reduced motility, causing the ‘twitch-once’ phenotype. We review current knowledge regarding zebrafish ‘accordion’ and ‘twitch-once’ mutants, including beo and sho, and report the identification of a new α2 subunit that revises the phylogeny of zebrafish GlyRs.
doi:10.3389/neuro.02.026.2009
PMCID: PMC2813725  PMID: 20161699
glycine; synapse; receptor; transporter; zebrafish; behavior; motility
6.  A MusD Retrotransposon Insertion in the Mouse Slc6a5 Gene Causes Alterations in Neuromuscular Junction Maturation and Behavioral Phenotypes 
PLoS ONE  2012;7(1):e30217.
Glycine is the major inhibitory neurotransmitter in the spinal cord and some brain regions. The presynaptic glycine transporter, GlyT2, is required for sustained glycinergic transmission through presynaptic reuptake and recycling of glycine. Mutations in SLC6A5, encoding GlyT2, cause hereditary hyperekplexia in humans, and similar phenotypes in knock-out mice, and variants are associated with schizophrenia. We identified a spontaneous mutation in mouse Slc6a5, caused by a MusD retrotransposon insertion. The GlyT2 protein is undetectable in homozygous mutants, indicating a null allele. Homozygous mutant mice are normal at birth, but develop handling-induced spasms at five days of age, and only survive for two weeks, but allow the study of early activity-regulated developmental processes. At the neuromuscular junction, synapse elimination and the switch from embryonic to adult acetylcholine receptor subunits are hastened, consistent with a presumed increase in motor neuron activity, and transcription of acetylcholine receptors is elevated. Heterozygous mice, which show no reduction in lifespan but nonetheless have reduced levels of GlyT2, have a normal thermal sensitivity with the hot-plate test, but differences in repetitive grooming and decreased sleep time with home-cage monitoring. Open-field and elevated plus-maze tests did not detect anxiety-like behaviors; however, the latter showed a hyperactivity phenotype. Importantly, grooming and hyperactivity are observed in mouse schizophrenia models. Thus, mutations in Slc6a5 show changes in neuromuscular junction development as homozygotes, and behavioral phenotypes as heterozygotes, indicating their usefulness for studies related to glycinergic dysfunction.
doi:10.1371/journal.pone.0030217
PMCID: PMC3260239  PMID: 22272310
7.  Identification of a novel missense GLRA1 gene mutation in hyperekplexia: a case report 
Introduction
Hereditary hyperekplexia is a neurological disorder characterized by excessive startle responses with violent jerking to noise or touch, stiffening of the trunk and limbs, clenching of the fists and attacks of a high-frequency trembling. Hyperekplexia has a heterogeneous genetic background with several identified causative genes and demonstrates both dominant and recessive inheritance. Mutations in the glycine receptor alpha 1 subunit gene occur in about 30 percent of hyperekplexia cases.
Case presentation
In this study, we report the case of a Hungarian boy whose abnormal movements, muscle stiffness and convulsions were first noted when he was 4 days old. Neurological and electrophysiological investigation suggested the clinical diagnosis of hyperekplexia.
Conclusions
Direct sequencing of the coding regions and the flanking introns of the glycine receptor alpha 1 subunit gene revealed a novel heterozygous missense mutation (c.211A/T, p.Ile71Phe). Genetic screening of our patient’s family revealed that the clinically unaffected parents and sister do not carry the mutation, suggesting that the identified sequence change is a de novo mutation. Since hyperekplexia can have severe consequences, including sudden infant death due to laryngospasm and cardiorespiratory failure, identification of the causative genetic alteration(s) of the disease is high priority. Such knowledge is necessary for prenatal diagnosis, which would allow informed family planning and greater parental sensitivity to hyperekplexia 1-associated risks.
