Positive Allosteric Modulators of Glycine Receptors and Their Potential Use in Pain Therapies.

Glycine receptors are ligand-gated ion channels that mediate synaptic inhibition throughout the mammalian spinal cord, brainstem, and higher brain regions. They have recently emerged as promising targets for novel pain therapies due to their ability to produce antinociception by inhibiting nociceptive signals within the dorsal horn of the spinal cord. This has greatly enhanced the interest in developing positive allosteric modulators of glycine receptors. Several pharmaceutical companies and research facilities have attempted to identify new therapeutic leads by conducting large-scale screens of compound libraries, screening new derivatives from natural sources, or synthesizing novel compounds that mimic endogenous compounds with antinociceptive activity. Advances in structural techniques have also led to the publication of multiple high-resolution structures of the receptor, highlighting novel allosteric binding sites and providing additional information for previously identified binding sites. This has greatly enhanced our understanding of the functional properties of glycine receptors and expanded the structure activity relationships of novel pharmacophores. Despite this, glycine receptors are yet to be used as drug targets due to the difficulties in obtaining potent, selective modulators with favorable pharmacokinetic profiles that are devoid of side effects. This review presents a summary of the structural basis for how current compounds cause positive allosteric modulation of glycine receptors and discusses their therapeutic potential as analgesics. SIGNIFICANCE STATEMENT: Chronic pain is a major cause of disability, and in Western societies, this will only increase as the population ages. Despite the high level of prevalence and enormous socioeconomic burden incurred, treatment of chronic pain remains limited as it is often refractory to current analgesics, such as opioids. The National Institute for Drug Abuse has set finding effective, safe, nonaddictive strategies to manage chronic pain as their top priority. Positive allosteric modulators of glycine receptors may provide a therapeutic option.

Clinical, genetic, and functional characterization of the glycine receptor ß-subunit A455P variant in a family affected by hyperekplexia syndrome.

Hyperekplexia is a rare neurological disorder characterized by exaggerated startle responses affecting newborns with the hallmark characteristics of hypertonia, apnea, and noise or touch-induced nonepileptic seizures. The genetic causes of the disease can vary, and several associated genes and mutations have been reported to affect glycine receptors (GlyRs); however, the mechanistic links between GlyRs and hyperekplexia are not yet understood. Here, we describe a patient with hyperekplexia from a consanguineous family. Extensive genetic screening using exome sequencing coupled with autozygome analysis and iterative filtering supplemented by in silico prediction identified that the patient carries the homozygous missense mutation A455P in GLRB, which encodes the GlyR ß-subunit. To unravel the physiological and molecular effects of A455P on GlyRs, we used electrophysiology in a heterologous system as well as immunocytochemistry, confocal microscopy, and cellular biochemistry. We found a reduction in glycine-evoked currents in N2A cells expressing the mutation compared to WT cells. Western blot analysis also revealed a reduced amount of GlyR ß protein both in cell lysates and isolated membrane fractions. In line with the above observations, coimmunoprecipitation assays suggested that the GlyR α1-subunit retained coassembly with ßA455P to form membrane-bound heteromeric receptors. Finally, structural modeling showed that the A455P mutation affected the interaction between the GlyR ß-subunit transmembrane domain 4 and the other helices of the subunit. Taken together, our study identifies and validates a novel loss-of-function mutation in GlyRs whose pathogenicity is likely to cause hyperekplexia in the affected individual.

Dominant-negative variants in CBX1 cause a neurodevelopmental disorder.

PURPOSE: This study aimed to establish variants in CBX1, encoding heterochromatin protein 1ß (HP1ß), as a cause of a novel syndromic neurodevelopmental disorder. METHODS: Patients with CBX1 variants were identified, and clinician researchers were connected using GeneMatcher and physician referrals. Clinical histories were collected from each patient. To investigate the pathogenicity of identified variants, we performed in vitro cellular assays and neurobehavioral and cytological analyses of neuronal cells obtained from newly generated Cbx1 mutant mouse lines. RESULTS: In 3 unrelated individuals with developmental delay, hypotonia, and autistic features, we identified heterozygous de novo variants in CBX1. The identified variants were in the chromodomain, the functional domain of HP1ß, which mediates interactions with chromatin. Cbx1 chromodomain mutant mice displayed increased latency-to-peak response, suggesting the possibility of synaptic delay or myelination deficits. Cytological and chromatin immunoprecipitation experiments confirmed the reduction of mutant HP1ß binding to heterochromatin, whereas HP1ß interactome analysis demonstrated that the majority of HP1ß-interacting proteins remained unchanged between the wild-type and mutant HP1ß. CONCLUSION: These collective findings confirm the role of CBX1 in developmental disabilities through the disruption of HP1ß chromatin binding during neurocognitive development. Because HP1ß forms homodimers and heterodimers, mutant HP1ß likely sequesters wild-type HP1ß and other HP1 proteins, exerting dominant-negative effects.

