Molecular dissection of an immunodominant epitope in Kv1.2-exclusive autoimmunity.

Introduction: Subgroups of autoantibodies directed against voltage-gated potassium channel (Kv) complex components have been associated with immunotherapy-responsive clinical syndromes. The high prevalence and the role of autoantibodies directly binding Kv remain, however, controversial. Our objective was to determine Kv autoantibody binding requirements and to clarify their contribution to the observed immune response. Methods: Binding epitopes were studied in sera (n = 36) and cerebrospinal fluid (CSF) (n = 12) from a patient cohort positive for Kv1.2 but negative for 32 common neurological autoantigens and controls (sera n = 18 and CSF n = 5) by phospho and deep mutational scans. Autoantibody specificity and contribution to the observed immune response were resolved on recombinant cells, cerebellum slices, and nerve fibers. Results: 83% of the patients (30/36) within the studied cohort shared one out of the two major binding epitopes with Kv1.2-3 reactivity. Eleven percent (4/36) of the serum samples showed no binding. Fingerprinting resolved close to identical sequence requirements for both shared epitopes. Kv autoantibody response is directed against juxtaparanodal regions in peripheral nerves and the axon initial segment in central nervous system neurons and exclusively mediated by the shared epitopes. Discussion: Systematic mapping revealed two shared autoimmune responses, with one dominant Kv1.2-3 autoantibody epitope being unexpectedly prevalent. The conservation of the molecular binding requirements among these patients indicates a uniform autoantibody repertoire with monospecific reactivity. The enhanced sensitivity of the epitope-based (10/12) compared with that of the cell-based detection (7/12) highlights its use for detection. The determined immunodominant epitope is also the primary immune response visible in tissue, suggesting a diagnostic significance and a specific value for routine screening.

Different pain phenotypes are associated with anti-Caspr2 autoantibodies.

Autoantibodies against contactin-associated protein 2 (Caspr2) not only induce limbic autoimmune encephalitis but are also associated with pain conditions. Here, we analyzed clinical data on pain in a large cohort of patients included into the German Network for Research in Autoimmune Encephalitis. Out of 102 patients in our cohort, pain was a frequent symptom (36% of all patients), often severe (63.6% of the patients with pain) and/or even the major symptom (55.6% of the patients with pain). Pain phenotypes differed between patients. Cluster analysis revealed two major phenotypes including mostly distal-symmetric burning pain and widespread pain with myalgia and cramps. Almost all patients had IgG4 autoantibodies and some additional IgG1, 2, and/or 3 autoantibodies, but IgG subclasses, titers, and presence or absence of intrathecal synthesis were not associated with the occurrence of pain. However, certain pre-existing risk factors for chronic pain like diabetes mellitus, peripheral neuropathy, or preexisting chronic back pain tended to occur more frequently in patients with anti-Caspr2 autoantibodies and pain. Our data show that pain is a relevant symptom in patients with anti-Caspr2 autoantibodies and support the idea of decreased algesic thresholds leading to pain. Testing for anti-Caspr2 autoantibodies needs to be considered in patients with various pain phenotypes.

Glycine Receptor ß-Targeting Autoantibodies Contribute to the Pathology of Autoimmune Diseases.

BACKGROUND AND OBJECTIVES: Stiff-person syndrome (SPS) and progressive encephalomyelitis with rigidity and myoclonus (PERM) are rare neurologic disorders of the CNS. Until now, exclusive GlyRα 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 GlyRß subunits has not yet been shown. Autoantibodies against GlyRα1 target the large extracellular N-terminal domain. This domain shares a high degree of sequence homology with GlyRß making it not unlikely that GlyRß-specific autoantibody (aAb) exist and contribute to the disease pathology. METHODS: In this study, we investigated serum samples from 58 patients for aAb specifically detecting GlyRß. Studies in microarray format, cell-based assays, and primary spinal cord neurons and spinal cord tissue immunohistochemistry were performed to determine specific GlyRß 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 GlyRß aAb binding were resolved by whole-cell patch-clamp recordings. RESULTS: Among 58 samples investigated, cell-based assays, tissue analysis, and preadsorption approaches revealed 2 patients with high specificity for GlyRß aAb. Quantitative protein cluster analysis demonstrated aAb binding to synaptic GlyRß colocalized with the scaffold protein gephyrin independent of the presence of GlyRα1. At the functional level, binding of GlyRß aAb from both patients to its target impair glycine efficacy. DISCUSSION: Our study establishes GlyRß as novel target of aAb in patients with SPS/PERM. In contrast to exclusively GlyRα1-positive sera, which alter glycine potency, aAbs against GlyRß impair receptor efficacy for the neurotransmitter glycine. Imaging and functional analyses showed that GlyRß aAbs antagonize inhibitory neurotransmission by affecting receptor function rather than localization.