doi:10.1186/1752-1947-8-233
PMCID: PMC4096538  PMID: 24969041
Stiff-baby syndrome; Hereditary hyperekplexia; GLRA1 gene; Missense mutation
8.  Glycine receptor mutants of the mouse: what are possible routes of inhibitory compensation? 
Defects in glycinergic inhibition result in a complex neuromotor disorder in humans known as hyperekplexia (OMIM 149400) with similar phenotypes in rodents characterized by an exaggerated startle reflex and hypertonia. Analogous to genetic defects in humans single point mutations, microdeletions, or insertions in the Glra1 gene but also in the Glrb gene underlie the pathology in mice. The mutations either localized in the α (spasmodic, oscillator, cincinnati, Nmf11) or the β (spastic) subunit of the glycine receptor (GlyR) are much less tolerated in mice than in humans, leaving the question for the existence of different regulatory elements of the pathomechanisms in humans and rodents. In addition to the spontaneous mutations, new insights into understanding of the regulatory pathways in hyperekplexia or glycine encephalopathy arose from the constantly increasing number of knock-out as well as knock-in mutants of GlyRs. Over the last five years, various efforts using in vivo whole cell recordings provided a detailed analysis of the kinetic parameters underlying glycinergic dysfunction. Presynaptic compensation as well as postsynaptic compensatory mechanisms in these mice by other GlyR subunits or GABAA receptors, and the role of extra-synaptic GlyRs is still a matter of debate. A recent study on the mouse mutant oscillator displayed a novel aspect for compensation of functionality by complementation of receptor domains that fold independently. This review focuses on defects in glycinergic neurotransmission in mice discussed with the background of human hyperekplexia en route to strategies of compensation.
doi:10.3389/fnmol.2012.00098
PMCID: PMC3484359  PMID: 23118727
GlyRs; synaptic inhibition; spontaneous mouse mutants; knockout mice; hyperekplexia; rescue
9.  Transient neuromotor phenotype in transgenic spastic mice expressing low levels of glycine receptor β-subunit: an animal model of startle disease 
Startle disease or hereditary hyperekplexia has been shown to result from mutations in the α1-subunit gene of the inhibitory glycine receptor (GlyR). In hyperekplexia patients, neuromotor symptoms generally become apparent at birth, improve with age, and often disappear in adulthood. Loss-of-function mutations of GlyR α or β-subunits in mice show rather severe neuromotor phenotypes. Here, we generated mutant mice with a transient neuromotor deficiency by introducing a GlyR β transgene into the spastic mouse (spa/spa), a recessive mutant carrying a transposon insertion within the GlyR β-subunit gene. In spa/spa TG456 mice, one of three strains generated with this construct, which expressed very low levels of GlyR β transgene-dependent mRNA and protein, the spastic phenotype was found to depend upon the transgene copy number. Notably, mice carrying two copies of the transgene showed an age-dependent sensitivity to tremor induction, which peaked at ∼ 3–4 weeks postnatally. This closely resembles the development of symptoms in human hyperekplexia patients, where motor coordination significantly improves after adolescence. The spa/spa TG456 line thus may serve as an animal model of human startle disease.
doi:10.1046/j.1460-9568.2000.00877.x
PMCID: PMC3655541  PMID: 10651857
glycine receptor; hereditary hyperekplexia; spa/spa TG456 mice; startle syndrome
10.  A Balanced Chromosomal Translocation Disrupting ARHGEF9 Is Associated With Epilepsy, Anxiety, Aggression, and Mental Retardation 
Human mutation  2009;30(1):61-68.
Clustering of inhibitory γ-aminobutyric acidA (GABAA) and glycine receptors at synapses is thought to involve key interactions between the receptors, a “scaffolding” protein known as gephyrin and the RhoGEF collybistin. We report the identification of a balanced chromosomal translocation in a female patient presenting with a disturbed sleep-wake cycle, late-onset epileptic seizures, increased anxiety, aggressive behavior, and mental retardation, but not hyperekplexia. Fine mapping of the breakpoint indicates disruption of the collybistin gene (ARHGEF9) on chromosome Xq11, while the other breakpoint lies in a region of 18q11 that lacks any known or predicted genes. We show that defective collybistin transcripts are synthesized and exons 7–10 are replaced by cryptic exons from chromosomes X and 18. These mRNAs no longer encode the pleckstrin homology (PH) domain of collybistin, which we now show binds phosphatidylinositol-3-phosphate (PI3P/ PtdIns-3-P), a phosphoinositide with an emerging role in membrane trafficking and signal transduction, rather than phosphatidylinositol 3,4,5-trisphosphate (PIP3/PtdIns-3,4,5-P) as previously suggested in the “membrane activation model” of gephyrin clustering. Consistent with this finding, expression of truncated collybistin proteins in cultured neurons interferes with synaptic localization of endogenous gephyrin and GABAA receptors. These results suggest that collybistin has a key role in membrane trafficking of gephyrin and selected GABAA receptor subtypes involved in epilepsy, anxiety, aggression, insomnia, and learning and memory.
doi:10.1002/humu.20814
PMCID: PMC3621145  PMID: 18615734
GABAA receptors; glycine receptors; collybistin; ARHGEF9; gephyrin; clustering; anxiety; epilepsy; mental retardation; aggression
11.  Hyperekplexia and stiff-man syndrome: abnormal brainstem reflexes suggest a physiological relationship 
Background and objectives: Hyperekplexia and the stiff-man syndrome (SMS) are both conditions with exaggerated startle suggesting abnormal brainstem function. Investigation of brainstem reflexes may provide insight into disturbed reflex excitation and inhibition underlying these movement disorders.