De Novo ZMYND8 variants result in an autosomal dominant neurodevelopmental disorder with cardiac malformations.

PURPOSE: ZMYND8 encodes a multidomain protein that serves as a central interactive hub for coordinating critical roles in transcription regulation, chromatin remodeling, regulation of super-enhancers, DNA damage response and tumor suppression. We delineate a novel neurocognitive disorder caused by variants in the ZMYND8 gene. METHODS: An international collaboration, exome sequencing, molecular modeling, yeast two-hybrid assays, analysis of available transcriptomic data and a knockdown Drosophila model were used to characterize the ZMYND8 variants. RESULTS: ZMYND8 variants were identified in 11 unrelated individuals; 10 occurred de novo and one suspected de novo; 2 were truncating, 9 were missense, of which one was recurrent. The disorder is characterized by intellectual disability with variable cardiovascular, ophthalmologic and minor skeletal anomalies. Missense variants in the PWWP domain of ZMYND8 abolish the interaction with Drebrin and missense variants in the MYND domain disrupt the interaction with GATAD2A. ZMYND8 is broadly expressed across cell types in all brain regions and shows highest expression in the early stages of brain development. Neuronal knockdown of the DrosophilaZMYND8 ortholog results in decreased habituation learning, consistent with a role in cognitive function. CONCLUSION: We present genomic and functional evidence for disruption of ZMYND8 as a novel etiology of syndromic intellectual disability.

MED27, SLC6A7, and MPPE1 Variants in a Complex Neurodevelopmental Disorder with Severe Dystonia.

BACKGROUND: Despite advances in next generation sequencing technologies, the identification of variants of uncertain significance (VUS) can often hinder definitive diagnosis in patients with complex neurodevelopmental disorders. OBJECTIVE: The objective of this study was to identify and characterize the underlying cause of disease in a family with two children with severe developmental delay associated with generalized dystonia and episodic status dystonicus, chorea, epilepsy, and cataracts. METHODS: Candidate genes identified by autozygosity mapping and whole-exome sequencing were characterized using cellular and vertebrate model systems. RESULTS: Homozygous variants were found in three candidate genes: MED27, SLC6A7, and MPPE1. Although the patients had features of MED27-related disorder, the SLC6A7 and MPPE1 variants were functionally investigated. SLC6A7 variant in vitro overexpression caused decreased proline transport as a result of reduced cell-surface expression, and zebrafish knockdown of slc6a7 exhibited developmental delay and fragile motor neuron morphology that could not be rescued by L-proline transporter-G396S RNA. Lastly, patient fibroblasts displayed reduced cell-surface expression of glycophosphatidylinositol-anchored proteins linked to MPPE1 dysfunction. CONCLUSIONS: We report a family harboring a homozygous MED27 variant with additional loss-of-function SLC6A7 and MPPE1 gene variants, which potentially contribute to a blended phenotype caused by multilocus pathogenic variants. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Drug dose and animal welfare: important considerations in the treatment of wildlife.

