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1.  Startle disease in Irish wolfhounds associated with a microdeletion in the glycine transporter GlyT2 gene 
Neurobiology of Disease  2011;43(1):184-189.
Defects in glycinergic synaptic transmission in humans, cattle, and rodents result in an exaggerated startle reflex and hypertonia in response to either acoustic or tactile stimuli. Molecular genetic studies have determined that mutations in the genes encoding the postsynaptic glycine receptor (GlyR) α1 and β subunits (GLRA1 and GLRB) and the presynaptic glycine transporter GlyT2 (SLC6A5) are the major cause of these disorders. Here, we report the first genetically confirmed canine cases of startle disease. A litter of seven Irish wolfhounds was identified in which two puppies developed muscle stiffness and tremor in response to handling. Although sequencing of GLRA1 and GLRB did not reveal any pathogenic mutations, analysis of SLC6A5 revealed a homozygous 4.2 kb microdeletion encompassing exons 2 and 3 in both affected animals. This results in the loss of part of the large cytoplasmic N-terminus and all subsequent transmembrane domains due to a frameshift. This genetic lesion was confirmed by defining the deletion breakpoint, Southern blotting, and multiplex ligation-dependent probe amplification (MLPA). This analysis enabled the development of a rapid genotyping test that revealed heterozygosity for the deletion in the dam and sire and three other siblings, confirming recessive inheritance. Wider testing of related animals has identified a total of 13 carriers of the SLC6A5 deletion as well as non-carrier animals. These findings will inform future breeding strategies and enable a rational pharmacotherapy of this new canine disorder.
Research Highlights
► Startle disease is caused by mutations in glycine receptor and transporter genes. ► We report the first genetically confirmed cases of canine startle disease. ► We found a novel microdeletion in SLC6A5 encoding the glycine transporter GlyT2. ► We can now offer diagnostic testing of carriers and potentially treat this disorder. ► Deletion detection techniques should be applied in human startle disease genetics.
doi:10.1016/j.nbd.2011.03.010
PMCID: PMC4068303  PMID: 21420493
Glycine transporter; GlyT2; Hyperekplexia; Irish wolfhound; SLC6A5; Startle disease
2.  GRIN2B Mutations in West Syndrome and Intellectual Disability with Focal Epilepsy 
Annals of Neurology  2014;75(1):147-154.
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 Mg2+ block and higher Ca2+ permeability, leading to a dramatically increased Ca2+ 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.
doi:10.1002/ana.24073
PMCID: PMC4223934  PMID: 24272827
3.  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
4.  Distinct phenotypes in zebrafish models of human startle disease☆ 
Neurobiology of Disease  2013;60(100):139-151.
Startle disease is an inherited neurological disorder that causes affected individuals to suffer noise- or touch-induced non-epileptic seizures, excessive muscle stiffness and neonatal apnea episodes. Mutations known to cause startle disease have been identified in glycine receptor subunit (GLRA1 and GLRB) and glycine transporter (SLC6A5) genes, which serve essential functions at glycinergic synapses. Despite the significant successes in identifying startle disease mutations, many idiopathic cases remain unresolved. Exome sequencing in these individuals will identify new candidate genes. To validate these candidate disease genes, zebrafish is an ideal choice due to rapid knockdown strategies, accessible embryonic stages, and stereotyped behaviors. The only existing zebrafish model of startle disease, bandoneon (beo), harbors point mutations in glrbb (one of two zebrafish orthologs of human GLRB) that cause compromised glycinergic transmission and touch-induced bilateral muscle contractions. In order to further develop zebrafish as a model for startle disease, we sought to identify common phenotypic outcomes of knocking down zebrafish orthologs of two known startle disease genes, GLRA1 and GLRB, using splice site-targeted morpholinos. Although both morphants were expected to result in phenotypes similar to the zebrafish beo mutant, our direct comparison demonstrated that while both glra1 and glrbb morphants exhibited embryonic spasticity, only glrbb morphants exhibited bilateral contractions characteristic of beo mutants. Likewise, zebrafish over-expressing a dominant startle disease mutation (GlyR α1R271Q) exhibited spasticity but not bilateral contractions. Since GlyR βb can interact with GlyR α subunits 2–4 in addition to GlyR α1, loss of the GlyR βb subunit may produce more severe phenotypes by affecting multiple GlyR subtypes. Indeed, immunohistochemistry of glra1 morphants suggests that in zebrafish, alternate GlyR α subunits can compensate for the loss of the GlyR α1 subunit. To address the potential for interplay among GlyR subunits during development, we quantified the expression time-course for genes known to be critical to glycinergic synapse function. We found that GlyR α2, α3 and α4a are expressed in the correct temporal pattern and could compensate for the loss of the GlyR α1 subunit. Based on our findings, future studies that aim to model candidate startle disease genes in zebrafish should include measures of spasticity and synaptic development.
