Biology subjects

Schwarz, G.

Publications and source records attributed to Schwarz, G..

13 recordsLinked to original sources

bioRxiv2026

Impaired gephyrin G-domain trimerization and phase separation in a patient with developmental epileptic encephalopathy

Epilepsy, a common neurological disorder is frequently linked to genetic variants in synaptic proteins. Here, we describe a de novo pathogenic missense variant in the gephyrin G-domain (G134R) identified in an epileptic patient with developmental delay and seizures. Functional analyses reveal that G134R disrupts higher-order oligomerization, leading to impaired liquid-liquid phase separation (LLPS) and synaptic clustering. Recombinant G134R-gephyrin variant forms lower oligomers with reduced molybdenum cofactor (Moco) synthesis. In non-neuronal cells, G134R fails to oligomerize beyond dimers and loses Moco synthesis function. In neurons, G134R is unable to form synaptic clusters and exerts a dominant-negative effect on WT-gephyrin, severely disrupting inhibitory synapse formation. Our findings highlight a critical role for the G-domain in gephyrin self-assembly and LLPS, shifting the focus from the E-domain-centric view of gephyrin function and providing a novel molecular mechanism for epilepsy linked to G-domain mutations.

neuroscience↗

bioRxiv2025

GephyrinΔ199-233 - an epileptogenic microdeletion

Gephyrin, as the main organizer of inhibitory synapses, is crucial for inhibitory signal transmission, and implicated in various neurological disorders. Various studies have identified gephyrin microdeletions in conditions of autism, schizophrenia, and epilepsy. Those deletions affected the N-terminal G-domain and/or the central C-domain of gephyrin while the receptor binding C-terminal E-domain was not affected. Here, we investigated the importance of a specific microdeletion ({Delta}199-233) within the C-domain using a full-body knock-in mouse model. Homozygous mice displayed a severe phenotype characterized by reduced fertility, increased mortality, and neurological deficits at early developmental stages. Analyses in dissociated hippocampal neurons demonstrated disrupted synaptic targeting of gephyrin {Delta}199-233 that harbors the functionally important S-palmitoylation site at Cys212. Simultaneously, we found adaptations at the excitatory synapse, with smaller, but more numerous clusters of the excitatory scaffolding protein PSD95. Although, gephyrin {Delta}199-233 showed unexpectedly a facilitated receptor interaction, inhibitory signal transmission was reduced. We hypothesize, that the gephyrin {Delta}199-233-mediated reduction of inhibition triggers compensatory excitation, which possibly fails and/or disrupts the excitation/inhibition ratio in our mouse model. These findings highlight the critical role of the gephyrin C-domain and its post-translational modifications in synaptic function and neuronal health, offering a novel mouse model for the development of potential therapeutic targets addressing gephyrin-associated neurological disorders.

neuroscience↗

bioRxiv2025

Phosphoinositide- and Collybistin-Dependent Synaptic Clustering of Gephyrin

Gephyrin is the main scaffolding protein at inhibitory synapses clustering glycine and GABA type A receptors. At specific GABAergic synapses, the nucleotide exchange factor collybistin recruits gephyrin to the postsynaptic membrane via interaction with phosphoinositides. However, the molecular mechanisms underlying the formation, maintenance and regulation of collybistin-dependent gephyrin clusters remain poorly understood. This study sheds light on the molecular mechanism of gephyrin cluster formation based on gephyrin self-oligomerization induced by collybistin, leading to the formation of a high-molecular weight (>5 MDa) gephyrin-collybistin complex, which is regulated in two ways: First, plasma-membrane phosphoinositides promote complex formation demonstrating their critical role in membrane targeting and stabilization of gephyrin-collybistin clusters at postsynaptic sites. Second, gephyrin phosphorylation at Ser325 abolishes complex formation with collybistin thus impairing collybistin-dependent gephyrin clustering at GABAergic synapses. Collectively, our data demonstrates a molecular mechanism for synaptic clustering of gephyrin which involves collybistin- and phosphoinositide-dependent formation of high-molecular gephyrin oligomers.

biochemistry↗

bioRxiv2024

Endocytic adaptor AP-2 maintains Purkinje cell function by balancing cerebellar parallel and climbing fiber synapses