Role of the Glycine Receptor ß Subunit in Synaptic Localization and Pathogenicity in Severe Startle Disease.

Startle disease is due to the disruption of recurrent inhibition in the spinal cord. Most common causes are genetic variants in genes (GLRA1, GLRB) encoding inhibitory glycine receptor (GlyR) subunits. The adult GlyR is a heteropentameric complex composed of α1 and ß subunits that localizes at postsynaptic sites and replaces embryonically expressed GlyRα2 homomers. The human GlyR variants of GLRA1 and GLRB, dominant and recessive, have been intensively studied in vitro. However, the role of unaffected GlyRß, essential for synaptic GlyR localization, in the presence of mutated GlyRα1 in vivo is not fully understood. Here, we used knock-in mice expressing endogenous mEos4b-tagged GlyRß that were crossed with mouse Glra1 startle disease mutants. We explored the role of GlyRß under disease conditions in mice carrying a missense mutation (shaky) or resulting from the loss of GlyRα1 (oscillator). Interestingly, synaptic targeting of GlyRß was largely unaffected in both mouse mutants. While synaptic morphology appears unaltered in shaky animals, synapses were notably smaller in homozygous oscillator animals. Hence, GlyRß enables transport of functionally impaired GlyRα1 missense variants to synaptic sites in shaky animals, which has an impact on the efficacy of possible compensatory mechanisms. The observed enhanced GlyRα2 expression in oscillator animals points to a compensation by other GlyRα subunits. However, trafficking of GlyRα2ß complexes to synaptic sites remains functionally insufficient, and homozygous oscillator mice still die at 3 weeks after birth. Thus, both functional and structural deficits can affect glycinergic neurotransmission in severe startle disease, eliciting different compensatory mechanisms in vivo.

Dual Role of Dysfunctional Asc-1 Transporter in Distinct Human Pathologies, Human Startle Disease, and Developmental Delay.

Human startle disease is associated with mutations in distinct genes encoding glycine receptors, transporters or interacting proteins at glycinergic synapses in spinal cord and brainstem. However, a significant number of diagnosed patients does not carry a mutation in the common genes GLRA1, GLRB, and SLC6A5 Recently, studies on solute carrier 7 subfamily 10 (SLC7A10; Asc-1, alanine-serine-cysteine transporter) knock-out (KO) mice displaying a startle disease-like phenotype hypothesized that this transporter might represent a novel candidate for human startle disease. Here, we screened 51 patients from our patient cohort negative for the common genes and found three exonic (one missense, two synonymous), seven intronic, and single nucleotide changes in the 5' and 3' untranslated regions (UTRs) in Asc-1. The identified missense mutation Asc-1G307R from a patient with startle disease and developmental delay was investigated in functional studies. At the molecular level, the mutation Asc-1G307R did not interfere with cell-surface expression, but disrupted glycine uptake. Substitution of glycine at position 307 to other amino acids, e.g., to alanine or tryptophan did not affect trafficking or glycine transport. By contrast, G307K disrupted glycine transport similar to the G307R mutation found in the patient. Structurally, the disrupted function in variants carrying positively charged residues can be explained by local structural rearrangements because of the large positively charged side chain. Thus, our data suggest that SLC7A10 may represent a rare but novel gene associated with human startle disease and developmental delay.

The beer component hordenine inhibits alcohol addiction-associated behaviours in mice.