Graphical abstract
Highlights
•Morpholinos and dominant-negative mutants disrupted GlyR α1 subunit function.•Spasticity and delayed onset of rhythmic motor behaviors were observed.•GlyR βb subunit morphants exhibit both spasticity and bilateral muscle contractions.•Bilateral contractions likely result from disrupting multiple GlyR subtypes.•Spasticity may provide a common zebrafish ‘startle’ phenotype.
doi:10.1016/j.nbd.2013.09.002
PMCID: PMC3972633  PMID: 24029548
glra1; glrb; Glycine receptor; Startle disease; Hyperekplexia; Zebrafish
5.  Glycine Receptor α2 Subunit Activation Promotes Cortical Interneuron Migration 
Cell Reports  2013;4(4):738-750.
Summary
Glycine receptors (GlyRs) are detected in the developing CNS before synaptogenesis, but their function remains elusive. This study demonstrates that functional GlyRs are expressed by embryonic cortical interneurons in vivo. Furthermore, genetic disruption of these receptors leads to interneuron migration defects. We discovered that extrasynaptic activation of GlyRs containing the α2 subunit in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx, which further modulates actomyosin contractility to fine-tune nuclear translocation during migration. Taken together, our data highlight the molecular events triggered by GlyR α2 activation that control cortical tangential migration during embryogenesis.
Graphical Abstract
Highlights
•Migrating cortical interneurons express functional α2 subunit GlyRs•GlyRs activation controls interneuron migration by fine-tuning nucleokinesis•GlyR α2 activation promotes calcium oscillations and myosin II phosphorylation•GlyR α2 activation regulates actomyosin contractility during nucleokinesis
Glycine receptors (GlyRs) are detected in the developing CNS before synaptogenesis, but their function remains elusive. By combining in vitro with in vivo analyses, Rigo, Nguyen, and colleagues expand the known physiological functions of glycine and GlyRs in cerebral cortex development by demonstrating that endogenous activation of GlyR homomers composed of α2 subunits controls cortical interneuron migration.
doi:10.1016/j.celrep.2013.07.016
PMCID: PMC3763372  PMID: 23954789
6.  Subtle functional defects in the Arf-specific guanine nucleotide exchange factor IQSEC2 cause non-syndromic X-linked intellectual disability 
Small GTPases  2010;1(2):98-103.
Mutations in IQSEC2, a guanine nucleotide exchange factor for the ADP-ribosylation factor (Arf) family of small GTPases have recently been shown to cause non-syndromic X-linked intellectual disability (ID), characterised by substantial limitations in intellectual functioning and adaptive behaviour. This discovery was revealed by a combination of large-scale resequencing of the X chromosome, and key functional assays that revealed a reduction, but not elimination, of IQSEC2 GEF activity for mutations affecting conserved amino acids in the IQ-like and Sec7 domains. Compromised GTP binding activity of IQSEC2 leading to reduced activation of selected Arf substrates (Arf1, Arf6) is expected to impact on cytoskeletal organization, dendritic spine morphology and synaptic organisation. This study highlights the need for further investigation of the IQSEC gene family and Arf GTPases in neuronal morphology and synaptic function, and suggests that the genes encoding the ArfGEFs IQSEC1 and IQSEC3 should be considered as candidates for screening in autosomal ID.
doi:10.4161/sgtp.1.2.13285
PMCID: PMC3116596  PMID: 21686261
Arf GTPases; BRAG1; IQSEC2; genetics; IQ-ArfGEF
7.  Glycine Receptor Autoimmune Spectrum With Stiff-Man Syndrome Phenotype 
JAMA neurology  2013;70(1):44-50.