Patients and methods: Using four-channel EMG, we examined four trigeminal brainstem reflexes (monosynaptic masseter, masseter inhibitory, glabella, and orbicularis oculi blink reflexes) and their spread into pericranial muscles in five patients with familial hyperekplexia (FH), two with acquired hyperekplexia (AH), 10 with SMS, and 15 healthy control subjects.
Results: Both FH/AH and SMS patients had abnormal propagation of brainstem reflexes into pericranial muscles. All patients with hyperekplexia showed an abnormal short-latency (15–20 ms) reflex in the trapezius muscle with a characteristic clinical appearance ("head retraction jerk") evoked by tactile or electrical stimulation of the trigeminal nerve, but normal monosynaptic masseter reflexes. Inhibitory brainstem reflexes were attenuated in some FH/AH patients. Four of 10 patients with SMS had similar short-latency reflexes in the neck muscles and frequently showed widespread enhancement of other excitatory reflexes, reflex spasms, and attenuation of inhibitory brainstem reflexes.
Conclusion: Reflex excitation is exaggerated and inhibition is attenuated in both stiff-man syndrome and familial or acquired hyperekplexia, indicating a physiological relationship. Reflex transmission in the brainstem appears biased towards excitation which may imply dysfunction of inhibitory glycinergic or GABAergic interneurons, or both.
doi:10.1136/jnnp.2003.018135
PMCID: PMC1739206  PMID: 15314112
12.  The impact of human hyperekplexia mutations on glycine receptor structure and function 
Molecular Brain  2014;7:2.
Hyperekplexia is a rare neurological disorder characterized by neonatal hypertonia, exaggerated startle responses to unexpected stimuli and a variable incidence of apnoea, intellectual disability and delays in speech acquisition. The majority of motor defects are successfully treated by clonazepam. Hyperekplexia is caused by hereditary mutations that disrupt the functioning of inhibitory glycinergic synapses in neuromotor pathways of the spinal cord and brainstem. The human glycine receptor α1 and β subunits, which predominate at these synapses, are the major targets of mutations. International genetic screening programs, that together have analysed several hundred probands, have recently generated a clear picture of genotype-phenotype correlations and the prevalence of different categories of hyperekplexia mutations. Focusing largely on this new information, this review seeks to summarise the effects of mutations on glycine receptor structure and function and how these functional alterations lead to hyperekplexia.
doi:10.1186/1756-6606-7-2
PMCID: PMC3895786  PMID: 24405574
Cys-loop receptor; Ligand-gated ion channel; Chloride channel; Startle disease; Glycinergic neurotransmission
13.  Hyperekplexia in neonates 
Postgraduate Medical Journal  2001;77(911):570-572.
Hyperekplexia (startle disease) is a rare non-epileptic disorder characterised by an exaggerated persistent startle reaction to unexpected auditory, somatosensory and visual stimuli, generalised muscular rigidity, and nocturnal myoclonus. The genetic basis is a mutation usually of the arginine residue 271 leading to neuronal hyperexcitability by impairing glycinergic inhibition. Hyperekplexia is usually familial, most often autosomal dominant with complete penetrance and variable expression. It can present in fetal life as abnormal intrauterine movements, or later at any time from the neonatal period to adulthood. Early manifestations include abnormal responses to unexpected auditory, visual, and somatosensory stimuli such as sustained tonic spasm, exaggerated startle response, and fetal posture with clenched fists and anxious stare. The tonic spasms may mimic generalised tonic seizures, leading to apnoea and death. Consistent generalised flexor spasm in response to tapping of the nasal bridge (without habituation) is the clinical hallmark of hyperekplexia. Electroencephalography may show fast spikes initially during the tonic spasms, followed by slowing of background activity with eventual flattening corresponding to the phase of apnoea bradycardia and cyanosis. Electromyography shows a characteristic almost permanent muscular activity with periods of electrical quietness. Nerve conduction velocity is normal. No specific computed tomography findings have been reported yet. Clonazepam, a gamma aminobutyric acid (GABA) receptor agonist, is the treatment of choice for hypertonia and apnoeic episodes. It, however, may not influence the degree of stiffness significantly. A simple manoeuvre like forced flexion of the head and legs towards the trunk is known to be life saving when prolonged stiffness impedes respiration.