Alcohol consumption is a widespread behaviour that may eventually result in the development of alcohol use disorder (AUD). Alcohol, however, is rarely consumed in pure form but in fruit- or corn-derived preparations, like beer. These preparations add other compounds to the consumption, which may critically modify alcohol intake and AUD risk. We investigated the effects of hordenine, a barley-derived beer compound on alcohol use-related behaviours. We found that the dopamine D2 receptor agonist hordenine (50 mg/kg) limited ongoing alcohol consumption and prophylactically diminished relapse drinking after withdrawal in mice. Although not having reinforcing effects on its own, hordenine blocked the establishment of alcohol-induced conditioned place preference (CPP). However, it independently enhanced alcohol CPP retrieval. Hordenine had a dose-dependent inhibitory effect on locomotor activity. Chronic hordenine exposure enhanced monoamine tissue levels in many brain regions. Further characterization revealed monoaminergic binding sites of hordenine and found a strong binding on the serotonin and dopamine transporters, and dopamine D3 , and adrenergic α1A and α2A receptor activation but no effects on GABAA receptor or glycinergic signalling. These findings suggest that natural ingredients of beer, like hordenine, may work as an inhibitory and use-regulating factor by their modulation of monoaminergic signalling in the brain.

Different binding and pathogenic effect of neurofascin and contactin-1 autoantibodies in autoimmune nodopathies.

Introduction: IgG4 autoantibodies against paranodal proteins are known to induce acute-onset and often severe sensorimotor autoimmune neuropathies. How autoantibodies reach their antigens at the paranode in spite of the myelin barrier is still unclear. Methods: We performed in vitro incubation experiments with patient sera on unfixed and unpermeabilized nerve fibers and in vivo intraneural and intrathecal passive transfer of patient IgG to rats, to explore the access of IgG autoantibodies directed against neurofascin-155 and contactin-1 to the paranodes and their pathogenic effect. Results: We found that in vitro incubation resulted in weak paranodal binding of anti-contactin-1 autoantibodies whereas anti-neurofascin-155 autoantibodies bound to the nodes more than to the paranodes. After short-term intraneural injection, no nodal or paranodal binding was detectable when using anti-neurofascin-155 antibodies. After repeated intrathecal injections, nodal more than paranodal binding could be detected in animals treated with anti-neurofascin-155, accompanied by sensorimotor neuropathy. In contrast, no paranodal binding was visible in rats intrathecally injected with anti-contactin-1 antibodies, and animals remained unaffected. Conclusion: These data support the notion of different pathogenic mechanisms of anti-neurofascin-155 and anti-contactin-1 autoantibodies and different accessibility of paranodal and nodal structures.

Primary Glial Cell and Glioblastoma Morphology in Cocultures Depends on Scaffold Design and Hydrogel Composition.

3D cell cultures better replicate the in vivo environment compared to 2D models. Glioblastoma multiforme, a malignant brain tumor, highly profits from its cellular environment. Here, the U87 glioblastoma cell line in the presence/absence of primary astrocytes is studied. Thiolated hyaluronic acid (HA-SH) hydrogel reinforced with microfiber scaffolds is compared to Matrigel. Hyaluronic acid is a major extracellular matrix (ECM) component in the brain. Poly(ɛ-caprolactone) (PCL) scaffolds are written by meltelectrowriting in a box and triangular shaped design with pore sizes of 200 µm. Scaffolds are composed of 10-layers of PCL microfibers. It is found that scaffold design has an impact on cellular morphology in the absence of hydrogel. Moreover, the used hydrogels have profound influences on cellular morphology resulting in spheroid formation in HA-SH for both the tumor-derived cell line and astrocytes, while cell viability is high. Although cocultures of U87 and astrocytes exhibit cell-cell interactions, polynucleated spheroid formation is still present for U87 cells in HA-SH. Locally restricted ECM production or inability to secrete ECM proteins may underlie the observed cell morphologies. Thus, the 3D reinforced PCL-HA-SH composite with glioma-like cells and astrocytes constitutes a reproducible system to further investigate the impact of hydrogel modifications on cellular behavior and development.

Glycine receptor autoantibody binding to the extracellular domain is independent from receptor glycosylation.