Objectives
To determine whether glycine receptor α1 subunit-specific autoantibodies (GlyRα1-IgG) occur in a broader spectrum of brainstem and spinal hyperexcitability disorders than the progressive encephalomyelitis with rigidity and myoclonus phenotype recognized to date, and to ascertain disease specificity.
Design
Retrospective, case-control study.
Settings
Mayo Clinic, Rochester, Minnesota, and University of Barcelona, Spain.
Patients
Eighty-one patients with stiff-man syndrome phenotype, 80 neurologic control subjects, and 20 healthy control subjects.
Intervention
Glycine receptor α1–transfected cells to test serum or cerebrospinal fluid from cases and control subjects.
Main Outcome Measures
Frequency of GlyRα1-IgG positivity among stiff-man syndrome phenotype cases and control subjects. Comparison of GlyRα1-IgG seropositive and seronegative cases.
Results
Seropositive cases (12% of cases) included 9 with stiff-man syndrome (4 classic; 5 variant; 66% were glutamic acid decarboxylase 65–IgG positive) and 1 with progressive encephalomyelitis with rigidity and myoclonus. Immunotherapy responses were noted more frequently in GlyRα1-IgG–positive cases (6 of 7 improved) than in seronegative cases (7 of 25 improved; P=.02). The single seropositive control patient had steroid-responsive vision loss and optic atrophy with inflammatory cerebrospinal fluid.
Conclusions
Glycine receptor α1–IgG aids identification of autoimmune brainstem/spinal cord hyperexcitability disorders and may extend to the glycinergic visual system.
doi:10.1001/jamaneurol.2013.574
PMCID: PMC3718477  PMID: 23090334
8.  Mutations in the guanine nucleotide exchange factor gene IQSEC2 cause nonsyndromic intellectual disability 
Nature genetics  2010;42(6):486-488.
The first family identified as having a nonsyndromic intellectual disability was mapped in 1988. Here we show that a mutation of IQSEC2, encoding a guanine nucleotide exchange factor for the ADP-ribosylation factor family of small GTPases, caused this disorder. In addition to MRX1, IQSEC2 mutations were identified in three other families with X-linked intellectual disability. This discovery was made possible by systematic and unbiased X chromosome exome resequencing.
doi:10.1038/ng.588
PMCID: PMC3632837  PMID: 20473311
9.  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
10.  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
11.  A canine BCAN microdeletion associated with episodic falling syndrome 
Neurobiology of Disease  2012;45(1):130-136.
Episodic falling syndrome (EFS) is a canine paroxysmal hypertonicity disorder found in Cavalier King Charles spaniels. Episodes are triggered by exercise, stress or excitement and characterized by progressive hypertonicity throughout the thoracic and pelvic limbs, resulting in a characteristic 'deer-stalking' position and/or collapse. We used a genome-wide association strategy to map the EFS locus to a 3.48 Mb critical interval on canine chromosome 7. By prioritizing candidate genes on the basis of biological plausibility, we found that a 15.7 kb deletion in BCAN, encoding the brain-specific extracellular matrix proteoglycan brevican, is associated with EFS. This represents a compelling causal mutation for EFS, since brevican has an essential role in the formation of perineuronal nets governing synapse stability and nerve conduction velocity. Mapping of the deletion breakpoint enabled the development of Multiplex PCR and Multiplex Ligation-dependent Probe Amplification (MLPA) genotyping tests that can accurately distinguish normal, carrier and affected animals. Wider testing of a larger population of CKCS dogs without a history of EFS from the USA revealed that carriers are extremely common (12.9%). The development of molecular genetic tests for the EFS microdeletion will allow the implementation of directed breeding programs aimed at minimizing the number of animals with EFS and enable confirmatory diagnosis and pharmacotherapy of affected dogs.