The selective loss of cerebellar Purkinje cells is a hallmark of various neurodegenerative movement disorders, yet the precise mechanism driving their degeneration remains enigmatic. Here, we show that the endocytic adaptor protein complex 2 (AP-2) is essential for the survival of Purkinje cells. Employing a multidisciplinary approach encompassing mouse genetics, viral tracing, ex vivo calcium imaging, and kinematic analysis, we demonstrate that mice lacking the {micro}-subunit of AP-2 in cerebellar Purkinje cells exhibit early-onset ataxia associated with progressive Purkinje cell degeneration. Importantly, we uncover that synaptic input dysfunctions, characterized by a predominance of parallel fiber (PF) over climbing fiber (CF) synapses, precede Purkinje cell loss. Mechanistically, we find that AP-2 localizes to Purkinje cell dendrites, where it interacts with the PF synapse-enriched protein GRID2IP. The loss of AP-2 results in proteasome-dependent degradation of GRID2IP and accumulation of the glutamate {delta}2 receptor (GLUR{delta}2) in distal Purkinje cell dendrites, leading to an excess of PF synapses while CF synapses are drastically reduced. The overrepresentation of PF synaptic input induces Purkinje cell hyperexcitation, which can be alleviated by enhancing synaptic glutamate clearance using the antibiotic ceftriaxone. Our findings demonstrate the critical role of AP-2 in preventing motor gait dysfunctions by regulating GRID2IP levels in Purkinje cells, thereby preserving the equilibrium of PF and CF synaptic inputs in a cell-autonomous manner.

neuroscience↗

bioRxiv2024

Novel, postnatal manifestation of an epidermal barrier defect in a mouse model of isolated sulfite oxidase deficiency

Sulfite oxidase deficiency is a rare inborn error in metabolism leading to early childhood death due to rapidly progressing neurodegeneration. A new mouse model of sulfite oxidase deficiency carrying a homozygous deletion in the Suox gene resembles the human pathology in terms of neonatal death and elevation of sulfite and thiosulfate in plasma and urine, respectively. Homozygous Suox-/- mice are initially born healthy, display growth retardation starting at postnatal day 4 and die in average at day 9.6. Here we report that Suox-/- mice develop dry and scaly skin early postnatally, showing that sulfite oxidase is essential to maintain a functional skin barrier after birth. At postnatal day 5 Suox-/- mice develop altered epidermal morphology and dysregulated early and late keratinocyte differentiation accompanied by increased stress response. We propose a sulfite-induced cleavage of disulfide bonds in key epidermal proteins essential for a functional barrier.

biochemistry↗

bioRxiv2024

Redox-dependent synaptic clustering of gephyrin

Reactive oxygen species (ROS) play a central role in enhancing inhibitory signal transmission, thus extending their role beyond oxidative stress in disease and aging. However, the underlying molecular mechanisms mediating these functions have remained elusive. At inhibitory synapses, the scaffolding protein gephyrin clusters glycine and GABA type A receptors. Since gephyrin harbors multiple surface-exposed cysteines, we investigated the regulatory influence of ROS on gephyrin. We show that H2O2-induced oxidation of gephyrin cysteines triggered reversible, synaptic multimerization through disulfide bridge formation, which provided more receptor binding sites, lead to proteolytic protection and enhanced liquid-liquid phase separation. We identified mitochondria-derived ROS as a physiological source and observed oxidized gephyrin multimers in vivo, indicating that gephyrin can be regulated by the redox environment. Collectively, our findings suggest that cysteines in gephyrin modulate synaptic localization and clustering as regulatory redox-switches thereby establishing a link between neuronal and mitochondrial activity.

neuroscience↗

bioRxiv2024

Sulfite oxidase deficiency causes persulfidation loss and H2S release

Sulfite oxidase (SOX) deficiency is a rare inborn error of cysteine metabolism resulting in severe neurological damage. In patients, sulfite accumulates to toxic levels causing a raise in downstream products S-sulfocysteine (SSC), mediating excitotoxicity, and thiosulfate, a catabolic intermediate/product of H2S metabolism. Here, we report a full-body knock-out mouse model for SOX deficiency (SOXD) with a severely impaired phenotype. Amongst the urinary biomarkers, thiosulfate showed a 45-fold accumulation in SOXD mice representing the major excreted S-metabolite. Consistently, we found increased plasma H2S, which was derived from sulfite-induced release from persulfides as demonstrated in vitro and in vivo. Mass spectrometric analysis of total protein persulfidome identified a major loss of persulfidation in 20% of the proteome affecting enzymes in amino acids and fatty acid metabolism. Urinary amino acid profiles indicate metabolic rewiring suggesting partial reversal of the TCA cycle thus identifying a novel contribution of H2S metabolism and persulfidation in SOXD.

biochemistry↗

bioRxiv2024

Autophagy regulator ATG5 preserves cerebellar function by safeguarding its glycolytic activity