Glycine receptor (GlyR) autoantibodies are associated with stiff-person syndrome and the life-threatening progressive encephalomyelitis with rigidity and myoclonus in children and adults. Patient histories show variability in symptoms and responses to therapeutic treatments. A better understanding of the autoantibody pathology is required to develop improved therapeutic strategies. So far, the underlying molecular pathomechanisms include enhanced receptor internalization and direct receptor blocking altering GlyR function. A common epitope of autoantibodies against the GlyRα1 has been previously defined to residues 1A-33G at the N-terminus of the mature GlyR extracellular domain. However, if other autoantibody binding sites exist or additional GlyR residues are involved in autoantibody binding is yet unknown. The present study investigates the importance of receptor glycosylation for binding of anti-GlyR autoantibodies. The glycine receptor α1 harbors only one glycosylation site at the amino acid residue asparagine 38 localized in close vicinity to the identified common autoantibody epitope. First, non-glycosylated GlyRs were characterized using protein biochemical approaches as well as electrophysiological recordings and molecular modeling. Molecular modeling of non-glycosylated GlyRα1 did not show major structural alterations. Moreover, non-glycosylation of the GlyRα1N38Q did not prevent the receptor from surface expression. At the functional level, the non-glycosylated GlyR demonstrated reduced glycine potency, but patient GlyR autoantibodies still bound to the surface-expressed non-glycosylated receptor protein in living cells. Efficient adsorption of GlyR autoantibodies from patient samples was possible by binding to native glycosylated and non-glycosylated GlyRα1 expressed in living not fixed transfected HEK293 cells. Binding of patient-derived GlyR autoantibodies to the non-glycosylated GlyRα1 offered the possibility to use purified non-glycosylated GlyR extracellular domain constructs coated on ELISA plates and use them as a fast screening readout for the presence of GlyR autoantibodies in patient serum samples. Following successful adsorption of patient autoantibodies by GlyR ECDs, binding to primary motoneurons and transfected cells was absent. Our results indicate that the glycine receptor autoantibody binding is independent of the receptor's glycosylation state. Purified non-glycosylated receptor domains harbouring the autoantibody epitope thus provide, an additional reliable experimental tool besides binding to native receptors in cell-based assays for detection of autoantibody presence in patient sera.

Startle Disease: New Molecular Insights into an Old Neurological Disorder.

Startle disease (SD) is characterized by enhanced startle responses, generalized muscle stiffness, unexpected falling, and fatal apnea episodes due to disturbed feedback inhibition in the spinal cord and brainstem of affected individuals. Mutations within the glycine receptor (GlyR) subunit and glycine transporter 2 (GlyT2) genes have been identified in individuals with SD. Impaired inhibitory neurotransmission in SD is due to pre- and/or postsynaptic GlyR or presynaptic GlyT2 dysfunctions. Previous research has focused on mutated GlyRs and GlyT2 that impair ion channel/transporter function or trafficking. With insights provided by recently solved cryo-electron microscopy and X-ray structures of GlyRs, a detailed picture of structural transitions important for receptor gating has emerged, allowing a deeper understanding of SD at the molecular level. Moreover, studies on novel SD mutations have demonstrated a higher complexity of SD, with identification of additional clinical signs and symptoms and interaction partners representing key players for fine-tuning synaptic processes. Although our knowledge has steadily improved during the last years, changes in synaptic localization and GlyR or GlyT2 homeostasis under disease conditions are not yet completely understood. Combined proteomics, interactomics, and high-resolution microscopy techniques are required to reveal alterations in receptor dynamics at the synaptic level under disease conditions.

Anti-pan-neurofascin antibodies induce subclass-related complement activation and nodo-paranodal damage.