A recent publication in Parasitology Research by (Old et al. Parasitol Res 120:1077-1090, 2021) raises the topical and often controversial issue of the treatment of wildlife by personnel with little or no formal scientific training (e.g. wildlife carers). In a valuable contribution to the subject, Old and colleagues document a wide range of topical (pour-on) application doses and frequencies of moxidectin (Cydectin®) administered in situ to bare-nosed wombats (Vombatus ursinus) by members of the wildlife carer/treater community in southeast Australia to treat sarcoptic mange disease. This treatment occurred under minor use permits issued by the Australian Pesticides and Veterinary Management Authority (APVMA). These permits do not require veterinary supervision, although carers are registered and are expected to comply with the guidelines of this permit.The prevalence and severity of sarcoptic mange in wildlife is influenced by a variety of factors including mite biology, environmental conditions, population density, animal behaviour and immune susceptibility (Browne et al. Bioscience, 2021). In bare-nosed wombats, combinations of these elements play a substantial role in making the treatment of an already difficult disease more complex. (Moroni et al. Parasit Vectors 13:471, 2020) comment that any pharmacological treatment of free-ranging wildlife must consider these factors when assessing their feasibility and implications, especially in the context of emerging drug resistance and potential long-term ecological impacts. As individuals with significant interest in sarcoptic mange and representing a range of professional research and veterinary expertise, we see value in providing expert commentary on this issue.

Glycine Receptor Complex Analysis Using Immunoprecipitation-Blue Native Gel Electrophoresis-Mass Spectrometry.

The pentameric glycine receptor (GlyR), comprising the α1 and ß subunits, is a major inhibitory ionotropic receptor in brainstem and spinal cord. GlyRs interact with gephyrin (GPHN), a scaffold protein that anchors the GlyR in the plasma membrane and enables it to form clusters in glycinergic postsynapses. Using an interaction proteomics approach, evidence of the ArfGEFs IQ motif and Sec7 domain 3 (IQSEC3) and IQ motif and Sec7 domain 2 (IQSEC2) as two novel synaptic proteins interacting with GlyR complexes is provided. When the affinity-isolated GlyR complexes are fractionated by blue native gel electrophoresis and characterized by mass spectrometry, GlyR α1ß-GPHN appears as the most abundant complex with a molecular weight of ≈1 MDa, and GlyR α1ß-GPHN-IQSEC3 as a minor protein complex of ≈1.2 MDa. A third GlyR α1ß-GPHN-IQSEC2 complex exists at the lowest amount with a mass similar to the IQSEC3 containing complex. Using yeast two-hybrid it is demonstrated that IQSEC3 interacts with the GlyR complex by binding to the GPHN G domain at the N-terminal of the IQSEC3 IQ-like domain. The data provide direct evidence of the interaction of IQSEC3 with GlyR-GPHN complexes, underscoring a potential role of these ArfGEFs in the function of glycinergic synapses.

Effects of GluN2A and GluN2B gain-of-function epilepsy mutations on synaptic currents mediated by diheteromeric and triheteromeric NMDA receptors.

Mutations in synaptic NMDA receptors (NMDARs) are associated with epilepsy and neurodevelopmental disorders. The effects of several such mutations have been investigated in recombinantly-expressed NMDARs under conditions of steady-state activation. Such experiments provide only limited insight into how mutations affect NMDAR-mediated excitatory synaptic currents (EPSCs). The present study aimed to characterize the effects of the GluN2AN615K, GluN2BN615I and GluN2BV618G gain-of-function mutations on EPSCs mediated by diheteromeric GluN1/2A and GluN1/2B receptors and triheteromeric GluN1/2A/2B receptors, as these are the most abundant synaptic NMDARs in vivo. Subunit composition was controlled by studying 'artificial' synapses formed between cultured neurons (which provide presynaptic terminals) and HEK293 cells that express the NMDAR subunits of interest plus the synapse-promoting molecule, neuroligin-1B. When incorporated into diheteromeric receptors, all three mutations ablated voltage-dependent Mg2+ block of EPSCs, as previously shown. In addition, we were surprised to find that increasing external Mg2+ from 0 to 1 mM strongly enhanced the magnitude of EPSCs mediated by mutant diheteromers. In contrast, triheteromeric receptors exhibited normal voltage-dependent Mg2+ block. The GluN2AN615K mutation also slowed the decay of GluN1/2A/2B- but not GluN1/2A-mediated EPSCs. The GluN2BN615I mutation enhanced the magnitude of both GluN1/2B- and GluN1/2A/2B-mediated EPSCs. The GluN2BV618G mutation enhanced the magnitude of both GluN1/2B- and GluN1/2A/2B-mediated EPSCs, although these effects were partly compensated by a faster EPSC decay rate. The mutations also diminished the potency of the anti-epileptic pore-blocker, memantine, thus explaining the lack of memantine efficacy in patients with GluN2BN615I or GluN2BV618G mutations. Given these effects, the three mutations would be expected to enhance the cation influx rate and thereby contribute to epilepsy phenotypes.

