Introduction Subgroups of autoantibodies directed against voltage-gated potassium channel (K v ) complex components have been associated with immunotherapy-responsive clinical syndromes. The high prevalence and the role of autoantibodies directly binding K v remain, however, controversial. Our objective was to determine K v 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 K v 1.2 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 K v 1.2-3 reactivity. Eleven percent (4/36) of the serum samples showed no binding. Fingerprinting resolved close to identical sequence requirements for both shared epitopes. K v 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 K v 1.2-3 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.
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 GlyRβ 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 GlyRβ making it not unlikely that GlyRβ-specific autoantibody (aAb) exist and contribute to the disease pathology.
Abstract Patients with autoantibodies (aAbs) against the contactin-associated protein-like 2 (CASPR2) suffer from a variety of clinical syndromes including neuropathic pain, in some patients even as the only symptom. CASPR2 is an adhesion protein of the neurexin IV family and part of the voltage-gated potassium channel complex (VGKC) in neurons of dorsal root ganglia (DRG). The subsequent pathological mechanisms following the binding of CASPR2 aAbs and their association with pain are only partially understood. CASPR2 aAbs are mainly of the IgG4 subclass. Previous studies have neglected subclass-dependent effects. Here we investigated 49 subclassified patient serum samples positive for CASPR2 aAbs. To unravel underlying molecular mechanisms, we used a combination of super-resolution lattice structural illumination microscopy (SIM 2 ) and functional readouts by calcium imaging and electrophysiological recordings. CASPR2-positive patient sera subclassified in IgG4 together with at least one other IgG subclass (IgGX) and patients with only IgG4 were further subdivided into the pain and no pain group. Patient subclassification shed further light on the pathological mechanisms of CASPR2 aAbs. A decrease of CASPR2 expression after long-term exposure to CASPR2 aAbs was only observed for the patient group without pain. Upon withdrawal of the CASPR2 aAbs, CASPR2 expression returned to normal level. Structural alterations were obtained by increased distances between CASPR2 and associated potassium channels along DRG axons using high-resolution lattice SIM 2 microscopy but only following binding of CASPR2 aAbs from patients with pain. Similarly, 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 four CASPR2 aAbs subclassifications with the most prominent effect of serum samples harboring IgG4 aAbs. Notably, a patient serum sample lacking IgG4 did not alter Kv channel function. Withdrawal of aAbs rescued Kv channel function to normal levels suggesting that the affected potassium channel function is rather due to a functional block of the VGKC rather than altered structural integrity of the VGKC. Taken together, we found IgG4 aAbs to be a major modifier of potassium channel function. The increase in DRG excitability is primarily due to impaired Kv channel conductance as a consequence of CASPR2 aAbs binding but additional and so far unidentified signal pathways contribute to this process in patients with neuropathic pain.
Autoantibodies (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 α-subunits and, more rarely, also to the GlyR β-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. We 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. Several 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 GlyRα1 in some interneuron subpopulations, GlyRα2 and patient serum signals exhibited a significantly higher colocalization in samples with presynaptic preference. This finding identifies GlyRα2 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. In 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.
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 β subunits that localizes at postsynaptic sites and replaces embryonically expressed GlyRα2 homomers. Human GlyR variants of GLRA1 and GLRB , dominant and recessive, have been intensively studied in vitro . However, the role of unaffected GlyRβ, 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 GlyRβ that were crossed with mouse Glra1 startle disease mutants. We explored the role of GlyRβ under disease conditions in mice carrying a missense mutation ( shaky ) or resulting from the loss of GlyRα1 ( oscillator ). Interestingly, synaptic targeting of GlyRβ was largely unaffected in both mouse mutants. While synaptic morphology appears unaltered in shaky animals, synapses were notably smaller in homozygous oscillator animals. Hence, GlyRβ 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 GlyRα2β complexes to synaptic sites remains functionally insufficient and homozygous oscillator mice still die at three weeks after birth. Thus, both functional and structural deficits can affect glycinergic neurotransmission in severe startle disease, eliciting different compensatory mechanisms in vivo . Scientific statement Glycinergic dysfunction results in a neurological disorder called startle disease. The most common affected gene is GLRA1 that encodes the glycine receptor α1 subunit. A comprehensive analysis of the role of GlyRβ in startle disease has been hampered by the lack of reliable GlyRβ-specific antibodies. The novel mouse model Glrb eos has allowed us to identify compensatory processes during pathology. Targeting of heteromeric GlyRαβ receptors to synaptic sites is maintained in the presence of mutated GlyRα1 and even in the absence of GlyRα1. Thus, insufficient quality control of mutant heteromeric GlyRs in the endoplasmic reticulum underlies the ineffectiveness of functional compensation, even though the presence of a mature postsynaptic specialization could serve as a structural platform to overcome disease severity.