Autoimmune neuropathy associated with antibodies against pan-neurofascin is a new subtype of nodo-paranodopathy. It is relevant because it is associated with high morbidity and mortality. Affected patients often require intensive care unit treatment for several months, and data on the reversibility and long-term prognosis are limited. The pathogenicity including IgG subclass-associated mechanisms has not been unravelled, nor directly compared to anti-neurofascin-155 IgG4-related pathology. Understanding the underlying pathology might have a direct impact on treatment of these severely affected patients. By a multicentre combined prospective and retrospective approach, we provide clinical data of a large cohort of patients with anti-neurofascin-associated neuropathy (n = 18) including longitudinal titre and neurofilament light chain assessment via Ella® and relate clinical data to in vitro pathogenicity studies of anti-neurofascin antibodies. We assessed antibody binding characteristics and the pathogenic effects of anti-pan-neurofascin versus neurofascin-155 antibodies on living myelinating dorsal root ganglia co-cultures. Additionally, we analysed the IgG subclass profile and the complement binding capacity and effector functions considering the effects of intravenous immunoglobulin preparations via enzyme-linked immunosorbent and cell-based assays. In contrast to chronic neurofascin-155 IgG4-associated neuropathy, anti-pan-neurofascin-associated disease presented with a high morbidity and mortality, but as a monophasic and potentially reversible disorder. During follow-up, antibodies were no longer detectable in 8 of 11 patients. Anti-pan-neurofascin had direct access to the nodes of Ranvier in myelinating cultures titre-dependently, most probably inducing this severe phenotype. Antibody preincubation led to impaired paranode formation, destruction of paranodal architecture and alterations on paranodal myelin and sensory neurons in the cultures, with more severe effects than neurofascin-155 antibodies. Besides IgG4, subclass IgG3 was detected and associated with complement binding and cytotoxic effects in vitro. As a possible correlate of axonal damage in vivo, we detected highly increased serum neurofilament light chain levels (sNF-L), correlating to serum C3a. Still, sNF-L was not identified as a marker for poor prognosis, but rather as an intra- and interindividual marker for acuteness, severity and course, with a strong decrease during recovery. Our data provide evidence that anti-pan-neurofascin antibodies directly attack the node and induce severe and acute, but potentially reversible, nodo-paranodal pathology, possibly involving complement-mediated mechanisms. Screening for autoantibodies thus is crucial to identify this subset of patients who benefit from early antibody-depleting therapy. Titre and sNF-L might serve as valuable follow-up parameters. The prospect of a favourable outcome has high relevance for physicians, patients and relatives during months of critical care.

Reinforced Hyaluronic Acid-Based Matrices Promote 3D Neuronal Network Formation.

3D neuronal cultures attempt to better replicate the in vivo environment to study neurological/neurodegenerative diseases compared to 2D models. A challenge to establish 3D neuron culture models is the low elastic modulus (30-500 Pa) of the native brain. Here, an ultra-soft matrix based on thiolated hyaluronic acid (HA-SH) reinforced with a microfiber frame is formulated and used. Hyaluronic acid represents an essential component of the brain extracellular matrix (ECM). Box-shaped frames with a microfiber spacing of 200 µm composed of 10-layers of poly(ɛ-caprolactone) (PCL) microfibers (9.7 ± 0.2 µm) made via melt electrowriting (MEW) are used to reinforce the HA-SH matrix which has an elastic modulus of 95 Pa. The neuronal viability is low in pure HA-SH matrix, however, when astrocytes are pre-seeded below this reinforced construct, they significantly support neuronal survival, network formation quantified by neurite length, and neuronal firing shown by Ca2+ imaging. The astrocyte-seeded HA-SH matrix is able to match the neuronal viability to the level of Matrigel, a gold standard matrix for neuronal culture for over two decades. Thus, this 3D MEW frame reinforced HA-SH composite with neurons and astrocytes constitutes a reliable and reproducible system to further study brain diseases.

Loss, Gain and Altered Function of GlyR α2 Subunit Mutations in Neurodevelopmental Disorders.

Glycine receptors (GlyRs) containing the α2 subunit govern cell fate, neuronal migration and synaptogenesis in the developing cortex and spinal cord. Rare missense variants and microdeletions in the X-linked GlyR α2 subunit gene (GLRA2) have been associated with human autism spectrum disorder (ASD), where they typically cause a loss-of-function via protein truncation, reduced cell-surface trafficking and/or reduced glycine sensitivity (e.g., GLRA2Δex8-9 and extracellular domain variants p.N109S and p.R126Q). However, the GlyR α2 missense variant p.R323L in the intracellular M3-M4 domain results in a gain-of-function characterized by slower synaptic decay times, longer duration active periods and increases in channel conductance. This study reports the functional characterization of four missense variants in GLRA2 associated with ASD or developmental disorders (p.V-22L, p.N38K, p.K213E, p.T269M) using a combination of bioinformatics, molecular dynamics simulations, cellular models of GlyR trafficking and electrophysiology in artificial synapses. The GlyR α2V-22L variant resulted in altered predicted signal peptide cleavage and a reduction in cell-surface expression, suggestive of a partial loss-of-function. Similarly, GlyR α2N38K homomers showed reduced cell-surface expression, a reduced affinity for glycine and a reduced magnitude of IPSCs in artificial synapses. By contrast, GlyR α2K213E homomers showed a slight reduction in cell-surface expression, but IPSCs were larger, with faster rise/decay times, suggesting a gain-of-function. Lastly, GlyR α2T269M homomers exhibited a high glycine sensitivity accompanied by a substantial leak current, suggestive of an altered function that could dramatically enhance glycinergic signaling. These results may explain the heterogeneity of clinical phenotypes associated with GLRA2 mutations and reveal that missense variants can result in a loss, gain or alteration of GlyR α2 function. In turn, these GlyR α2 missense variants are likely to either negatively or positively deregulate cortical progenitor homeostasis and neuronal migration in the developing brain, leading to changes in cognition, learning, and memory.

