Robert Harvey

Biallelic loss-of-function variants in NRROS (Negative regulator of reactive oxygen species, previously LRRC33) cause a progressive neurodegenerative disorder characterized by early-onset seizures with brain calcifications. To date, 16 affected individuals from 13 unrelated families have been reported for this condition. We describe two siblings from a consanguineous family with this disorder, carrying a homozygous NRROS missense variant (c.1222A>C, p.(Asn408His), NM_198565.3). The mutational spectrum of NRROS disorder is characterized primarily by loss-of-function variants, with only four missense variants identified in affected individuals to date p.(Leu10Pro), p.(Leu62Pro), p.(Leu86Pro), and p.(Asn408His). We characterized these missense variants using silico predictions, molecular modelling and in-vitro cellular localization studies. In silico tools predict that p.(Leu10Pro) alters signal peptide cleavage, while p.(Asn408His) impedes N-linked glycosylation. Molecular modelling suggests that p.(Leu62Pro) and p.(Leu86Pro) variants may destabilize NRROS structure, while p.(Asn408His) interferes with NRROS glycosylation. In-vitro studies overexpressing HA-tagged NRROS wild type and missense variants in HEK293 cells showed altered subcellular distribution patterns for the variant proteins. In summary, we report a homozygous missense variant in NRROS associated with severe infantile epilepsy syndrome with intracranial calcifications, and neurodegeneration.
Biallelic loss-of-function variants in NRROS cause SENEBAC. Here, we report two affected siblings from a consanguineous family with a homozygous missense variant in NRROS. Preliminary functional assays in HEK293 cells for all the pathogenic NRROS missense variants, including the newly identified one, showed cellular mislocalization compared to the wild-type protein.

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.

Objective
Landau–Kleffner syndrome (LKS), is a rare, poorly-understood epileptic encephalopathy with spike–wave activation in sleep associated with mutations in GRIN2A, encoding the N-Methyl-D-Aspartate receptor (NMDAR) GluN2A subunit. Physicians rely on empirical treatments, with scarce information on treatment efficacy and outcomes. This study aims to improve the understanding and clinical management of LKS.

Methods
Fifty-two patients with LKS were recruited via one quaternary referral center. Case-notes review delineated clinical features, long-term outcomes, and prognostic factors. Generalized estimating equations were used to determine the longitudinal association among electroencephalogram abnormalities, steroid therapy, and neuropsychological findings. After genetic screening, the impact of identified GRIN2A missense variants on NMDAR function was assessed using homology modeling, cell-surface trafficking assays, and electrophysiology in artificial synapses. Whole exome/genome sequencing was performed on GRIN2A-negative patients to identify novel gene associations.

Results
LKS is complex with significant clinical and genetic heterogeneity. Besides speech and language impairment, many patients had other co-morbidities and almost half have long-term disability. Early age at disease onset was associated with worse outcomes. There was no reliable correlation between electroencephalogram findings and developmental scores. Steroid therapy improved language outcomes independently of electroencephalogram findings. GRIN2A mutations were identified in 15.5% of the cohort. Likely pathogenic variants in GABBR2, SCN1A, TRPC1, ERRFI1, CTXN3, IRX6, and IQCA1 were identified in 7 GRIN2A-negative individuals.

Interpretation
For LKS, early intervention is important for long-term outcomes. Furthermore, management should not be based solely on electroencephalogram findings. Genetic and functional investigations offer insights into disease pathophysiology and facilitate development of future targeted therapies. ANN NEUROL 2025

Hypertrophic scarring of the skin is a cause of pain, disfigurement, and restricted mobility. Excessive TGF-β1 signalling leads to SMAD3 phosphorylation, which is implicated in hypertrophic scarring. In this study, we examined the mechanism of action of tomentosenol A, a small compound that we isolated from the propolis of the Australian stingless bee Tetragonula carbonaria. Cultured adult human dermal fibroblasts and HEK293 cells were stimulated with TGF-β1, with or without tomentosenol A, and were assessed for phosphorylation of SMADs 2/3 (Western blot, AlphaLISA assay), SMAD signalling (HEK293 cells expressing a SMAD3 reporter gene), and profibrotic gene transcription using RTqPCR for ACTA2 (smooth muscle α-actin), COL1A1 and COL3A (collagens), CCN2 (connective tissue growth factor) and FN1 (fibronectin). Protein expression was measured using ELISA (fibronectin) and visualised via confocal microscopy (smooth muscle α-actin). TGF-β1 increased SMAD3 phosphorylation by 44.3-fold above baseline levels, and this effect was inhibited by tomentosenol A in a concentration-dependent manner (IC50, 99.0 nM). TGF-β1 stimulated SMAD3 reporter gene expression and upregulated ACTA2, COL1A1, COL3A1, FN1 and CCN2 transcription; fibronectin protein expression; and smooth muscle α-actin filament formation in fibroblasts. These responses were inhibited by 6.25 μM tomentosenol A. These findings indicate that tomentosenol A inhibits TGF-β1/SMAD3 signalling and downstream profibrotic gene transcription and protein expression. As this pathway is implicated in hypertrophic scarring of the skin, tomentosenol A can be developed as a novel therapy for the management of scars caused by deep dermal injuries that are associated with surgery, trauma and burns.

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.

Rare neurodevelopmental disorders (NDDs) are one of the most significant unmet challenges in healthcare due to their lifelong nature, high management costs, and recurrence within families. This review will focus on newly-emerging genetic forms of NDDs resulting from variants in the glycine receptor (GlyR) α2 subunit gene. Studies using Glra2 knockout mice have convincingly demonstrated that GlyR α2 is essential for cortical interneuron migration and progenitor homeostasis. Genetic inactivation of GlyR α2 impairs the capacity of apical progenitors to generate basal progenitors, resulting in an overall reduction of projection neurons in the cerebral cortex. As a result, microcephaly is observed in newborn Glra2 knockout mice, as well as defects in neuronal morphology, increased susceptibility to seizures, and defects in novel object recognition, motor memory consolidation, righting reflexes, novelty-induced locomotion in the open field test, and motivational reward tasks. Consistent with these findings, we and others have identified missense variants and microdeletions in the human GlyR α2 subunit gene (GLRA2) in individuals with autism spectrum disorder (ASD), developmental delay (DD) and/or intellectual disability (ID), often accompanied by microcephaly, language delay and epilepsy. In this review, we highlight the critical role of the GlyR α2 subunit revealed by knockout mice and our current understanding of GlyR α2 pathomechanisms in human NDDs. Finally, we will consider the current gaps in our knowledge, which include: (i) Limited functional validation for GlyR α2 missense variants associated with human NDDs; (ii) The lack of gain-of-function GlyR α2 mouse models; (iii) Our limited knowledge of GlyR α2 interacting proteins. We also highlight potential future developments in the field, including routes to personalized medicines for individuals with GlyR α2 mutations.