Robert Harvey

Rare genetic variants in the glycine receptor (GlyR) α2 subunit gene ( GLRA2 ) are associated with autism spectrum disorder, developmental delay, and intellectual disability, often accompanied by microcephaly, language delay or epilepsy. We report detailed structure-function analyses of nine previously uncharacterised GlyR α2 missense variants, including a novel de novo change (p.S285P) linked to epileptic encephalopathy. Using molecular modelling/dynamics simulations, electrophysiology, and immunocytochemistry, we assessed effects of GlyR α2 variants on agonist potency, efficacy, channel gating, and cell-surface trafficking. Five missense variants caused a partial loss-of-function via reduced glycine potency (p.F20S, p.A261T, p.R418Q), reduced glycine efficacy (p.F20S), or faster channel deactivation (p.R323C, p.P369T). By contrast, p.R225C abolished cell-surface expression resulting in a complete loss-of-function . The p.A261T variant also significantly reduced picrotoxin binding, resolving ambiguity in GlyR-PTX interaction models. Additional variants showed an alteration-of-function (p.I232M) or a gain-of-function (p.S285P), combining reduced glycine efficacy with increased potency and spontaneous leak currents. Two variants within the intracellular M3-M4 domain (p.R323C and p.P369T) had enhanced channel deactivation consistent with a loss-of-function , while p.P373L showed no detectable functional deficit. These findings expand the clinical and mechanistic spectrum of GlyR α2 variants, identify a key determinant of picrotoxin binding, and highlight unresolved roles of intracellular protein-protein interaction motifs in GlyR α2 function.

The honeybee mite, Varroa destructor, finally breached Australia’s biosecurity defences four years ago, and is here to stay. Even more concerning, our standard treatments – such as specialised pesticides – are already failing.

What does this mean for Australians, and what can we do about it?

Roughly the size of a pinhead, the parasitic mite is regarded as the most destructive pest of honeybees worldwide. It feeds on bees, weakening colonies and causing their collapse.

For decades, Australia was the only continent free of the mite. That changed in 2022, when Varroa was detected in sentinel hives at the Port of Newcastle, New South Wales.

An ambitious eradication campaign was launched, but abandoned by 2023. Today, Varroa is established across much of Australia’s eastern and southern states. The focus has shifted from eradication to management, and we now face a new threat – treatment-resistant mites.

Pentameric glycine receptors (GlyRs) are key modulators of inhibitory neurotransmission, yet visualization of their activity across neuronal compartments has remained a challenge. Current methods that employ intracellularly tagged genetically encoded fluorescent proteins are prone to artefacts, as the tags can disrupt protein interactions that regulate receptor trafficking and positioning within the cell. We developed a novel, genetically encoded GlyRα2 activity reporter by fusing a chloride-sensitive fluorescent protein, mClYFP, to the extracellular N-terminus of GlyRα2. This chimeric receptor allows real-time nanoscopic visualization of the receptor and glycine-induced chloride concentration changes using total internal reflection fluorescence microscopy and ratio image analysis. Simultaneous electrophysiological and fluorescence measurements validated the functionality of both the ion channel and mClYFP components of our GlyRα2 activity reporter. The GlyRα2 ion channel characteristics are preserved, and the extracellular mClYFP tag reports chloride concentration changes in the physiological range. Therefore, mClYFP-GlyRα2 allowed us to detect receptor activity of chloride-permeable ionotropic receptors. In addition, we demonstrate that mClYFP-GlyRα2 can be effectively expressed in physiologically relevant striatal neurons. We present an extracellularly located, receptor-specific sensor that enables surface-accessible tracking of chloride ion dynamics in live cells. Our approach enables spatially resolved, non-invasive monitoring of chloride permeable receptor signaling, offering a powerful tool to investigate pentameric receptor function at the nanoscale.

Glycine receptors (GlyRs) are typically known for mediating inhibitory synaptic transmission within the spinal cord and brainstem, but they also have key roles in embryonic brain development, learning/memory, inflammatory pain sensitization, and rhythmic breathing. GlyR dysfunction has been implicated in multiple neurological disease states, including startle disease (GlyR α1β) and neurodevelopmental disorders (NDDs) including autism spectrum disorder (ASD), intellectual disability (ID), developmental delay (DD) and epilepsy (GlyR α2). However, GlyRs do not operate in isolation but depend upon stable and transient protein–protein interactions (PPIs) that influence synaptic localization, homeostasis, signaling pathways, and receptor function. Despite the affinity purification of GlyRs using the antagonist strychnine over four decades ago, we still have much to learn about native GlyR stoichiometry and accessory proteins. In contrast to other neurotransmitter receptors, < 20 potential GlyR interactors have been identified to date. These include some well-known proteins that are vital to inhibitory synapse function, such as the postsynaptic scaffolding protein gephyrin and the RhoGEF collybistin. However, the majority of known interactors either bind to the GlyR α1 and β subunits, or the binding partner in the GlyR complex is unknown. Several potential GlyR interactors are not found at inhibitory synapses and/or have no clear functional role. Moreover, other GlyR interactors are secondary interactors that bind indirectly, for example, via gephyrin. In this review, we provide a critical evaluation of known GlyR interacting proteins and methodological limitations to date. We also provide a road map for the use of innovative and emerging interaction proteomic techniques that will unlock the GlyR interactome. With the emergence of disease-associated missense mutations in the α1, α2 and β subunit intracellular domains in startle disease and NDDs, understanding the identity and roles of GlyR accessory proteins is vital in understanding GlyR function and dysfunction in health and disease.