IQSEC2 mutation update and review of the female-specific phenotype spectrum including intellectual disability and epilepsy.

The IQSEC2- related disorders represent a spectrum of X-chromosome phenotypes with intellectual disability (ID) as the cardinal feature. Here, we review the increasing number of reported families and isolated cases have been reported with a variety of different pathogenic variants. The spectrum of clinical features is expanding with early-onset seizures as a frequent comorbidity in both affected male and female patients. There is a growing number of female patients with de novo loss-of-function variants in IQSEC2 have a more severe phenotype than the heterozygous state would predict, particularly if IQSEC2 is thought to escape X-inactivation. Interestingly, these findings highlight that the classical understanding of X-linked inheritance does not readily explain the emergence of these affected females, warranting further investigations into the underlying mechanisms.

Disruption of a Structurally Important Extracellular Element in the Glycine Receptor Leads to Decreased Synaptic Integration and Signaling Resulting in Severe Startle Disease.

Functional impairments or trafficking defects of inhibitory glycine receptors (GlyRs) have been linked to human hyperekplexia/startle disease and autism spectrum disorders. We found that a lack of synaptic integration of GlyRs, together with disrupted receptor function, is responsible for a lethal startle phenotype in a novel spontaneous mouse mutant shaky, caused by a missense mutation, Q177K, located in the extracellular ß8-ß9 loop of the GlyR α1 subunit. Recently, structural data provided evidence that the flexibility of the ß8-ß9 loop is crucial for conformational transitions during opening and closing of the ion channel and represents a novel allosteric binding site in Cys-loop receptors. We identified the underlying neuropathological mechanisms in male and female shaky mice through a combination of protein biochemistry, immunocytochemistry, and both in vivo and in vitro electrophysiology. Increased expression of the mutant GlyR α1Q177K subunit in vivo was not sufficient to compensate for a decrease in synaptic integration of α1Q177Kß GlyRs. The remaining synaptic heteromeric α1Q177Kß GlyRs had decreased current amplitudes with significantly faster decay times. This functional disruption reveals an important role for the GlyR α1 subunit ß8-ß9 loop in initiating rearrangements within the extracellular-transmembrane GlyR interface and that this structural element is vital for inhibitory GlyR function, signaling, and synaptic clustering.SIGNIFICANCE STATEMENT GlyR dysfunction underlies neuromotor deficits in startle disease and autism spectrum disorders. We describe an extracellular GlyR α1 subunit mutation (Q177K) in a novel mouse startle disease mutant shaky Structural data suggest that during signal transduction, large transitions of the ß8-ß9 loop occur in response to neurotransmitter binding. Disruption of the ß8-ß9 loop by the Q177K mutation results in a disruption of hydrogen bonds between Q177 and the ligand-binding residue R65. Functionally, the Q177K change resulted in decreased current amplitudes, altered desensitization decay time constants, and reduced GlyR clustering and synaptic strength. The GlyR ß8-ß9 loop is therefore an essential regulator of conformational rearrangements during ion channel opening and closing.

Cerebral Cortical Circuitry Formation Requires Functional Glycine Receptors.

The development of the cerebral cortex is a complex process that requires the generation, migration, and differentiation of neurons. Interfering with any of these steps can impair the establishment of connectivity and, hence, function of the adult brain. Neurotransmitter receptors have emerged as critical players to regulate these biological steps during brain maturation. Among them, α2 subunit-containing glycine receptors (GlyRs) regulate cortical neurogenesis and the present work demonstrates the long-term consequences of their genetic disruption on neuronal connectivity in the postnatal cerebral cortex. Our data indicate that somatosensory cortical neurons of Glra2 knockout mice (Glra2KO) have more dendritic branches with an overall increase in total spine number. These morphological defects correlate with a disruption of the excitation/inhibition balance, thereby increasing network excitability and enhancing susceptibility to epileptic seizures after pentylenetetrazol tail infusion. Taken together, our findings show that the loss of embryonic GlyRα2 ultimately impairs the formation of cortical circuits in the mature brain.

