A 3D cellular breast‐to‐brain tumor model is developed using thiolated‐hyaluronic‐acid‐based hydrogel cross‐linked with extracellular matrix (ECM) proteins collagen type IV, laminin‐211, and fibronectin to mimic the native‐brain ECM. The 3D model resembles the basement membrane around the blood vessels, which surrounds the brain parenchyma and is important for initial survival of breast tumor cells passing the blood–brain barrier. Brain metastasis in breast cancer patients is one of the leading causes of mortality. While, supplemented ECM proteins do not significantly alter the nominal stress response of the hydrogel, the analysis of the tumor microenvironment interactions displays a significant impact of neuron and astrocyte presence on ECM secretion. Additional ECM supplementation does not further promote proliferation of breast tumor cells in a natively extrinsic surrounding. MDA‐MB‐361 exhibits numerous contacts with collagen type IV. Neuronal network activity is significantly increased in the presence of MDA‐MB‐361 cells. Increased colocalization of the vesicular glutamate transporter 1 on neurons and connexin 43 on astrocytes argues for beneficial interactions for the tumor's intrinsic proliferation capacity via pseudosynaptic contacts and gap junctions as shown in vivo. Such 3D disease models represent a suitable platform to further investigate tumor treatment options in a systematic manner.

Three-dimensional (3D) models to study human disease mechanisms have demonstrated that the third dimension is an essential component for neuronal maturation and function. However, 3D neuronal cell culture is challenging due to their ultra-soft nature and specific extracellular matrix (ECM) organization. This study presents a microfiber reinforcement approach, combining primary mouse spinal cord neurons (SCNs) in a purpose-adaptable hyaluronic-acid-based matrix with melt electrowritten (MEW) frames to study disease mechanisms. The importance of laminins (LNs) is evaluated, which are vital for neuronal adhesion and maturation. Astrocytes (ACs) are mandatory in brain development and function by secretion of signal molecules, maintaining ion homeostasis, clearing of neurotransmitters, and by actively modulating neuronal activity. Three combinations are compared (i) isolated primary SCN, (ii) SCN with ACs (SCN-AC) and (iii) SCN-AC and LNs (SCN-AC-LN). Multimodal analysis by comparing protein expression, dendrite length, complex mechanical properties, and network functionality via Ca2+-imaging allows validation of structural and functional neuronal network development. The suitability of the 3D model to study pathomechanisms is demonstrated for Stiff Person Syndrome (SPS). For the first time, functional impairment of spinal cord neurons by patient-derived autoantibodies characteristic for SPS is recapitulated in a 3D spinal cord model platform adaptable to other disease types.

IntroductionPatients with glycine receptor (GlyR) aAbs suffer from various diseases, including stiff-person syndrome (SPS), and currently, no cure exists. Several treatment options exist; however, these treatment options lack specificity. To date, only one common epitope has been mapped for GlyR aAbs in the far N-terminal region of the GlyRα1 subunit. However, some patient sera also bind GlyRα2, GlyRα3, or GlyRβ. Therefore, more than one common epitope may exist. Unraveling these epitopes will help generate more specific treatment approaches.MethodsHere, we constructed GlyRa1 and GlyRa3 variants by site-directed mutagenesis using amino acid differences between these two subunits within their extracellular domains. Peptide microarrays, which have shown that an epitope including the binding site of a commercial pan-a antibody (96PDLFFANEKS105) and its surrounding residues is highly relevant for aAb binding, were utilized to identify additional residues important for aAb binding. Two overlapping peptides (93LWKPDLFFANEKSAN107 and 98LFFANEKSANFHDVT112) were used for aAb neutralization in cell-based assays.ResultsThe GlyRa1 and GlyRa3 variants helped to identify which amino acid sequences in the extracellular domain of GlyRs represent additional aAb epitopes or are involved in aAb binding. Using both generated peptides for aAb neutralization with a patient serum containing GlyRb aAbs that bind specifically to this region 96PDLFFANEKSANFHDV111, successful neutralization was demonstrated. In contrast, when using patient sera that reliably target the extracellular domain including 96PDLFFANEKS105 of the GlyRa subunits, the overlapping peptides reduced aAb binding but failed to fully neutralize the aAbs.DiscussionIn conclusion, our data demonstrate that GlyR aAbs are polyclonal or bind to structural epitopes. These results define single residues important for aAb binding and help explain why no further common aAb binding site has been identified so far. Hence, patient-specific pattern for GlyR aAbs exist, emphasizing the importance of epitope characterization as basis for future therapeutic testing or even complete neutralization of the aAbs.