Purpose
Sulfate is vital for many physiological processes, including the structural and functional maintenance of macromolecules and formation of sulfur-containing compounds essential for cartilage and bone development. SLC13A1 is a sodium-sulfate co-transporter primarily expressed in the kidney, where it mediates sulfate reabsorption and maintenance of circulating sulfate levels. In this study, we characterized the clinical, biochemical, and functional impact of biallelic SLC13A1 nonsense and/or missense variants in individuals presenting with a skeletal phenotype.
Methods
Probands were identified by exome or genome sequencing and GeneMatcher. Sulfate levels were quantified using ion chromatography. SLC13A1 missense variants p.(Arg237Cys), p.(Gly448Asp), p.(Leu516Pro), and p.(Tyr582His) were characterized using bioinformatics, molecular modeling, and [35S]-sulfate uptake assays in MDCK cells.
Results
All probands presented with concern for short stature and were found to have scoliosis and/or skeletal dysplasia. A reduction in plasma sulfate level and/or increase in urinary sulfate excretion was detected in 2 of 2 probands evaluated. Functional studies were consistent with SLC13A1 variants resulting in complete loss of sulfate transport activity.
Conclusion
Biallelic loss-of-function variants in SLC13A1 are a novel cause of skeletal phenotypes in humans with a measurable biomarker. Sulfate measurements should be considered in the clinical interpretation of variants identified in SLC13A1.

Purpose
This study details a novel syndromic form of autosomal recessive intellectual disability resulting from recessive variants in GTF3C3, encoding a key component of the DNA-binding transcription factor IIIC, which has a conserved role in RNA polymerase III-mediated transcription.

Methods
Exome sequencing, minigene analysis, molecular modeling, RNA polymerase III reporter gene assays, and Drosophila knockdown models were utilized to characterize GTF3C3 variants.

Results
Twelve affected individuals from 7 unrelated families were identified with homozygous or compound heterozygous missense variants in GTF3C3 including c.503C>T p.(Ala168Val), c.1268T>C p.(Leu423Pro), c.1436A>G p.(Tyr479Cys), c.2419C>T p.(Arg807Cys), and c.2420G>A p.(Arg807His). The cohort presented with intellectual disability, variable nonfamilial facial features, motor impairments, seizures, and cerebellar/corpus callosum malformations. Consistent with disruptions in intra- and intermolecular interactions observed in molecular modeling, RNA polymerase III reporter assays confirmed that the majority of missense variants resulted in a loss of function. Minigene analysis of the recurrent c.503C>T p.(Ala168Val) variant confirmed the introduction of a cryptic donor site into exon 4, resulting in mRNA missplicing. Consistent with the clinical features of this cohort, neuronal loss of Gtf3c3 in Drosophila induced seizure-like behavior, motor impairment, and learning deficits.

Conclusion
These findings confirm that GTF3C3 variants result in an autosomal recessive form of syndromic intellectual disability.

Background
Sarcoptic mange is a serious animal welfare concern in bare-nosed wombats (Vombatus ursinus). Fluralaner (Bravecto®) is a novel acaricide that has recently been utilised for treating mange in wombats. The topical ‘spot-on’ formulation of fluralaner can limit treatment delivery options in situ, but dilution to a volume for ‘pour-on’ delivery is one practicable solution. This study investigated the in vitro acaricidal activity of Bravecto, a proposed essential oil-based diluent (Orange Power®), and two of its active constituents, limonene and citral, against Sarcoptes scabiei.

Methods
Sarcoptes scabiei were sourced from experimentally infested pigs. In vitro assays were performed to determine the lethal concentration (LC50) and survival time of the mites when exposed to varying concentrations of the test solutions.

Results
All compounds were highly effective at killing mites in vitro. The LC50 values of Bravecto, Orange Power, limonene and citral at 1 h were 14.61 mg/ml, 4.50%, 26.53% and 0.76%, respectively. The median survival times of mites exposed to undiluted Bravecto, Orange Power and their combination were 15, 5 and 10 min, respectively. A pilot survival assay of mites collected from a mange-affected wombat showed survival times of < 10 min when exposed to Bravecto and Orange Power and 20 min when exposed to moxidectin.

Conclusions
These results confirm the acaricidal properties of Bravecto, demonstrate acaricidal properties of Orange Power and support the potential suitability of Orange Power and its active constituents as a diluent for Bravecto. As well as killing mites via direct exposure, Orange Power could potentially enhance the topical delivery of Bravecto to wombats by increasing drug penetration in hyperkeratotic crusts. Further research evaluating the physiochemical properties and modes of action of Orange Power and its constituents as a formulation vehicle would be of value.

Mitochondrial fission is critically important for controlling mitochondrial morphology, function, quality and transport. Drp1 is the master regulator driving mitochondrial fission, but exactly how Drp1 is regulated remains unclear. Here, we identified Drosophila Clueless and its mammalian orthologue CLUH as key regulators of Drp1. As with loss of drp1, depletion of clueless or CLUH results in mitochondrial elongation, while as with drp1 overexpression, clueless or CLUH overexpression leads to mitochondrial fragmentation. Importantly, drp1 overexpression rescues adult lethality, tissue disintegration and mitochondrial defects of clueless null mutants in Drosophila. Mechanistically, Clueless and CLUH promote recruitment of Drp1 to mitochondria from the cytosol. This involves CLUH binding to mRNAs encoding Drp1 receptors MiD49 and Mff, and regulation of their translation. Our findings identify a crucial role of Clueless and CLUH in controlling mitochondrial fission through regulation of Drp1.

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 Drosophila ZMYND8 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.