Murine startle mutant Nmf11 affects the structural stability of the glycine receptor and increases deactivation.

KEY POINTS: Hyperekplexia or startle disease is a serious neurological condition affecting newborn children and usually involves dysfunctional glycinergic neurotransmission. Glycine receptors (GlyRs) are major mediators of inhibition in the spinal cord and brainstem. A missense mutation, replacing asparagine (N) with lysine (K), at position 46 in the GlyR α1 subunit induced hyperekplexia following a reduction in the potency of the transmitter glycine; this resulted from a rapid deactivation of the agonist current at mutant GlyRs. These effects of N46K were rescued by mutating a juxtaposed residue, N61 on binding Loop D, suggesting these two asparagines may interact. Asparagine 46 is considered to be important for the structural stability of the subunit interface and glycine binding site, and its mutation represents a new mechanism by which GlyR dysfunction induces startle disease. ABSTRACT: Dysfunctional glycinergic inhibitory transmission underlies the debilitating neurological condition, hyperekplexia, which is characterised by exaggerated startle reflexes, muscle hypertonia and apnoea. Here we investigated the N46K missense mutation in the GlyR α1 subunit gene found in the ethylnitrosourea (ENU) murine mutant, Nmf11, which causes reduced body size, evoked tremor, seizures, muscle stiffness, and morbidity by postnatal day 21. Introducing the N46K mutation into recombinant GlyR α1 homomeric receptors, expressed in HEK cells, reduced the potencies of glycine, ß-alanine and taurine by 9-, 6- and 3-fold respectively, and that of the competitive antagonist strychnine by 15-fold. Replacing N46 with hydrophobic, charged or polar residues revealed that the amide moiety of asparagine was crucial for GlyR activation. Co-mutating N61, located on a neighbouring ß loop to N46, rescued the wild-type phenotype depending on the amino acid charge. Single-channel recording identified that burst length for the N46K mutant was reduced and fast agonist application revealed faster glycine deactivation times for the N46K mutant compared with the WT receptor. Overall, these data are consistent with N46 ensuring correct alignment of the α1 subunit interface by interaction with juxtaposed residues to preserve the structural integrity of the glycine binding site. This represents a new mechanism by which GlyR dysfunction induces startle disease.

Control of Inhibition by the Direct Action of Cannabinoids on GABAA Receptors.

Cannabinoids are known to regulate inhibitory synaptic transmission via activation of presynaptic G protein-coupled cannabinoid CB1 receptors (CB1Rs). Additionally, recent studies suggest that cannabinoids can also directly interact with recombinant GABAA receptors (GABAARs), potentiating currents activated by micromolar concentrations of γ-aminobutyric acid (GABA). However, the impact of this direct interaction on GABAergic inhibition in central nervous system is unknown. Here we report that currents mediated by recombinant GABAARs activated by high (synaptic) concentrations of GABA as well as GABAergic inhibitory postsynaptic currents (IPSCs) at neocortical fast spiking (FS) interneuron to pyramidal neuron synapses are suppressed by exogenous and endogenous cannabinoids in a CB1R-independent manner. This IPSC suppression may account for disruption of inhibitory control of pyramidal neurons by FS interneurons. At FS interneuron to pyramidal neuron synapses, endocannabinoids induce synaptic low-pass filtering of GABAAR-mediated currents evoked by high-frequency stimulation. The CB1R-independent suppression of inhibition is synapse specific. It does not occur in CB1R containing hippocampal cholecystokinin-positive interneuron to pyramidal neuron synapses. Furthermore, in contrast to synaptic receptors, the activity of extrasynaptic GABAARs in neocortical pyramidal neurons is enhanced by cannabinoids in a CB1R-independent manner. Thus, cannabinoids directly interact differentially with synaptic and extrasynaptic GABAARs, providing a potent novel context-dependent mechanism for regulation of inhibition.

GLRB is the third major gene of effect in hyperekplexia.