Keywords: hyperekplexia; neonates; startle
doi:10.1136/pmj.77.911.570
PMCID: PMC1757896  PMID: 11524514
14.  The GLRA1 Missense Mutation W170S Associates Lack of Zn2+ Potentiation with Human Hyperekplexia 
The Journal of Neuroscience  2013;33(45):17675-17681.
Hyperekplexia is a neurological disorder associated primarily with mutations in the α1 subunit of glycine receptors (GlyRs) that lead to dysfunction of glycinergic inhibitory transmission. To date, most of the identified mutations result in disruption of surface expression or altered channel properties of α1-containing GlyRs. Little evidence has emerged to support an involvement of allosteric GlyR modulation in human hyperekplexia. Here, we report that recombinant human GlyRs containing α1 or α1β subunits with a missense mutation in the α1 subunit (W170S), previously identified from familial hyperekplexia, caused remarkably reduced potentiation and enhanced inhibition by Zn2+. Interestingly, mutant α1W170Sβ GlyRs displayed no significant changes in potency or maximum response to glycine, taurine, or β-alanine. By temporally separating the potentiating and the inhibitory effects of Zn2+, we found that the enhancement of Zn2+ inhibition resulted from a loss of Zn2+-mediated potentiation. The W170S mutation on the background of H107N, which was previously reported to selectively disrupt Zn2+ inhibition, showed remarkable attenuation of Zn2+-mediated potentiation and thus indicated that W170 is an important residue for the Zn2+-mediated GlyR potentiation. Moreover, overexpressing the α1W170S subunit in cultured rat neurons confirmed the results from heterologous expression. Together, our results reveal a new zinc potentiation site on α1 GlyRs and a strong link between Zn2+ modulation and human disease.
doi:10.1523/JNEUROSCI.3240-13.2013
PMCID: PMC3873627  PMID: 24198360
15.  β Subunit M2–M3 Loop Conformational Changes Are Uncoupled from α1 β Glycine Receptor Channel Gating: Implications for Human Hereditary Hyperekplexia 
PLoS ONE  2011;6(11):e28105.
Hereditary hyperekplexia, or startle disease, is a neuromotor disorder caused mainly by mutations that either prevent the surface expression of, or modify the function of, the human heteromeric α1 β glycine receptor (GlyR) chloride channel. There is as yet no explanation as to why hyperekplexia mutations that modify channel function are almost exclusively located in the α1 to the exclusion of β subunit. The majority of these mutations are identified in the M2–M3 loop of the α1 subunit. Here we demonstrate that α1 β GlyR channel function is less sensitive to hyperekplexia-mimicking mutations introduced into the M2–M3 loop of the β than into the α1 subunit. This suggests that the M2–M3 loop of the α subunit dominates the β subunit in gating the α1 β GlyR channel. A further attempt to determine the possible mechanism underlying this phenomenon by using the voltage-clamp fluorometry technique revealed that agonist-induced conformational changes in the β subunit M2–M3 loop were uncoupled from α1 β GlyR channel gating. This is in contrast to the α subunit, where the M2–M3 loop conformational changes were shown to be directly coupled to α1 β GlyR channel gating. Finally, based on analysis of α1 β chimeric receptors, we demonstrate that the structural components responsible for this are distributed throughout the β subunit, implying that the β subunit has evolved without the functional constraint of a normal gating pathway within it. Our study provides a possible explanation of why hereditary hyperekplexia-causing mutations that modify α1 β GlyR channel function are almost exclusively located in the α1 to the exclusion of the β subunit.
doi:10.1371/journal.pone.0028105
PMCID: PMC3222680  PMID: 22132222
16.  Analysis of GLRA1 in hereditary and sporadic hyperekplexia: a novel mutation in a family cosegregating for hyperekplexia and spastic paraparesis. 
Journal of Medical Genetics  1996;33(5):435-436.
Hyperekplexia is a rare condition characterised by the presence of neonatal hypertonia and an exaggerated startle response. Mutations have been described in GLRA1, the gene encoding the alpha 1 subunit of the glycine receptor, in dominant families with hyperekplexia and in a single sporadic case, thought to represent an autosomal recessive form of the disease. In this study the coding region of the GLRA1 was analysed in eight probands with hyperekplexia by restriction digest and sequencing. Two familial cases were found to possess the previously described G1192A (R271Q) mutation in exon 6. In an additional family in which hyperekplexia cosegregates with spastic paraparesis, a novel A to G transversion at nucleotide 1206 in exon 6 was detected that changes a lysine at amino acid 276 to a glutamate (K276E). In four sporadic cases no mutations were found. In addition, one familial case did not have a mutation in the coding region of the gene.
