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Synaptic inhibition is essential for shaping the dynamics of neuronal networks, and aberrant inhibition is linked to epilepsy. Gephyrin (Geph) is the principal scaffolding protein at inhibitory synapses and is essential for postsynaptic clustering of glycine (GlyRs) and GABA type A receptors. Consequently, gephyrin is crucial for maintaining the relationship between excitation and inhibition in normal brain function and mutations in the gephyrin gene (GPHN) are associated with neurodevelopmental disorders and epilepsy. We identified bi-allelic variants in the GPHN gene, namely the missense mutation c.1264G > A and splice acceptor variant c.1315-2A > G, in a patient with developmental and epileptic encephalopathy. We demonstrate that the splice acceptor variant leads to nonsense-mediated mRNA decay. Furthermore, the missense variant (D422N) alters gephyrin structure, as examined by analytical size exclusion chromatography and circular dichroism-spectroscopy, thus leading to reduced receptor clustering and sensitivity towards calpain-mediated cleavage. In addition, both alterations contribute to an observed reduction of inhibitory signal transmission in neurons, which likely contributes to the pathological encephalopathy., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Sulfite oxidase (SO) is encoded by the nuclear SUOX gene and catalyzes the final step in cysteine catabolism thereby oxidizing sulfite to sulfate. Oxidation of sulfite is dependent on two cofactors within SO, a heme and the molybdenum cofactor (Moco), the latter forming the catalytic site of sulfite oxidation. SO localizes to the intermembrane space of mitochondria where both-pre-SO processing and cofactor insertion-are essential steps during SO maturation. Isolated SO deficiency (iSOD) is a rare inborn error of metabolism caused by mutations in the SUOX gene that lead to non-functional SO. ISOD is characterized by rapidly progressive neurodegeneration and death in early infancy. We diagnosed an iSOD patient with homozygous mutation of SUOX at c.1084G>A replacing Gly362 to serine. To understand the mechanism of disease, we expressed patient-derived G362S SO in Escherichia coli and surprisingly found full catalytic activity, while in patient fibroblasts no SO activity was detected, suggesting differences between bacterial and human expression. Moco reconstitution of apo-G362S SO was found to be approximately 90-fold reduced in comparison to apo-WT SO in vitro. In line, levels of SO-bound Moco in cells overexpressing G362S SO were significantly reduced compared to cells expressing WT SO providing evidence for compromised maturation of G362S SO in cellulo. Addition of molybdate to culture medium partially rescued impaired Moco binding of G362S SO and restored SO activity in patient fibroblasts. Thus, this study demonstrates the importance of the orchestrated maturation of SO and provides a first case of Moco-responsive iSOD., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Molybdenum cofactor deficiency (MoCD) is an autosomal recessive inborn error of metabolism characterized by neurodegeneration and death in early childhood. The rapid and progressive neurodegeneration in MoCD presents a major clinical challenge and may relate to the poor understanding of the molecular mechanisms involved. Recently, we reported that treating patients with cyclic pyranopterin monophosphate (cPMP) is a successful therapy for a subset of infants with MoCD and prevents irreversible brain damage. Here, we studied S-sulfocysteine (SSC), a structural analog of glutamate that accumulates in the plasma and urine of patients with MoCD, and demonstrated that it acts as an N-methyl D-aspartate receptor (NMDA-R) agonist, leading to calcium influx and downstream cell signaling events and neurotoxicity. SSC treatment activated the protease calpain, and calpain-dependent degradation of the inhibitory synaptic protein gephyrin subsequently exacerbated SSC-mediated excitotoxicity and promoted loss of GABAergic synapses. Pharmacological blockade of NMDA-R, calcium influx, or calpain activity abolished SSC and glutamate neurotoxicity in primary murine neurons. Finally, the NMDA-R antagonist memantine was protective against the manifestation of symptoms in a tungstate-induced MoCD mouse model. These findings demonstrate that SSC drives excitotoxic neurodegeneration in MoCD and introduce NMDA-R antagonists as potential therapeutics for this fatal disease.