Glycinergic neurotransmission is a major inhibitory influence in the CNS and its disruption triggers a paediatric and adult startle disorder, hyperekplexia. The postsynaptic α(1)-subunit (GLRA1) of the inhibitory glycine receptor (GlyR) and the cognate presynaptic glycine transporter (SLC6A5/GlyT2) are well-established genes of effect in hyperekplexia. Nevertheless, 52% of cases (117 from 232) remain gene negative and unexplained. Ligand-gated heteropentameric GlyRs form chloride ion channels that contain the α(1) and ß-subunits (GLRB) in a 2α(1):3ß configuration and they form the predominant population of GlyRs in the postnatal and adult human brain, brainstem and spinal cord. We screened GLRB through 117 GLRA1- and SLC6A5-negative hyperekplexia patients using a multiplex-polymerase chain reaction and Sanger sequencing approach. The screening identified recessive and dominant GLRB variants in 12 unrelated hyperekplexia probands. This primarily yielded homozygous null mutations, with nonsense (n = 3), small indel (n = 1), a large 95 kb deletion (n = 1), frameshifts (n = 1) and one recurrent splicing variant found in four cases. A further three cases were found with two homozygous and one dominant GLRB missense mutations. We provide strong evidence for the pathogenicity of GLRB mutations using splicing assays, deletion mapping, cell-surface biotinylation, expression studies and molecular modelling. This study describes the definitive assignment of GLRB as the third major gene for hyperekplexia and impacts on the genetic stratification and biological causation of this neonatal/paediatric disorder. Driven principally by consanguineous homozygosity of GLRB mutations, the study reveals long-term additive phenotypic outcomes for affected cases such as severe apnoea attacks, learning difficulties and developmental delay.

Control of ethanol sensitivity of the glycine receptor α3 subunit by transmembrane 2, the intracellular splice cassette and C-terminal domains.

Previous studies have shown that the effect of ethanol onglycine receptors (GlyRs) containing the a1 subunit is affected by interaction with heterotrimeric G proteins (Gßγ). GlyRs containing the α3 subunit are involved in inflammatory pain sensitization and rhythmic breathing and have received much recent attention. For example, it is unknown whether ethanol affects the function of this important GlyR subtype. Electrophysiologic experiments showed that GlyR α3 subunits were not potentiated by pharmacologic concentrations of ethanol or by Gßγ. Thus, we studied GlyR α1­α3 chimeras and mutants to determine the molecular properties that confer ethanol insensitivity. Mutation of corresponding glycine 254 in transmembrane domain 2 (TM2) found in a1 in the α3(A254G) ­α1 chimera induced a glycine-evoked current that displayed potentiation during application of ethanol (46 ± 5%, 100 mM) and Gßγ activation (80 ± 17%). Interestingly,insertion of the intracellular α3L splice cassette into GlyR α1 abolished the enhancement of the glycine-activated current by ethanol (5 ± 6%) and activation by Gßγ (21 6 7%). In corporation of the GlyR α1 C terminus into the ethanol-resistant α3S(A254G) mutant produced a construct that displayed potentiation of the glycine-activated current with 100 mM ethanol (40 ± 6%)together with a current enhancement after G protein activation (68 ± 25%). Taken together, these data demonstrate that GlyRα3 subunits are not modulated by ethanol. Residue A254 in TM2, the α3L splice cassette, and the C-terminal domain of α3GlyRs are determinants for low ethanol sensitivity and form the molecular basis of subtype-selective modulation of GlyRs by alcohol.

GRIN2B mutations in West syndrome and intellectual disability with focal epilepsy.

OBJECTIVE: To identify novel epilepsy genes using a panel approach and describe the functional consequences of mutations. METHODS: Using a panel approach, we screened 357 patients comprising a vast spectrum of epileptic disorders for defects in genes known to contribute to epilepsy and/or intellectual disability (ID). After detection of mutations in a novel epilepsy gene, we investigated functional effects in Xenopus laevis oocytes and screened a follow-up cohort. RESULTS: We revealed de novo mutations in GRIN2B encoding the NR2B subunit of the N-methyl-D-aspartate (NMDA) receptor in 2 individuals with West syndrome and severe developmental delay as well as 1 individual with ID and focal epilepsy. The patient with ID and focal epilepsy had a missense mutation in the extracellular glutamate-binding domain (p.Arg540His), whereas both West syndrome patients carried missense mutations within the NR2B ion channel-forming re-entrant loop (p.Asn615Ile, p.Val618Gly). Subsequent screening of 47 patients with unexplained infantile spasms did not reveal additional de novo mutations, but detected a carrier of a novel inherited GRIN2B splice site variant in close proximity (c.2011-5_2011-4delTC). Mutations p.Asn615Ile and p.Val618Gly cause a significantly reduced Mg(2+) block and higher Ca(2+) permeability, leading to a dramatically increased Ca(2+) influx, whereas p.Arg540His caused less severe disturbance of channel function, corresponding to the milder patient phenotype. INTERPRETATION: We identified GRIN2B gain-of-function mutations as a cause of West syndrome with severe developmental delay as well as of ID with childhood onset focal epilepsy. Severely disturbed channel function corresponded to severe clinical phenotypes, underlining the important role of facilitated NMDA receptor signaling in epileptogenesis.