PMCID: PMC1050620  PMID: 8733061
17.  Development of glycinergic innervation to the murine LSO and SPN in the presence and absence of the MNTB 
Neurons in the superior olivary complex (SOC) integrate excitatory and inhibitory inputs to localize sounds in space. The majority of these inhibitory inputs have been thought to arise within the SOC from the medial nucleus of the trapezoid body (MNTB). However, recent work demonstrates that glycinergic innervation of the SOC persists in Egr2; En1CKO mice that lack MNTB neurons, suggesting that there are other sources of this innervation (Jalabi et al., 2013). To study the development of MNTB- and non-MNTB-derived glycinergic SOC innervation, we compared immunostaining patterns of glycine transporter 2 (GlyT2) at several postnatal ages in control and Egr2; En1CKO mice. GlyT2 immunostaining was present at birth (P0) in controls and reached adult levels by P7 in the superior paraolivary nucleus (SPN) and by P12 in the lateral superior olive (LSO). In Egr2; En1CKO mice, glycinergic innervation of the LSO developed at a similar rate but was delayed by one week in the SPN. Conversely, consistent reductions in the number of GlyT2+ boutons located on LSO somata were seen at all ages in Egr2; En1CKO mice, while these numbers reached control levels in the SPN by adulthood. Dendritic localization of GlyT2+ boutons was unaltered in both the LSO and SPN of adult Egr2; En1CKO mice. On the postsynaptic side, adult Egr2; En1CKO mice had reduced glycine receptor α1 (GlyRα1) expression in the LSO but normal levels in the SPN. GlyRα2 was not expressed by LSO or SPN neurons in either genotype. These findings contribute important information for understanding the development of MNTB- and non-MNTB-derived glycinergic pathways to the mouse SOC.
doi:10.3389/fncir.2014.00109
PMCID: PMC4162373  PMID: 25309335
hearing; deafness; mouse models; brain development; auditory system
18.  Pontine hyperperfusion in sporadic hyperekplexia 
Objective
To explore with neuroimaging techniques the anatomical and functional correlates of sporadic hyperekplexia.
Methods
Two elderly women with sporadic hyperekplexia underwent neurophysiological assessment, MRI of the brain and proton magnetic resonance spectroscopy (1H‐MRS) of the brainstem and frontal lobes. Regional cerebral blood flow was investigated with single photon emission tomography (SPECT) during evoked startles and at rest.
Results
Both patients showed excessively large and non‐habituating startle responses. In both patients, MRI showed impingement of the brainstem by the vertebrobasilar artery, lack of frontal or brainstem abnormalities on 1H‐MRS and hyperperfusion in the dorsal pons and cingulate cortex, and superior frontal gyrus at SPECT during evoked startles.
Conclusions
In our patients with hyperekplexia, the vertebrobasilar arteries were found to impinge on the brainstem. Neurophysiological findings and neurofunctional imaging of evoked startles indicated a pontine origin of the movement disorder modulated by activation in cortical, especially frontal, areas. The neurofunctional correlates of evoked startles in human sporadic hyperekplexia are similar to those observed for the startle circuit in animals.
doi:10.1136/jnnp.2006.113837
PMCID: PMC2117864  PMID: 17702784
19.  Novel missense mutations in the glycine receptor β subunit gene (GLRB) in startle disease 
Neurobiology of Disease  2013;52(C):137-149.
Startle disease is a rare, potentially fatal neuromotor disorder characterized by exaggerated startle reflexes and hypertonia in response to sudden unexpected auditory, visual or tactile stimuli. Mutations in the GlyR α1 subunit gene (GLRA1) are the major cause of this disorder, since remarkably few individuals with mutations in the GlyR β subunit gene (GLRB) have been found to date. Systematic DNA sequencing of GLRB in individuals with hyperekplexia revealed new missense mutations in GLRB, resulting in M177R, L285R and W310C substitutions. The recessive mutation M177R results in the insertion of a positively-charged residue into a hydrophobic pocket in the extracellular domain, resulting in an increased EC50 and decreased maximal responses of α1β GlyRs. The de novo mutation L285R results in the insertion of a positively-charged side chain into the pore-lining 9′ position. Mutations at this site are known to destabilize the channel closed state and produce spontaneously active channels. Consistent with this, we identified a leak conductance associated with spontaneous GlyR activity in cells expressing α1βL285R GlyRs. Peak currents were also reduced for α1βL285R GlyRs although glycine sensitivity was normal. W310C was predicted to interfere with hydrophobic side-chain stacking between M1, M2 and M3. We found that W310C had no effect on glycine sensitivity, but reduced maximal currents in α1β GlyRs in both homozygous (α1βW310C) and heterozygous (α1ββW310C) stoichiometries. Since mild startle symptoms were reported in W310C carriers, this may represent an example of incomplete dominance in startle disease, providing a potential genetic explanation for the ‘minor’ form of hyperekplexia.