Insulin-like growth factor 5 associates with human Aß plaques and promotes cognitive impairment.

Risk factors such as dysregulation of Insulin-like growth factor (IGF) signaling have been linked to Alzheimer's disease. Here we show that Insulin-like Growth Factor Binding Protein 5 (Igfbp5), an inhibitory binding protein for insulin-like growth factor 1 (Igf-1) accumulates in hippocampal pyramidal neurons and in amyloid plaques in brains of Alzheimer patients. We investigated the pathogenic relevance of this finding with transgenic mice overexpressing Igfbp5 in pyramidal neurons of the brain. Neuronal overexpression of Igfbp5 prevents the training-induced increase of hippocampal and cortical Bdnf expression and reduces the effects of exercise on memory retention, but not on learning acquisition. Hence, elevated IGFBP5 expression could be responsible for some of the early cognitive deficits that occur during the course of Alzheimer's disease.

A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity.

Visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques, and usually relies on genetic manipulation or the use of antibodies that underperform in tissue immunofluorescence. Starting from an endogenous ligand of gephyrin, a universal marker of the inhibitory synapse, we developed a short peptidic binder and dimerized it, significantly increasing affinity and selectivity. We further tailored fluorophores to the binder, yielding "Sylite"-a probe with outstanding signal-to-background ratio that outperforms antibodies in tissue staining with rapid and efficient penetration, mitigation of staining artifacts, and simplified handling. In super-resolution microscopy Sylite precisely localizes the inhibitory synapse and enables nanoscale measurements. Sylite profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the midbrain and combined with complimentary tracing techniques reveals the synaptic connectivity.

Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy.

Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft.

Identification of a stereotypic molecular arrangement of endogenous glycine receptors at spinal cord synapses.

Precise quantitative information about the molecular architecture of synapses is essential to understanding the functional specificity and downstream signaling processes at specific populations of synapses. Glycine receptors (GlyRs) are the primary fast inhibitory neurotransmitter receptors in the spinal cord and brainstem. These inhibitory glycinergic networks crucially regulate motor and sensory processes. Thus far, the nanoscale organization of GlyRs underlying the different network specificities has not been defined. Here, we have quantitatively characterized the molecular arrangement and ultra-structure of glycinergic synapses in spinal cord tissue using quantitative super-resolution correlative light and electron microscopy. We show that endogenous GlyRs exhibit equal receptor-scaffold occupancy and constant packing densities of about 2000 GlyRs µm-2 at synapses across the spinal cord and throughout adulthood, even though ventral horn synapses have twice the total copy numbers, larger postsynaptic domains, and more convoluted morphologies than dorsal horn synapses. We demonstrate that this stereotypic molecular arrangement is maintained at glycinergic synapses in the oscillator mouse model of the neuromotor disease hyperekplexia despite a decrease in synapse size, indicating that the molecular organization of GlyRs is preserved in this hypomorph. We thus conclude that the morphology and size of inhibitory postsynaptic specializations rather than differences in GlyR packing determine the postsynaptic strength of glycinergic neurotransmission in motor and sensory spinal cord networks.

Novel Functional Properties of Missense Mutations in the Glycine Receptor ß Subunit in Startle Disease.

