Robert J. Harvey

Description: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.

Description: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.

Description: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.

Description: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 cotransporter 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. 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 Madin-Darby canine kidney cells. 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 the complete loss of sulfate transport activity. 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.

Description:Objective: 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. Conclusions: These findings confirm that GTF3C3 variants result in an autosomal recessive form of syndromic intellectual disability.