Molybdenum cofactor (MoCo) deficiency is a rare, autosomal-recessive disorder, mainly caused by mutations in MOCS1 (MoCo deficiency type A) or MOCS2 (MoCo deficiency type B) genes; the absence of active MoCo results in a deficiency in all MoCo-dependent enzymes. Patients with MoCo deficiency present with neonatal seizures, feeding difficulties, severe developmental delay, brain atrophy and early childhood death. Although substitution therapy with cyclic pyranopterin monophosphate (cPMP) has been successfully used in both Mocs1 knockout mice and in patients with MoCo deficiency type A, there is currently no Mocs2 knockout mouse and no curative therapy for patients with MoCo deficiency type B. Therefore, we generated and characterized a Mocs2-null mouse model of MoCo deficiency type B. Expression analyses of Mocs2 revealed a ubiquitous expression pattern; however, at the cellular level, specific cells show prominent Mocs2 expression, e.g., neuronal cells in cortex, hippocampus and brainstem. Phenotypic analyses demonstrated that Mocs2 knockout mice failed to thrive and died within 11 days after birth. None of the tested MoCo-dependent enzymes were active in Mocs2-deficient mice, leading to elevated concentrations of purines, such as hypoxanthine and xanthine, and non-detectable levels of uric acid in the serum and urine. Moreover, elevated concentrations of S-sulfocysteine were measured in the serum and urine. Increased levels of xanthine resulted in bladder and kidney stone formation, whereas increased concentrations of toxic sulfite triggered neuronal apoptosis. In conclusion, Mocs2-deficient mice recapitulate the severe phenotype observed in humans and can now serve as a model for preclinical therapeutic approaches for MoCo deficiency type B.

Molybdenum cofactor deficiency (MoCD) is a rare inherited metabolic disorder characterized by severe and progressive neurological damage mainly caused by the loss of sulfite oxidase activity. Elevated urinary levels of sulfite, thiosulfate, and S-sulfocysteine (SSC) are hallmarks in the diagnosis of MoCD and sulfite oxidase deficiency (SOD). Recently, a first successful treatment of a human MoCD type A patient based on a substitution therapy with the molybdenum cofactor precursor cPMP has been reported, resulting in nearly complete normalization of MoCD biomarkers. Knowing the rapid progression of the disease symptoms in nontreated patients, an early diagnosis of MoCD as well as a sensitive method to monitor daily changes in SSC levels, a key marker of sulfite toxicity, is crucial for treatment outcome. Here, we describe a fast and sensitive method for the analysis of SSC in human urine samples using high performance liquid chromatography (HPLC). The analysis is based on precolumn derivatization with O-phthaldialdehyde (OPA) and separation on a C18 reverse phase column coupled to UV detection. The method was extended to human serum analysis and no interference with endogenous amino acids was found. Finally, SSC values from 45 pediatric urine, 75 adult urine, and 24 serum samples from control individuals as well as MoCD patients are reported. Our method represents a cost-effective technique for routine diagnosis of MoCD and SOD, and can be used also to monitor treatment efficiency in those sulfite toxicity disorders on a daily basis.

The molybdenum cofactor (Moco), a highly conserved pterin compound coordinating molybdenum (Mo), is required for the activity of all Mo-dependent enzymes with the exception of nitrogenase. Moco is synthesized by a unique and evolutionary old multi-step pathway with two intermediates identified so far, the sulfur-free and metal-free pterin derivative precursor Z and molybdopterin, a pterin with an enedithiolate function essential for Mo ligation. The latter pterin component is believed to form a tetrahydropyranopterin similar to the one found for Moco in the crystal structure of Mo as well as tungsten (W) enzymes. Here we report the spectroscopic characterization and structure elucidation of precursor Z purified from Escherichia coli overproducing MoaA and MoaC, two proteins essential for bacterial precursor Z synthesis. We have shown that purified precursor Z is as active as precursor Z present in E. coli cell extracts, demonstrating that no modifications during the purification procedure have occurred. High resolution electrospray ionization mass spectrometry afforded a [M + H]+ ion compatible with a molecular formula of C10H15N5O8P. Consequently 1H NMR spectroscopy not allowed structural characterization of the molecule but confirmed that this intermediate undergoes direct oxidation to the previously well characterized non-productive follow-up product compound Z. The 1H chemical shift and coupling constant data are incompatible with previous structural proposals and indicate that precursor Z already is a tetrahydropyranopterin system and carries a geminal diol function in the C1' position.