Dysfunctions in autophagy, a highly conserved cellular mechanism responsible for the degradation of intracellular components within lysosomes, often result in neurodegeneration. The neuroprotective effect of autophagy varies across neuronal subtypes, and the mechanisms of selective vulnerability of neurons to autophagy dysfunction are currently unknown. Utilizing a mouse model of ATG5 deficiency in inhibitory neurons and a comprehensive approach, including PET imaging, metabolomics, stable-isotope labeling studies, and live cell imaging, we establish that autophagy contributes to the survival of cerebellar Purkinje cells (PCs) by safeguarding their glycolytic activity. We show that the core autophagy protein ATG5 downregulates the levels of the glucose transporter 2 (GLUT2) during brain maturation. Autophagy-deficient PCs exhibit increased glucose uptake, along with elevated levels of glycolytic intermediates and methylglyoxal-modified proteins. We propose lysophosphatidic acid and serine as glycolytic intermediates inducing PC death and demonstrate that deletion of GLUT2 in ATG5-deficient mice mitigates PC neurodegeneration and restores their ataxic gait. Taken together, this study reveals a novel neuroprotective role of autophagy in preventing excessive glycolytic metabolism in the brain.

neuroscience↗

bioRxiv2022

Parallel Activation of Src and Hif1α Increases Localized Glycolytic ATP Generation for Re-assembly of Endothelial Adherens Junctions

Endothelial adherens junctions (AJs) are critical for the regulation of vascular barrier integrity and undergo dis-assembly during inflammatory injury, thus causing vascular leakiness. AJ re-assembly is thus necessary for restoration of the endothelial barrier following the initial injury. Here we examine the metabolic underpinnings that drive restoration of vascular integrity. In response to inflammatory stimuli, the glycolysis regulatory enzyme PFKFB3 is activated, resulting in a rapid and sustained increase of intracellular glycolytic ATP, especially in the proximity of AJs at the plasma membrane. We engineered a novel chemo-genetic construct (RapT) which allowed for precise temporal control of PFKFB3 recruitment to the plasma membrane. Activation of RapT by rapamycin during the barrier restoration phase increased regional ATP and accelerated AJ re-assembly. Mechanistically, we observed that PFKFB3 is activated through two modes. Src-mediated post-translational phosphorylation rapidly increases PFKFB3 activity. Using another chemo-genetic approach to temporally control Src activity, we found that Src activates PFKFB3 by binding to and phosphorylating it at residues Y175, Y334, and Y363. Tyrosine-phospho-deficient mutants of PFKFB3 at these residues block the glycolytic activation upon inflammatory stimuli. In parallel, elevated reactive oxygen species generated during inflammatory stimulation create pockets of regional hypoxia and allow for increased Hif1-mediated transcription of PFKFB3, leading to sustained glycolytic activation. Moreover, inhibition of PFKFB3 delays AJ reassembly and restoration of vascular integrity both in vitro and in vivo. In conclusion, we show that while inflammatory activation acutely compromises the endothelial barrier, inflammatory signaling also concomitantly generates a metabolic milieu in anticipation of the subsequent re-assembly of AJs and restoration of the vascular barrier.

cell biology↗

bioRxiv2022

Pentameric assembly of glycine receptor intracellular domains provides insights into gephyrin clustering

Pentameric ligand-gated ion channels represent a large family of receptors comprising an extracellular domain, four transmembrane helices and a cytosolic intracellular domain (ICD). ICDs play important roles in receptor localization and trafficking, thus regulating synaptic activity and plasticity. Glycine and GABA type A receptor ICDs bind to the scaffolding protein gephyrin, a master regulator of inhibitory synapses. Here we report the use of yeast lumazine synthase as soluble pentameric protein scaffold for the study of receptor ICDs derived from GlyR 1- and {beta}-subunits. We were able to create ICDs assemblies in a homo- (LS-{beta}ICD) and hetero-pentameric state (LS-{beta}ICD) and provide first-in-class structural insights on their high structural flexibility using small angle X-ray scattering. We report a high-affinity interaction between the LS-{beta}ICD and gephyrin leading to the in vitro formation of high-molecular mega-Dalton complexes composed of three gephyrin trimers and three pentamers as basic building block. Depending on the stoichiometric ratios between gephyrin and LS-ICDs the formed complexes grow or shrink in size. In cells, LS-ICDs efficiently recruited gephyrin and were able to accumulate gephyrin at GABAergic synapses in neurons. Our findings collectively propose a new, potentially general, mechanistic concept for a gephyrin-dependent bridging of GlyRs at the inhibitory synapse.

biochemistry↗

bioRxiv2021

A systematic analysis of diet-induced nephroprotection reveals overlapping and conserved changes in cysteine catabolism