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.

Infection with SARS-CoV-2 continues to be a threat to human health. Despite successful immunization campaigns, effective treatment of COVID-19 remains an essential need to help patients and prevent the spread of new virus strains. Viroporins are intracellular ion channels that are essential for virus replication and release, thus presenting promising pharmaceutical targets. Mutations found in variants of concern (VOC) are expected to increase the virulence of the new virus strains. Recognizing the effects of these mutations at the molecular level is essential for the development of improved therapies. Here, we characterized the putative viroporin ORF3a found in VOCs of SARS-CoV-2, using expression constructs containing a myc-tag for identification, and an optional membrane-directing signal peptide. Additionally, constructs containing N-terminal fluorescence protein tags were prepared. Expression and cell surface transport in HEK-293 cells were studied using Western blot and dot blot assays, and the cellular distribution of fluorescent-marked ORF3a was studied using subcellular organelle markers and high-resolution fluorescence microscopy. Viroporin activity of all ORF3a constructs was assessed using cell viability and metabolic assays, as well as patch-clamp recordings of recombinant ORF3a. All ORF3a mutants were expressed well in the recombinant system, and the presence of a signal peptide increased expression on the cellular surface. Intracellular distribution was similar for all variants. The VOC mutants ORF3a-S171L and ORF3a-Q57H showed reduced cytotoxic activity and sensitivity to the viroporin inhibitor rimantadine, respectively, suggesting these positions to be relevant for ORF3a function and a starting point for the search of novel antiviral drugs.

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.

Patients diagnosed with the rare autoimmune disease Stiff Person Syndrome (SPS) or the more severe form Progressive Encephalomyelitis with Rigidity and Myoclonus (PERM) as well as patients with encephalitis or epilepsy may harbor autoantibodies against synaptic molecules, for example the glycine receptor (GlyR). These autoantibodies interfere with inhibitory signal transmission, which causes a variety of symptoms. How the underlying autoantibody associated pathomechanisms contribute to the variability of clinical presentations, is so far not understood. In this study, binding patterns of GlyR autoantibodies from patients with SPS, PERM and epilepsy to murine central nervous system (CNS) tissue samples were analyzed for disease- and patient-specificity patterns. Twelve GlyR autoantibody positive patients were grouped by the patients’ primary diagnoses. Serum samples from these SPS, PERM and epilepsy patients were evaluated for autoantibody binding on transfected HEK-293 cells and murine spinal cord and various brain tissue samples. Autoantibody binding was further verified by co-localization with commercial antibodies binding to the GlyR and the synaptic marker synaptophysin. Immunochemistry revealed GlyRα1-specific autoantibody binding for all included patients on transfected HEK-293 cells and in the grey matter of murine spinal cord sections. Other CNS regions of enhanced autoantibody accumulation however varied among the groups of SPS, PERM and epilepsy patients and also within groups. Similarly, autoantibody deposits were detected in GlyR expressing higher brain areas for each patient. Even if variations between labeled areas and cell layers were rather patient-specific than group-specific, functionality of the labeled areas aligned with the patients’ impaired functions. Labeled areas and cell layers differing between patients could thereby explain the variability of symptomatology between and within the diseases. The observed diversity suggests a necessity for a personalized approach correlating patient-specific autoantibody properties, phenotype and treatment approaches.