Highlights
► We report novel missense mutations in the GlyR β subunit gene causing startle disease. ► Mutation M177R in the extracellular domain decreases GlyR agonist affinity. ► Mutation L285R in TM2 produces spontaneously active channels. ► Mutation W310C in TM3 affects hydrophobic stacking and shows incomplete dominance. ► Mutations in GLRB have unique pathogenic mechanisms and modes of inheritance.
doi:10.1016/j.nbd.2012.12.001
PMCID: PMC3581774  PMID: 23238346
GLRA1; GLRB; Glycine receptor; Hyperekplexia; Startle disease
20.  PKCβ–dependent phosphorylation of the glycine transporter 1 
Neurochemistry international  2011;59(8):1123-1132.
The extracellular levels of the neurotransmitter glycine in the brain are tightly regulated by the glycine transporter 1 (GlyT1) and the clearance rate for glycine depends on its rate of transport and the levels of cell surface GlyT1. Over the years, it has been shown that PKC tightly regulates the activity of several neurotransmitter transporters. In the present work, by stably expressing three N-terminus GlyT1 isoforms in porcine aortic endothelial cells and assaying for [32P]-orthophosphate metabolic labeling, we demonstrated that the isoforms GlyT1a, GlyT1b, and GlyT1c were constitutively phosphorylated, and that phosphorylation was dramatically enhanced, in a time dependent fashion, after PKC activation by phorbol ester. The phosphorylation was PKC-dependent, since pre-incubation of the cells with bisindolylmaleimide I, a selective PKC inhibitor, abolished the phorbol ester-induced phosphorylation. Blotting with specific anti-phospho-tyrosine antibodies did not yield any signal that could correspond to GlyT1 tyrosine phosphorylation, suggesting that the phosphorylation occurs at serine and/or threonine residues. In addition, a 23-40% -inhibition on Vmax was obtained by incubation with phorbol ester without a significant change on the apparent Km value. Furthermore, pre-incubation of the cells with the selective PKCα/β inhibitor Gö6976 abolished the downregulation effect of phorbol ester on uptake and phosphorylation, whereas the selective PKCβ inhibitors (PKCβ inhibitor or LY333531) prevented the phosphorylation without affecting glycine uptake, defining a specific role of classical PKC on GlyT1 uptake and phosphorylation. Taken together, these data suggest that phosphorylation that conventional PKCα/β regulates the uptake of glycine, whereas PKCβ is responsible for GlyT1 phosphorylation.
doi:10.1016/j.neuint.2011.08.006
PMCID: PMC3226844  PMID: 21864610
phosphorylation; glycine; transporter; PKC
21.  Serotonin receptor 1A–modulated phosphorylation of glycine receptor α3 controls breathing in mice 
The Journal of Clinical Investigation  2010;120(11):4118-4128.
Rhythmic breathing movements originate from a dispersed neuronal network in the medulla and pons. Here, we demonstrate that rhythmic activity of this respiratory network is affected by the phosphorylation status of the inhibitory glycine receptor α3 subtype (GlyRα3), which controls glutamatergic and glycinergic neuronal discharges, subject to serotonergic modulation. Serotonin receptor type 1A–specific (5-HTR1A–specific) modulation directly induced dephosphorylation of GlyRα3 receptors, which augmented inhibitory glycine-activated chloride currents in HEK293 cells coexpressing 5-HTR1A and GlyRα3. The 5-HTR1A–GlyRα3 signaling pathway was distinct from opioid receptor signaling and efficiently counteracted opioid-induced depression of breathing and consequential apnea in mice. Paradoxically, this rescue of breathing originated from enhanced glycinergic synaptic inhibition of glutamatergic and glycinergic neurons and caused disinhibition of their target neurons. Together, these effects changed respiratory phase alternations and ensured rhythmic breathing in vivo. GlyRα3-deficient mice had an irregular respiratory rhythm under baseline conditions, and systemic 5-HTR1A activation failed to remedy opioid-induced respiratory depression in these mice. Delineation of this 5-HTR1A–GlyRα3 signaling pathway offers a mechanistic basis for pharmacological treatment of opioid-induced apnea and other breathing disturbances caused by disorders of inhibitory synaptic transmission, such as hyperekplexia, hypoxia/ischemia, and brainstem infarction.
doi:10.1172/JCI43029
PMCID: PMC2964980  PMID: 20978350
22.  Synthesis and evaluation of 2-chloro N-[(S)-{(S)-1-[11 C]methylpiperidin-2-yl} (phenyl)methyl]3-trifluoromethyl-benzamide ([11 C]N-methyl-SSR504734) as a PET radioligand for glycine transporter 1 
EJNMMI Research  2012;2:37.