Highlights
► Episodic falling is a canine autosomal recessive paroxysmal hypertonicity disorder. ► We mapped the episodic falling locus to a 3.48 Mb region on canine chromosome 7. ► We found a novel microdeletion in BCAN encoding the proteoglycan brevican. ► Multiplex PCR and MLPA tests can distinguish normal, carrier and affected animals. ► BCAN mutations may underlie human exercise-induced hypertonicity disorders.
doi:10.1016/j.nbd.2011.07.014
PMCID: PMC3898273  PMID: 21821125
Brevican; BCAN; Episodic falling syndrome; Cavalier King Charles spaniels; Microdeletion
12.  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
14.  PINK1 cleavage at position A103 by the mitochondrial protease PARL 
Human Molecular Genetics  2010;20(5):867-879.
Mutations in PTEN-induced kinase 1 (PINK1) cause early onset autosomal recessive Parkinson's disease (PD). PINK1 is a 63 kDa protein kinase, which exerts a neuroprotective function and is known to localize to mitochondria. Upon entry into the organelle, PINK1 is cleaved to produce a ∼53 kDa protein (ΔN-PINK1). In this paper, we show that PINK1 is cleaved between amino acids Ala-103 and Phe-104 to generate ΔN-PINK1. We demonstrate that a reduced ability to cleave PINK1, and the consequent accumulation of full-length protein, results in mitochondrial abnormalities reminiscent of those observed in PINK1 knockout cells, including disruption of the mitochondrial network and a reduction in mitochondrial mass. Notably, we assessed three N-terminal PD-associated PINK1 mutations located close to the cleavage site and, while these do not prevent PINK1 cleavage, they alter the ratio of full-length to ΔN-PINK1 protein in cells, resulting in an altered mitochondrial phenotype. Finally, we show that PINK1 interacts with the mitochondrial protease presenilin-associated rhomboid-like protein (PARL) and that loss of PARL results in aberrant PINK1 cleavage in mammalian cells. These combined results suggest that PINK1 cleavage is important for basal mitochondrial health and that PARL cleaves PINK1 to produce the ΔN-PINK1 fragment.
doi:10.1093/hmg/ddq526
PMCID: PMC3033179  PMID: 21138942
15.  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
16.  TUBA1A mutations cause wide spectrum lissencephaly (smooth brain) and suggest that multiple neuronal migration pathways converge on alpha tubulins 
Human Molecular Genetics  2010;19(14):2817-2827.
We previously showed that mutations in LIS1 and DCX account for ∼85% of patients with the classic form of lissencephaly (LIS). Some rare forms of LIS are associated with a disproportionately small cerebellum, referred to as lissencephaly with cerebellar hypoplasia (LCH). Tubulin alpha1A (TUBA1A), encoding a critical structural subunit of microtubules, has recently been implicated in LIS. Here, we screen the largest cohort of unexplained LIS patients examined to date to determine: (i) the frequency of TUBA1A mutations in patients with lissencephaly, (ii) the spectrum of phenotypes associated with TUBA1A mutations and (iii) the functional consequences of different TUBA1A mutations on microtubule function. We identified novel and recurrent TUBA1A mutations in ∼1% of children with classic LIS and in ∼30% of children with LCH, making this the first major gene associated with the rare LCH phenotype. We also unexpectedly found a TUBA1A mutation in one child with agenesis of the corpus callosum and cerebellar hypoplasia without LIS. Thus, our data demonstrate a wider spectrum of phenotypes than previously reported and allow us to propose new recommendations for clinical testing. We also provide cellular and structural data suggesting that LIS-associated mutations of TUBA1A operate via diverse mechanisms that include disruption of binding sites for microtubule-associated proteins (MAPs).
doi:10.1093/hmg/ddq182
PMCID: PMC2893812  PMID: 20466733
17.  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
19.  Glycine and Glycine Receptor Signalling in Non-Neuronal Cells 
Glycine is an inhibitory neurotransmitter acting mainly in the caudal part of the central nervous system. Besides this neurotransmitter function, glycine has cytoprotective and modulatory effects in different non-neuronal cell types. Modulatory effects were mainly described in immune cells, endothelial cells and macroglial cells, where glycine modulates proliferation, differentiation, migration and cytokine production. Activation of glycine receptors (GlyRs) causes membrane potential changes that in turn modulate calcium flux and downstream effects in these cells. Cytoprotective effects were mainly described in renal cells, hepatocytes and endothelial cells, where glycine protects cells from ischemic cell death. In these cell types, glycine has been suggested to stabilize porous defects that develop in the plasma membranes of ischemic cells, leading to leakage of macromolecules and subsequent cell death. Although there is some evidence linking these effects to the activation of GlyRs, they seem to operate in an entirely different mode from classical neuronal subtypes.