BACKGROUND AND OBJECTIVES:Patients with autoantibodies (aAbs) against the contactin-associated protein-like 2 (CASPR2) suffer from a variety of clinical syndromes including neuropathic pain. CASPR2 is an adhesion protein of the neurexin family and part of the voltage-gated potassium channel complex (VGKC complex) in dorsal root ganglia (DRG) neurons. The pathologic mechanisms following the binding of CASPR2 aAbs and their association with pain are only partially understood. CASPR2 aAbs are mainly of the IgG4 subclass; however, previous studies have neglected subclass-dependent effects. METHODS:We investigated 49 subclassified patient serum samples positive for CASPR2 aAbs combining superresolution lattice structural illumination microscopy (SIM2) and functional readouts by calcium imaging and electrophysiologic recordings on cultured DRG neurons. CASPR2-positive patient sera subclassified in IgG4 together with at least 1 other IgG subclass (IgGX) and patients with only IgG4 were further subdivided into the pain and no pain groups. RESULTS:A decrease of CASPR2 expression along the axons after exposure to CASPR2 aAbs was observed for all patient groups except the group without pain and IgG4. Moreover, binding of CASPR2 aAbs from patients with pain increased the distance between CASPR2 and associated potassium channels along DRG axons determined by SIM2 microscopy. CASPR2 aAbs of patients with pain significantly increased overall neuronal excitability of cultured DRG neurons as measured by calcium imaging. Patch-clamp recordings revealed significantly decreased current amplitudes of voltage-gated potassium (Kv) channels after incubation with all 4 CASPR2 aAb subclassifications with the most prominent effect of serum samples harboring IgG4 aAbs only. Replacement of patient aAbs by healthy control serum rescued Kv channel function to normal levels suggesting that the affected potassium channel function is due to structural blockage and disrupted interactions within the VGKC complex. The last might also be rescued on novel protein synthesis and membrane trafficking of CASPR2. DISCUSSION:IgG4 aAbs seem to be the major modifier of potassium channel function. The DRG hyperexcitability is primarily due to impaired Kv channel conductance as a consequence of CASPR2 aAb binding. However, additional unidentified signal pathways contribute to this process in patients with neuropathic pain.

Glioblastoma multiforme is the most devastating brain tumor without cure. Although in vitro and in vivo research on glioblastoma multiforme have demonstrated its complexity, including interactions with brain cells and the tumor microenvironment, 3D models resembling those key features and allowing to study therapeutic interventions of this aggressive tumor are scarce. Here, a 3D glioblastoma model is developed that establishes a tumor microenvironment including a hyaluronic acid-based hydrogel cross-linked with laminin, both of which are key components of the brain's extracellular matrix. This hydrogel mimics the mechanical properties of the brain's extracellular matrix at the macroscopic and mesoscopic levels, as evaluated by stiffness, viscosity using rheological and nanoindentation measurements. The ultra-soft hydrogel with a storage modulus of 100 Pa is reinforced by 3D printed microfiber scaffolds which allow the setup of a multicellular 3D model including primary cortical neurons and astrocytes and glioblastoma cells. Tumor microenvironment interactions are characterized through nanoindentation and confocal shadow imaging with the 3D in vitro disease model resembling in vivo properties of glioblastoma tumor entities characterized by functional interactions with the surrounding astrocytes and neurons and the tumor's hijacking capability using neuronal signaling to promote its own proliferation.