Background
Dysfunction of the glycine transporter 1 (GlyT1) has been suggested to be involved in psychiatric disorders such as schizophrenia. GlyT1 inhibitors have therefore been considered to have antipsychotic therapeutic potential. Positron emission tomography (PET) imaging probes for GlyT1 are, consequently, expected to be useful for investigating the mechanism of such disease conditions and for measuring occupancy of GlyT1 inhibitors in vivo. The aim of this study was to assess the potential of 2-chloro N-[(S)-{(S)-1-[11 C]methylpiperidin-2-yl} (phenyl)methyl] 3-trifluoromethyl-benzamide ([11 C]N-methyl-SSR504734) as a PET imaging agent for GlyT1.
Methods
[11 C]N-methyl-SSR504734 was synthesized by N-[11 C]methylation of SSR504734 via [11 C]CH3OTf. In vitro brain distribution of [11 C]N-methyl-SSR504734 was tested in whole-hemisphere autoradiography (ARG) on human brain slices. Initial PET studies were performed using a cynomolgus monkey at baseline and after pretreatment with 0.1 to 1.5 mg/kg of SSR504734. Then, PET studies using rhesus monkeys were performed with arterial blood sampling at baseline and after pretreatment with 1.5 to 4.5 mg/kg SSR504734. Distribution volumes (VT) were calculated with a two-tissue compartment model, and GlyT1 occupancy by SSR504734 was estimated using a Lassen plot approach.
Results
[11 C]N-methyl-SSR504734 was successfully synthesized in moderate radiochemical yield and high specific radioactivity. In the ARG experiments, [11 C]N-methyl-SSR504734 showed specific binding in the white matter and pons. In the initial PET experiments in a cynomolgus monkey, [11 C]N-methyl-SSR504734 showed high brain uptake and consistent distribution with previously reported GlyT1 expression in vivo (thalamus, brainstem > cerebellum > cortical regions). However, the brain uptake increased after pretreatment with SSR504734. Further PET studies in rhesus monkeys showed a similar increase of brain uptake after pretreatment with SSR504734. However, the VT of [11 C]N-methyl-SSR504734 was found to decrease after pretreatment of SSR504734 in a dose-dependent manner. GlyT1 occupancy was calculated to be 45% and 73% at 1.5 and 4.5 mg/kg of SSR504734, respectively.
Conclusions
[11 C]N-methyl-SSR504734 is demonstrated to be a promising PET radioligand for GlyT1 in nonhuman primates. The present results warrant further PET studies in human subjects.
doi:10.1186/2191-219X-2-37
PMCID: PMC3531252  PMID: 22776065
Glycine transporter 1; SSR504734; 11 C; Positron emission tomography; Schizophrenia
23.  A Confusing Coincidence: Neonatal Hypoglycemic Seizures and Hyperekplexia 
Case Reports in Medicine  2014;2014:595412.
Hyperekplexia is a rare, nonepileptic, genetic, or sporadic neurologic disorder characterized by startle responses to acoustic, optic, or tactile stimuli. Genetic defects in glycine receptors as well as encephalitis, tumors, inflammation, and disgenesis are among the etiologic causes of the disease. The main problem in hyperekplexia is the incomplete development of inhibitory mechanisms or exaggerated stimulation of excitatory mediators. Hyperekplexia is often confused with epileptic seizures. Here we present a case with hypoglycemic convulsions coexisting with hyperekplexia, causing diagnostic difficulty.
doi:10.1155/2014/595412
PMCID: PMC3982260  PMID: 24782896
24.  Both Rare and De Novo Copy Number Variants Are Prevalent in Agenesis of the Corpus Callosum but Not in Cerebellar Hypoplasia or Polymicrogyria 
PLoS Genetics  2013;9(10):e1003823.