Mutations in the gene encoding GlyT2 (SLC6A5) define a presynaptic component of human startle disease.

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) alpha1 subunit (GLRA1). Genetic heterogeneity has been confirmed in rare sporadic cases, with mutations affecting other postsynaptic glycinergic proteins including the GlyR beta subunit (GLRB), gephyrin (GPHN) and RhoGEF collybistin (ARHGEF9). However, many individuals diagnosed with sporadic hyperekplexia do not carry mutations in these genes. Here we show that missense, nonsense and frameshift mutations in SLC6A5 (ref. 8), encoding the presynaptic glycine transporter 2 (GlyT2), also cause hyperekplexia. Individuals with mutations in SLC6A5 present with hypertonia, an exaggerated startle response to tactile or acoustic stimuli, and life-threatening neonatal apnea episodes. SLC6A5 mutations result in defective subcellular GlyT2 localization, decreased glycine uptake or both, with selected mutations affecting predicted glycine and Na+ binding sites.

Defective escape behavior in DEAH-box RNA helicase mutants improved by restoring glycine receptor expression.

RNA helicases regulate RNA metabolism, but their substrate specificity and in vivo function remain largely unknown. We isolated spontaneous mutant zebrafish that exhibit an abnormal dorsal bend at the beginning of tactile-evoked escape swimming. Similar behavioral defects were observed in zebrafish embryos treated with strychnine, which blocks glycine receptors (GlyRs), suggesting that the abnormal motor response in mutants may be attributable to a deficit in glycinergic synaptic transmission. We identified a missense mutation in the gene encoding RNA helicase Dhx37. In Dhx37 mutants, ribosomal RNA levels were unchanged, whereas GlyR α1, α3, and α4a subunit mRNA levels were decreased due to a splicing defect. We found that Dhx37 can interact with GlyR α1, α3, and α4a transcripts but not with the GlyR α2 subunit mRNA. Overexpression of GlyR α1, α3, or α4a subunits in Dhx37-deficient embryos restored normal behavior. Conversely, antisense-mediated knockdown of multiple GlyR α subunits in wild-type embryos was required to recapitulate the Dhx37 mutant phenotype. These results indicate that Dhx37 is specifically required for the biogenesis of a subset of GlyR α subunit mRNAs, thereby regulating glycinergic synaptic transmission and associated motor behaviors. To our knowledge, this is the first identification of pathologically relevant substrates for an RNA helicase.

The mitochondrial protease HtrA2 is regulated by Parkinson's disease-associated kinase PINK1.

In mice, targeted deletion of the serine protease HtrA2 (also known as Omi) causes mitochondrial dysfunction leading to a neurodegenerative disorder with parkinsonian features. In humans, point mutations in HtrA2 are a susceptibility factor for Parkinson's disease (PARK13 locus). Mutations in PINK1, a putative mitochondrial protein kinase, are associated with the PARK6 autosomal recessive locus for susceptibility to early-onset Parkinson's disease. Here we determine that HtrA2 interacts with PINK1 and that both are components of the same stress-sensing pathway. HtrA2 is phosphorylated on activation of the p38 pathway, occurring in a PINK1-dependent manner at a residue adjacent to a position found mutated in patients with Parkinson's disease. HtrA2 phosphorylation is decreased in brains of patients with Parkinson's disease carrying mutations in PINK1. We suggest that PINK1-dependent phosphorylation of HtrA2 might modulate its proteolytic activity, thereby contributing to an increased resistance of cells to mitochondrial stress.