Pathological deposition of hyperphosphorylated tau in the brain closely correlates with the course of Alzheimer’s disease (AD). Tau pathology occurs in axons of affected neurons and tau removal from axons might thus be an early intervention strategy. We investigated the role of the RNA-binding protein hnRNP R in axonal localization and local translation of Mapt mRNA in neurons cultured from hnRNP R knockout mice. hnRNP R knockout mice were crossed with 5×FAD mice, an AD mouse model, and the effects of hnRNP R loss on the deposition of phospho-tau and amyloid-β plaques were evaluated. We designed antisense oligonucleotides (MAPT-ASOs) to block the binding of hnRNP R to Mapt mRNA. Cultured mouse and human neurons were treated with MAPT-ASOs and axonal Mapt mRNA and tau protein levels were quantified. MAPT-ASO was injected intracerebroventricularly into 5×FAD mice followed by quantification of phospho-tau aggregates and amyloid-β plaques in their brains. Protein changes in brains of 5×FAD mice treated with the MAPT-ASO were measured by mass spectrometry. Mapt mRNA and tau protein were reduced in axons but not cell bodies of primary neurons cultured from hnRNP R knockout mice. Brains of 5×FAD mice deficient for hnRNP R contained less phospho-tau aggregates and amyloid-β plaques in the cortex and hippocampus. Treatment of neurons with MAPT-ASOs to block hnRNP R binding to Mapt similarly reduced axonal tau levels. Intracerebroventricular injection of a MAPT-ASO reduced the phospho-tau and plaque load and prevented neurodegeneration in the brains of 5×FAD mice, accompanied by rescue of proteome alterations. Lowering of tau selectively in axons thus represents an innovative therapeutic perspective for treatment of AD and other tauopathies.

The spatiotemporal organization of the postsynaptic density (PSD) is a fundamental determinant of synaptic transmission, information processing, and storage in the brain. The major bottleneck that prevents the direct and precise representation of the nanometer-scaled organization of excitatory glutamatergic synapses is the size of antibodies, nanobodies, and the genetically encoded fluorescent tags. Here, we introduce small, high affinity synthetic probes for simplified, high contrast visualization of excitatory synapses without the limitations of larger biomolecules. In vitro binding quantification together with microscopy-based evaluation identified eSylites, a series of fluorescent bivalent peptides comprising a dye, linker, and sequence composition that show remarkable cellular target selectivity. Applied on primary neurons or brain slices at nanomolar concentrations, eSylites specifically report PSD-95, the key orchestrator of glutamate receptor nanodomains juxtaposed to the presynaptic glutamate release sites that mediate fast synaptic transmission. The eSylite design minimizes a spatial dye offset and thereby enables visualization of PSD-95 with improved localization precision and further time-resolved discrimination. In particular, we find that individual dendritic spines can contain separate nanodomains enriched for either PSD-95 or its closest homologues, PSD-93 or SAP102. Collectively, these data establish eSylites as a broadly applicable tool for simplified excitatory synapse visualization, as well as a high-end microscopy compatible probe for resolving the PSD organization with unprecedented resolution.

Background and ObjectivesAutoantibodies (aAbs) against glycine receptors (GlyRs) are mainly associated with the rare neurologic diseases stiff person syndrome (SPS) and progressive encephalomyelitis with rigidity and myoclonus (PERM). GlyR aAbs are also found in other neurologic diseases such as epilepsy. The aAbs bind to different GlyR alpha-subunits and, more rarely, also to the GlyR beta-subunit. So far, studies on the pathogenic effects of the aAbs have focused on postsynaptic, heteromeric GlyRs, reporting a loss of ion channel function and receptor internalization upon aAb binding. We asked whether the aAbs also affect expression and functionality of presynaptic homomeric GlyRs.MethodsWe established interneuron cultures from mouse embryonic spinal cord neurons and used protein biochemistry and super-resolution microscopy to determine aAb binding to presynaptic GlyRs in a uniform neuronal subpopulation. Brainstem slice recordings were used to detect functional alterations.ResultsSeveral days-long exposure of spinal cord cultures with GlyR aAbs did not change expression levels of proteins building a functional glycinergic synapse. A notable exception was the enhanced expression of presynaptic glycine transporter 2 (GlyT2), possibly reflecting an adaptation to altered synaptic properties. Super-resolution microscopy revealed rather similar binding of patient-derived aAbs to postsynaptic vs presynaptic sites with individual binding preferences. Although characterization of interneurons showed absence of GlyR alpha 1 in some interneuron subpopulations, GlyR alpha 2 and patient serum signals exhibited a significantly higher colocalization in samples with presynaptic preference. This finding identifies GlyR alpha 2 as the hitherto unknown predominant presynaptic GlyR subunit in the spinal cord and a target of patient aAbs. Whole-cell recordings from glycinergic neurons in mouse brainstem slices underscored the functional relevance of presynaptic aAb binding demonstrated by a significant reduction in the frequency of spontaneous and miniature inhibitory postsynaptic potentials.DiscussionIn summary, our study is the first to implicate presynaptic defects in the pathophysiology of autoimmune diseases such as SPS and PERM, which are associated with GlyR aAbs. Individually tuned binding preferences for presynaptic and postsynaptic targets thus underlie the rather diverse appearance of clinical symptoms and different therapeutic responses in patients suffering from GlyR autoimmunity.