doi:10.3389/neuro.02.009.2009
PMCID: PMC2737430  PMID: 19738917
glycine receptor; cytoprotection; glia; immune cells; renal cells; hepatocytes; endothelial cells
20.  A Selective Role for α3 Subunit Glycine Receptors in Inflammatory Pain 
GlyR α3 has previously been found to play a critical role in pain hypersensitivity following spinal PGE2 injection, complete Freund's adjuvant (CFA) and zymosan induced peripheral inflammation. In this study, although all models displayed typical phenotypic behaviours, no significant differences were observed when comparing the pain behaviours of Glra3−/− and wild-type littermates following the injection of capsaicin, carrageenan, kaolin/carrageenan or monosodium iodoacetate, models of rheumatoid and osteoarthritis, respectively. However, clear differences were observed following CFA injection (p < 0.01). No significant differences were observed in the pain behaviours of Glra3−/− and wild-type littermates following experimentally induced neuropathic pain (partial sciatic nerve ligation). Similarly, Glra3−/− and wild-type littermates displayed indistinguishable visceromotor responses to colorectal distension (a model of visceral pain) and in vivo spinal cord dorsal horn electrophysiology revealed no differences in responses to multimodal suprathreshold stimuli, intensities which equate to higher pain scores such as those reported in the clinic. These data suggest that apart from its clear role in CFA- and zymosan-induced pain sensitisation, hypersensitivity associated with other models of inflammation, neuropathy and visceral disturbances involves mechanisms other than the EP2 receptor – GlyR α3 pathway.
doi:10.3389/neuro.02.014.2009
PMCID: PMC2776487  PMID: 19915732
glycine α3 subunit; prostaglandin E2 inflammation; visceral pain; arthritis; electrophysiology
21.  Glycinergic Transmission in the Mammalian Retina 
Glycine and γ-aminobutyric acid (GABA) are the major inhibitory neurotransmitters in the retina. Approximately half of the amacrine cells release glycine at their synapses with bipolar, other amacrine, and ganglion cells. Glycinergic amacrine cells are small-field amacrine cells with vertically oriented dendrites and comprise more than 10 different morphological types. The retinal distributions of glycine receptor (GlyR) α1, α2, α3 and α4 subtypes have been mapped with subunit-specific antibodies. GlyRs were clustered at postsynaptic hot spots which showed selective distributions for the different subunits. As a rule, only one α subunit was expressed at a given postsynaptic site. The kinetic properties of GlyRs were measured by recording spontaneous inhibitory postsynaptic currents (sIPSCs) from identified retinal neurons in wild-type, Glra1spd-ot, Glra2 and Glra3 knockout mice. From observed differences of sIPSCs in wild-type and mutant mice, the cell-type specific subunit composition of GlyRs could be defined. OFF-cone bipolar cells and A-type ganglion cells receive prominent glycinergic input with fast kinetics that is mainly mediated by α1β GlyRs (decay time constant τ ∼ 5 ms). By contrast, AII amacrine cells express α3β GlyRs with medium fast kinetics (τ ∼ 11 ms). Narrow-field (NF) and wide-field amacrine cells contain predominantly α2β GlyRs with slow kinetics (τ ∼ 27 ms). Lastly, ON-starburst, narrow-field and wide-field amacrine cells in Glra2 knockout mice express α4β GlyRs with very slow kinetics (τ ∼ 70 ms).
doi:10.3389/neuro.02.006.2009
PMCID: PMC2777502  PMID: 19924257
glycine receptors; sIPSCs; retina; synapses; Glra1spd-ot mice; Glra2−/− mice; Glra3−/− mice
22.  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
23.  Mutations in α-Tubulin Cause Abnormal Neuronal Migration in Mice and Lissencephaly in Humans 
Cell  2007;128(1):45-57.
