Cytoplasmic islet cell antibodies recognize distinct islet antigens in IDDM but not in stiff man syndrome

Cytoplasmic islet cell antibodies are well-established predictive markers of IDDM. Although target molecules of ICA have been suggested to be gangliosides, human monoclonal ICA of the immunoglobulin G class (MICA 1-6) produced from a patient with newly diagnosed IDDM recognized glutamate decarboxylase as a target antigen. Here we analyzed the possible heterogeneity of target antigens of ICA by subtracting the GAD-specific ICA staining from total ICA staining of sera. This was achieved 1) by preabsorption of ICA+ sera with recombinant GAD65 and/or GAD67 expressed in a baculovirus system and 2) by ICA analysis of sera on mouse pancreas, as GAD antibodies do not stain mouse islets in the immunofluorescence test. We show that 24 of 25 sera from newly diagnosed patients with IDDM recognize islet antigens besides GAD. In contrast, GAD was the only islet antigen recognized by ICA from 7 sera from patients with stiff man syndrome. Two of these sera, however, recognized antigens besides GAD in Purkinje cells. In patients with IDDM, non-GAD ICA were diverse. One group, found in 64% of the sera, stained human and mouse islets, whereas the other group of non-GAD ICA was human specific. Therefore, mouse islets distinguish two groups of non-GAD ICA and lack additional target epitopes of ICA besides GAD. Longitudinal analysis of 6 sera from nondiabetic ICA+ individuals revealed that mouse-reactive ICA may appear closer to clinical onset of IDDM in some individuals

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 1 A- 33 G 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 GlyRα1 N38Q 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

Two new cases of anti-Ca (anti-ARHGAP26/GRAF) autoantibody-associated cerebellar ataxia

Recently, we discovered a novel serum and cerebrospinal fluid (CSF) autoantibody (anti-Ca) to Purkinje cells in a patient with autoimmune cerebellar ataxia (ACA) and identified the RhoGTPase-activating protein 26 (ARHGAP26; alternative designations include GTPase regulator associated with focal adhesion kinase pp125, GRAF, and oligophrenin-1-like protein, OPHN1L) as the target antigen. Here, we report on two new cases of ARHGAP26 autoantibody-positive ACA that were first diagnosed after publication of the index case study. While the index patient developed ACA following an episode of respiratory infection with still no evidence for malignancy 52 months after onset, neurological symptoms heralded ovarian cancer in one of the patients described here. Our finding of anti-Ca/anti-ARHGAP26 antibodies in two additional patients supports a role of autoimmunity against ARHGAP26 in the pathogenesis of ACA. Moreover, the finding of ovarian cancer in one of our patients suggests that anti-Ca/anti-ARHGAP26-positive ACA might be of paraneoplastic aetiology in some cases. In conclusion, testing for anti-Ca/anti-ARHGAP26 should be included in the diagnostic work-up of patients with ACA, and an underlying tumour should be considered in patients presenting with anti-Ca/ARHGAP26 antibody-positive ACA

The novel hyperekplexia allele GLRA1(S267N) affects the ethanol site of the glycine receptor

Mutations in the GLRA1 gene, which encodes the a1-subunit of the inhibitory glycine receptor (GlyR), are the underlying causes in the majority of cases of hereditary startle disease (OMIM no. 149400). GlyRs are modulated by alcohols and volatile anesthetics, where a specific amino acid at position 267 has been implicated in receptor modulation. We describe a hyperekplexia family carrying the novel dominant missense allele GLRA1(S267N), that affects agonist responses and ethanol modulation of the mutant receptor. This study implies that a disease-related receptor allele carries the potential to alter drug responses in affected patients

Novel GLRA1 Missense Mutation (P250T) in Dominant Hyperekplexia Defines an Intracellular Determinant of Glycine Receptor Channel Gating

Missense mutations as well as a null allele of the human glycine receptor alpha 1 subunit gene GLRA1 result in the neurological disorder hyperekplexia [startle disease, stiff baby syndrome, Mendelian Inheritance in Man (MIM) #149400]. In a pedigree showing dominant transmission of hyperekplexia, we identified a novel point mutation C1128A of GLRA1. This mutation encodes an amino acid substitution (P250T) in the cytoplasmic loop linking transmembrane regions M1 and M2 of the mature alpha 1 polypeptide. After recombinant expression, homomeric alpha 1P250T subunit channels showed a strong reduction of maximum whole-cell chloride currents and an altered desensitization, consistent with a prolonged recovery from desensitization. Apparent glycine binding was less affected, yielding an approximately fivefold increase in Ki values. Topological analysis predicts that the substitution of proline 250 leads to the loss of an angular polypeptide structure, thereby destabilizing open channel conformations. Thus, the novel GLRA1 mutant allele P250T defines an intracellular determinant of glycine receptor channel gating

Glycine Receptor

Objective Impairment of glycinergic neurotransmission leads to complex movement and behavioral disorders. Patients harboring glycine receptor autoantibodies suffer from stiff‐person syndrome or its severe variant progressive encephalomyelitis with rigidity and myoclonus. Enhanced receptor internalization was proposed as the common molecular mechanism upon autoantibody binding. Although functional impairment of glycine receptors following autoantibody binding has recently been investigated, it is still incompletely understood. Methods A cell‐based assay was used for positive sample evaluation. Glycine receptor function was assessed by electrophysiological recordings and radioligand binding assays. The in vivo passive transfer of patient autoantibodies was done using the zebrafish animal model. Results Glycine receptor function as assessed by glycine dose–response curves showed significantly decreased glycine potency in the presence of patient sera. Upon binding of autoantibodies from 2 patients, a decreased fraction of desensitized receptors was observed, whereas closing of the ion channel remained fast. The glycine receptor N‐terminal residues 29A to 62G were mapped as a common epitope of glycine receptor autoantibodies. An in vivo transfer into the zebrafish animal model generated a phenotype with disturbed escape behavior accompanied by a reduced number of glycine receptor clusters in the spinal cord of affected animals. Interpretation Autoantibodies against the extracellular domain mediate alterations of glycine receptor physiology. Moreover, our in vivo data demonstrate that the autoantibodies are a direct cause of the disease, because the transfer of human glycine receptor autoantibodies to zebrafish larvae generated impaired escape behavior in the animal model compatible with abnormal startle response in stiff‐person syndrome or progressive encephalitis with rigidity and myoclonus patients. ANN NEUROL 2020;88:544–56