Introduction: Essential oils (EOs) are complex mixtures with different application areas in human medicine and health. Some harbor calming and anxiolytic effects, which has been attributed to GABAA receptor (GABAAR) modulation. Although GABAAR modulation has been demonstrated, most studies do not discriminate between different GABAAR subtypes present in the central nervous system. Objectives: We determined the modulatory effect of five EOs (lavender (Lavandula angustifolia Mill.), basil (Ocimum basilicum L.), lemon balm (Melissa officinalis L.), hops (Humulus lupulus L.) and chamomile (Matricaria chamomilla L.)) on recombinant α1β2 and α1β2γ2 GABAARs and characterized the chemical composition of the EOs. Methods: The chemical composition of the EOs was investigated by gas chromatography coupled with mass spectrometry. EOs were functionally investigated on transfected HEK293 cells with GABAARs by patch-clamp whole-cell recordings. Results: The chemical analysis revealed mono- and sesquiterpene substances being main constituents, yet no specific common compound being present at relevant levels in all five oils. All EOs investigated, displayed negative modulation of GABAARs lacking the γ2 subunit. For GABAARs containing the γ2 subunit, three oils (lavender, basil and chamomile) exhibited positive modulation of up to 98 % increased GABAergic responses while lemon balm and hops showed no significant effect. Conclusion: EOs which have not been investigated yet for their total modulatory potential on GABAARs exhibit specific receptor subtype dependent allosteric modulation. This modulatory potential of EOs together with its chemical identification offers a tool for the design of future studies with individual compounds or defined ratios of compounds.

BACKGROUND AND OBJECTIVES:Autoimmune nodopathy with anti-contactin1 (CNTN1) autoantibodies is a rare sensory-motor neuropathy characterized by subacute-onset sensory ataxia and variable disease courses. Comorbidities such as glomerulonephritis and diabetes mellitus are observed in some patients. The diversity in clinical presentation may reflect differences in the targeted CNTN1 epitopes. METHODS:To investigate the relationship between clinical features and the underlying epitopes, we analyzed serum samples from 16 anti-CNTN1-positive patients. Binding epitopes were assessed using cell-based assays with recombinant CNTN1 variants and peptide microarrays. RESULTS:Epitope mapping in 16 patients revealed that 13 harbored antibodies targeting CNTN1 immunoglobulin (Ig) domains while 3 recognized fibronectin (Fn) domains. Glomerulonephritis was exclusively observed in patients with autoantibodies binding to the Ig domain, whereas diabetes mellitus and a chronic course of disease were more prevalent in patients with binding to the fibronectin domain. DISCUSSION:Our findings highlight the heterogeneity of anti-contactin1 nodopathies, suggesting that distinct epitope-binding patterns may underlie different clinical manifestations and may be associated with different pathogenic mechanisms and comorbidities.