Agenesis of the corpus callosum (ACC), cerebellar hypoplasia (CBLH), and polymicrogyria (PMG) are severe congenital brain malformations with largely undiscovered causes. We conducted a large-scale chromosomal copy number variation (CNV) discovery effort in 255 ACC, 220 CBLH, and 147 PMG patients, and 2,349 controls. Compared to controls, significantly more ACC, but unexpectedly not CBLH or PMG patients, had rare genic CNVs over one megabase (p = 1.48×10−3; odds ratio [OR] = 3.19; 95% confidence interval [CI] = 1.89–5.39). Rare genic CNVs were those that impacted at least one gene in less than 1% of the combined population of patients and controls. Compared to controls, significantly more ACC but not CBLH or PMG patients had rare CNVs impacting over 20 genes (p = 0.01; OR = 2.95; 95% CI = 1.69–5.18). Independent qPCR confirmation showed that 9.4% of ACC patients had de novo CNVs. These, in comparison to inherited CNVs, preferentially overlapped de novo CNVs previously observed in patients with autism spectrum disorders (p = 3.06×10−4; OR = 7.55; 95% CI = 2.40–23.72). Interestingly, numerous reports have shown a reduced corpus callosum area in autistic patients, and diminished social and executive function in many ACC patients. We also confirmed and refined previously known CNVs, including significantly narrowing the 8p23.1-p11.1 duplication present in 2% of our current ACC cohort. We found six novel CNVs, each in a single patient, that are likely deleterious: deletions of 1p31.3-p31.1, 1q31.2-q31.3, 5q23.1, and 15q11.2-q13.1; and duplications of 2q11.2-q13 and 11p14.3-p14.2. One ACC patient with microcephaly had a paternally inherited deletion of 16p13.11 that included NDE1. Exome sequencing identified a recessive maternally inherited nonsense mutation in the non-deleted allele of NDE1, revealing the complexity of ACC genetics. This is the first systematic study of CNVs in congenital brain malformations, and shows a much higher prevalence of large gene-rich CNVs in ACC than in CBLH and PMG.
Author Summary
Here, we systematically test the genetic etiology of three common developmental brain malformations: agenesis of the corpus callosum (ACC), cerebellar hypoplasia (CBLH), and polymicrogyria (PMG) by copy number variation (CNV) analysis in a large cohort of brain malformation patients and controls. We found significantly more ACC but not CBLH or PMG patients with rare genic CNVs over one megabase and with rare CNVs impacting over 20 genes when compared with controls. De novo CNVs were found in 9.4% of ACC patients, and interestingly many such CNVs overlapped with de novo CNVs observed in autism. Notably, numerous studies have demonstrated a reduction in the corpus callosum area in autistic brains. Our analysis also refined previously known large CNVs that cause these malformations, and identified six novel CNVs that are likely deleterious. One ACC patient had inherited a deletion from the father which, through exome sequencing, was found to uncover a recessive nonsense mutation in NDE1 on the non-deleted allele inherited from the mother. Our study is the first to systematically evaluate the burden of rare genic CNVs in congenital brain malformations and shows that large gene-rich CNVs are more common in ACC than in CBLH and PMG.
doi:10.1371/journal.pgen.1003823
PMCID: PMC3789824  PMID: 24098143
25.  Localization of Glycine Receptors in the Human Forebrain, Brainstem, and Cervical Spinal Cord: An Immunohistochemical Review 
Inhibitory neurotransmitter receptors for glycine (GlyR) are heteropentameric chloride ion channels that are comprised of four functional subunits, alpha1–3 and beta and that facilitate fast-response, inhibitory neurotransmission in the mammalian brain and spinal cord. We have investigated the distribution of GlyRs in the human forebrain, brainstem, and cervical spinal cord using immunohistochemistry at light and confocal laser scanning microscopy levels. This review will summarize the present knowledge on the GlyR distribution in the human brain using our established immunohistochemical techniques. The results of our immunohistochemical labeling studies demonstrated GlyR immunoreactivity (IR) throughout the human basal ganglia, substantia nigra, various pontine regions, rostral medulla oblongata and the cervical spinal cord present an intense and abundant punctate IR along the membranes of the neuronal soma and dendrites. This work is part of a systematic study of inhibitory neurotransmitter receptor distribution in the human CNS, and provides a basis for additional detailed physiological and pharmacological studies on the inter-relationship of GlyR, GABAAR and gephyrin in the human brain. This basic mapping exercise, we believe, will provide important baselines for the testing of future pharmacotherapies and drug regimes that modulate neuroinhibitory systems. These findings provide new information for understanding the complexity of glycinergic functions in the human brain, which will translate into the contribution of inhibitory mechanisms in paroxysmal disorders and neurodegenerative diseases such as Epilepsy, Huntington's and Parkinson's Disease and Motor Neuron Disease.
doi:10.3389/neuro.02.025.2009
PMCID: PMC2776491  PMID: 19915682
human brain; glycine receptor; immunohistochemistry

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