Startle disease is a rare disorder associated with mutations in GLRA1 and GLRB, encoding glycine receptor (GlyR) α1 and ß subunits, which enable fast synaptic inhibitory transmission in the spinal cord and brainstem. The GlyR ß subunit is important for synaptic localization via interactions with gephyrin and contributes to agonist binding and ion channel conductance. Here, we have studied three GLRB missense mutations, Y252S, S321F, and A455P, identified in startle disease patients. For Y252S in M1 a disrupted stacking interaction with surrounding aromatic residues in M3 and M4 is suggested which is accompanied by an increased EC50 value. By contrast, S321F in M3 might stabilize stacking interactions with aromatic residues in M1 and M4. No significant differences in glycine potency or efficacy were observed for S321F. The A455P variant was not predicted to impact on subunit folding but surprisingly displayed increased maximal currents which were not accompanied by enhanced surface expression, suggesting that A455P is a gain-of-function mutation. All three GlyR ß variants are trafficked effectively with the α1 subunit through intracellular compartments and inserted into the cellular membrane. In vivo, the GlyR ß subunit is transported together with α1 and the scaffolding protein gephyrin to synaptic sites. The interaction of these proteins was studied using eGFP-gephyrin, forming cytosolic aggregates in non-neuronal cells. eGFP-gephyrin and ß subunit co-expression resulted in the recruitment of both wild-type and mutant GlyR ß subunits to gephyrin aggregates. However, a significantly lower number of GlyR ß aggregates was observed for Y252S, while for mutants S321F and A455P, the area and the perimeter of GlyR ß subunit aggregates was increased in comparison to wild-type ß. Transfection of hippocampal neurons confirmed differences in GlyR-gephyrin clustering with Y252S and A455P, leading to a significant reduction in GlyR ß-positive synapses. Although none of the mutations studied is directly located within the gephyrin-binding motif in the GlyR ß M3-M4 loop, we suggest that structural changes within the GlyR ß subunit result in differences in GlyR ß-gephyrin interactions. Hence, we conclude that loss- or gain-of-function, or alterations in synaptic GlyR clustering may underlie disease pathology in startle disease patients carrying GLRB mutations.

Spinal Cord Neuronal Network Formation in a 3D Printed Reinforced Matrix-A Model System to Study Disease Mechanisms.

3D cell cultures allow a better mimicry of the biological and mechanical environment of cells in vivo compared to 2D cultures. However, 3D cell cultures have been challenging for ultrasoft tissues such as the brain. The present study uses a microfiber reinforcement approach combining mouse primary spinal cord neurons in Matrigel with melt electrowritten (MEW) frames. Within these 3D constructs, neuronal network development is followed for 21 days in vitro. To evaluate neuronal development in 3D constructs, the maturation of inhibitory glycinergic synapses is analyzed using protein expression, the complex mechanical properties by assessing nonlinearity, conditioning, and stress relaxation, and calcium imaging as readouts. Following adaptation to the 3D matrix-frame, mature inhibitory synapse formation is faster than in 2D demonstrated by a steep increase in glycine receptor expression between days 3 and 10. The 3D expression pattern of marker proteins at the inhibitory synapse and the mechanical properties resemble the situation in native spinal cord tissue. Moreover, 3D spinal cord neuronal networks exhibit intensive neuronal activity after 14 days in culture. The spinal cord cell culture model using ultrasoft matrix reinforced by MEW fibers provides a promising tool to study and understand biomechanical mechanisms in health and disease.

Anti-contactin-1 Antibodies Affect Surface Expression and Sodium Currents in Dorsal Root Ganglia.

BACKGROUND AND OBJECTIVES: As autoantibodies to contactin-1 from patients with chronic inflammatory demyelinating polyradiculoneuropathy not only bind to the paranodes where they are supposed to cause conduction failure but also bind to other neuronal cell types, we aimed to investigate the effect of anti-contactin-1 autoantibodies on contactin-1 surface expression in cerebellar granule neurons, dorsal root ganglion neurons, and contactin-1-transfected human embryonic kidney 293 cells. METHODS: Immunocytochemistry including structured illumination microscopy and immunoblotting was used to determine expression levels of contactin-1 and/or sodium channels after long-term exposure to autoantibodies from 3 seropositive patients. For functional analysis of sodium channels, whole-cell recordings of sodium currents were performed on dorsal root ganglion neurons incubated with anti-contactin-1 autoantibodies. RESULTS: We found a reduction in contactin-1 expression levels on dorsal root ganglion neurons, cerebellar granule neurons, and contactin-1-transfected human embryonic kidney 293 cells and decreased dorsal root ganglion sodium currents after long-term exposure to anti-contactin-1 autoantibodies. Sodium channel density did not decrease. DISCUSSION: Our results demonstrate a direct effect of anti-contactin-1 autoantibodies on the surface expression of contactin-1 and sodium currents in dorsal root ganglion neurons. This may be the pathophysiologic correlate of sensory ataxia reported in these patients.