Summary
The development of the mammalian brain is dependent on extensive neuronal migration. Mutations in mice and humans that affect neuronal migration result in abnormal lamination of brain structures with associated behavioral deficits. Here, we report the identification of a hyperactive N-ethyl-N-nitrosourea (ENU)-induced mouse mutant with abnormalities in the laminar architecture of the hippocampus and cortex, accompanied by impaired neuronal migration. We show that the causative mutation lies in the guanosine triphosphate (GTP) binding pocket of α-1 tubulin (Tuba1) and affects tubulin heterodimer formation. Phenotypic similarity with existing mouse models of lissencephaly led us to screen a cohort of patients with developmental brain anomalies. We identified two patients with de novo mutations in TUBA3, the human homolog of Tuba1. This study demonstrates the utility of ENU mutagenesis in the mouse as a means to discover the basis of human neurodevelopmental disorders.
doi:10.1016/j.cell.2006.12.017
PMCID: PMC1885944  PMID: 17218254
24.  Anti-Human Immunodeficiency Virus Type 1 Activity of the Nonnucleoside Reverse Transcriptase Inhibitor GW678248 in Combination with Other Antiretrovirals against Clinical Isolate Viruses and In Vitro Selection for Resistance 
Antimicrobial Agents and Chemotherapy  2005;49(11):4465-4473.
GW678248, a novel nonnucleoside reverse transcriptase inhibitor, has been evaluated for anti-human immunodeficiency virus activity in a variety of in vitro assays against laboratory strains and clinical isolates. When GW678248 was tested in combination with approved drugs in the nucleoside and nucleotide reverse transcriptase inhibitor classes or the protease inhibitor class, the antiviral activities were either synergistic or additive. When GW678248 was tested in combination with approved drugs in the nonnucleoside reverse transcriptase inhibitor class, the antiviral activities were either additive or slightly antagonistic. Clinical isolates from antiretroviral drug-experienced patients were selected for evaluation of sensitivity to GW678248 in a recombinant virus assay. Efavirenz (EFV) and nevirapine (NVP) had ≥10-fold increases in their 50% inhibitory concentrations (IC50s) for 85% and 98% of the 55 selected isolates, respectively, whereas GW678248 had a ≥10-fold increase in the IC50 for only 17% of these isolates. Thus, 81 to 83% of the EFV- and/or NVP-resistant viruses from this data set were susceptible to GW678248. Virus populations resistant to GW678248 were selected by in vitro dose-escalating serial passage. Resistant progeny viruses recovered after eight passages had amino acid substitutions V106I, E138K, and P236L in the reverse transcriptase-coding region in one passage series and amino acid substitutions K102E, V106A, and P236L in a second passage series.
doi:10.1128/AAC.49.11.4465-4473.2005
PMCID: PMC1280120  PMID: 16251284
25.  The Human Cytomegalovirus UL44 Protein Is a Substrate for the UL97 Protein Kinase 
Journal of Virology  2003;77(14):7720-7727.
The human cytomegalovirus UL97 protein is an unusual protein kinase that is able to autophosphorylate and to phosphorylate certain exogenous substrates, including nucleoside analogs such as ganciclovir. However, no natural substrate of UL97 in infected cells has been identified. We report here that recombinant UL44 protein became radiolabeled when incubated with recombinant UL97 and [32P]ATP and that both proteins could be coimmunoprecipitated by an antibody that recognizes either protein. Subsequent studies showed that highly purified, recombinant UL97 phosphorylated purified, recombinant UL44. This phosphorylation occurred on serine and threonine residues and was sensitive to inhibition by maribavir and to a mutation that inactivates UL97 catalytic activity. Two-dimensional gel electrophoresis revealed the absence of specific phosphorylated forms of UL44 in immunoprecipitates from lysates of cells infected with a UL97 null mutant virus or with wild-type virus in the presence of maribavir. The results indicate that UL97 is sufficient to phosphorylate UL44 in vitro and is necessary for the normal phosphorylation of UL44 in infected cells. This strongly suggests that UL44 is a natural substrate of UL97.
doi:10.1128/JVI.77.14.7720-7727.2003
PMCID: PMC161957  PMID: 12829811

Results 1-25 (28)