ABSTRACTThe biological and mechanical environment of cells is better mimicked in 3D compared to 2D cell cultures. However, creating accurate 3D cell culture models particularly for ultra‐soft tissues like brain or spinal cord is challenging since the hydrogels that match these properties are mechanically fragile. Therefore, implementing reinforcing structures, such as microfibers, is essential to provide the necessary support. Particularly, fibrous systems are of interest since they offer natural fibrillar structures similar to the extracellular matrix. This study focuses on exploring the interactions between a motor neuron‐like cell line and multiple microfiber‐morphologies and mechanics. Monitoring cell‐microfiber interactions over time we unveiled various dynamic undetected behaviors and interactions happening upon contact depending on the used microfiber properties. These highly defined microfiber fragments were fabricated using multiple processes—electrospinning, Melt Electrowriting, and microfluidic spinning—with properties differing in size, mechanics, and surface chemistry. The excellent control over our microfiber systems enabled the investigation of single parameters in an isolated manner. In addition, we quantified the observed varying movement modes of the monitored cell‐microfiber tandems. The study demonstrates the significance of microfiber design for biological applications and establishes methodological foundations for the implementation of customized microfiber systems in the field of biofabrication.

Soft nano- and microfiber-based polymer scaffolds bear enormous potential for their use in cell culture and tissue engineering since they mimic natural collagen structures and may thus serve as biomimetic adhesive substrates. They have, however, so far been restricted to small-scale production in research labs with high batch-to-batch variation. They are commonly produced via electrospinning or melt electrowriting and their delicate nature poses obstacles in detachment, storage, and transportation. This study focuses on overcoming challenges in the high throughput production and practical handling, introducing new methods to reproducibly prepare such scaffolds suitable for quantitative cell culture applications. Attention is given to the seamless handling and transfer of samples without compromising structural integrity. Challenges in detaching fibers without damage as well as storage, and transport are addressed. Cell culture studies demonstrate the methodological advantages, emphasizing the potential for standardized testing and biological readouts of these delicate fiber materials. The developed methods are applicable across various electrospinning and melt electrowriting approaches and can essentially contribute to their utilization in laboratory research and commercial applications.

Background and ObjectivesStiff-person syndrome (SPS) and progressive encephalomyelitis with rigidity and myoclonus (PERM) are rare neurologic disorders of the CNS. Until now, exclusive GlyR alpha subunit-binding autoantibodies with subsequent changes in function and surface numbers were reported. GlyR autoantibodies have also been described in patients with focal epilepsy. Autoimmune reactivity against the GlyR beta subunits has not yet been shown. Autoantibodies against GlyR alpha 1 target the large extracellular N-terminal domain. This domain shares a high degree of sequence homology with GlyR beta making it not unlikely that GlyR beta-specific autoantibody (aAb) exist and contribute to the disease pathology.MethodsIn this study, we investigated serum samples from 58 patients for aAb specifically detecting GlyR beta. Studies in microarray format, cell-based assays, and primary spinal cord neurons and spinal cord tissue immunohistochemistry were performed to determine specific GlyR beta binding and define aAb binding to distinct protein regions. Preadsorption approaches of aAbs using living cells and the purified extracellular receptor domain were further used. Finally, functional consequences for inhibitory neurotransmission upon GlyR beta aAb binding were resolved by whole-cell patch-clamp recordings.ResultsAmong 58 samples investigated, cell-based assays, tissue analysis, and preadsorption approaches revealed 2 patients with high specificity for GlyR beta aAb. Quantitative protein cluster analysis demonstrated aAb binding to synaptic GlyR beta colocalized with the scaffold protein gephyrin independent of the presence of GlyR alpha 1. At the functional level, binding of GlyR beta aAb from both patients to its target impair glycine efficacy.DiscussionOur study establishes GlyR beta as novel target of aAb in patients with SPS/PERM. In contrast to exclusively GlyR alpha 1-positive sera, which alter glycine potency, aAbs against GlyR beta impair receptor efficacy for the neurotransmitter glycine. Imaging and functional analyses showed that GlyR beta aAbs antagonize inhibitory neurotransmission by affecting receptor function rather than localization.