Caloric Restriction (CR) extends lifespan and augments cellular stress-resistance from yeast to primates, making CR an attractive strategy for organ protection in the clinic. Translation of CR to patients is complex, due to problems regarding adherence, feasibility and safety concerns in frail patients. Novel tailored dietary regimens, which modulate the dietary composition of macro- and micronutrients rather than reducing calorie intake promise similar protective effects and increased translatability. However, a direct head-to-head comparison to identify the most potent approach for organ protection as well as overlapping metabolic consequences has not been performed. We systematically analyzed six dietary preconditioning protocols - fasting mimicking diet (FMD), ketogenic diet (KD), dietary restriction of branched chained amino acids (BCAA), two dietary regimens restricting sulfur-containing amino acids (SR80/100) and CR - in a rodent model of renal ischemia-reperfusion injury (IRI) to quantify diet-induced resilience in kidneys. Of the administered diets, FMD, SR80/100 and CR efficiently protect from kidney damage after IRI. Interestingly, these approaches show overlapping changes in oxidative and hydrogen sulfide (H2S)-dependent cysteine catabolism as a potential common mechanism of organ protection. Importantly, these metabolic changes can be recapitulated in patients preconditioned by a diet limiting sulfur-containing amino acids indicating conserved diet-induced mechanisms of stress-resistance that may ultimately lead to clinical application.

biochemistry↗

bioRxiv2020

Glycolysis Inhibition Regulates Endothelial Junctions by Perturbing Actin and Focal Adhesions

One of the central functions of the endothelium is to maintain a vascular barrier that prevents fluid leakiness and immune cell influx from the circulating blood into the tissue. The barrier integrity of the endothelium is largely controlled by adherens junctions (AJs) and the key AJ molecule VE-cadherin, which maintains cell cohesion via homotypic trans-interaction with VE-cadherin molecules on neighboring endothelial cells. Tension is required to maintain junction homeostasis, but little is known about the role of endothelial metabolism and bioenergetics in regulating junctional forces. Because glycolysis is the main source of ATP generation in endothelial cells, we examined the bioenergetic control of the mechanics of VE-cadherin junctions, by focusing on the glycolysis regulatory enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Results from traction force imbalance measurements and a VE-cadherin tension sensor revealed that inhibiting PFKFB3 significantly reduced the average junctional tension and the force on VE-cadherin complexes. The decrease in tension was largely due to mechanical changes distal from the cell-cell contacts. Specifically, inhibiting glycolysis perturbs focal adhesions and disrupts actin organization, directly impacting the net force on intercellular contacts. These findings identify a critical role of cellular metabolism for the mechanics and integrity of vascular endothelial junctions, by maintaining global cell mechanics. Statement of SignificanceThis study examines how forces at intercellular junctions are bioenergetically regulated. Results reveal altered mechanical force generation and transmission due to the breakdown of stress-transmitting fibers during lung injury. These junctions control the barrier function of the vascular endothelium, which requires tight inter cellular adhesions to prevent fluid and macromolecules from passing through the endothelial barrier. We determined how the availability of ATP affects the tension between human endothelial cells, by regulating forces produced remotely from the junctions. These global changes alter both the force at the junctions themselves, and the force transmitted across the entire cell through actin fibers.

biophysics↗

bioRxiv2018

Alternative splicing of bicistronic MOCS1 defines a novel mitochondrial protein maturation mechanism

Molybdenum cofactor biosynthesis is a conserved multistep pathway. The first step, the conversion of GTP to cyclic pyranopterin monophosphate (cPMP), requires bicsistronic MOCS1. Alternative splicing of MOCS1 in exons 1 and 9 produces four different N-terminal and three different C-terminal products (type I-III). Type I splicing results in bicistronic transcripts with two open reading frames, of which only the first, MOCS1A, is translated, whereas type II/III splicing produces two-domain MOCS1AB proteins. Here, we report and characterize the mitochondrial translocation of alternatively spliced MOCS1 proteins. While MOCS1A requires exon 1a for mitochondrial translocation, MOCS1AB variants target to mitochondria via an internal motif overriding the N-terminal targeting signal. Within mitochondria, MOCS1AB undergoes proteolytic cleavage resulting in mitochondrial matrix localization of the MOCS1B domain. In conclusion we found that MOCS1 produces two functional proteins, MOCS1A and MOCS1B, which follow different translocation routes before mitochondrial matrix import, where both proteins collectively catalyze cPMP biosynthesis. MOCS1 protein maturation provides a novel mechanism of alternative splicing ensuring the coordinated targeting of two functionally related mitochondrial proteins encoded by a single gene.

biochemistry↗