s A. Kleopa

Antibodies to Kv1 potassium channel-complex proteins leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 in limbic encephalitis, Morvan’s syndrome and acquired neuromyotonia

Antibodies that immunoprecipitate 125I-α-dendrotoxin-labelled voltage-gated potassium channels extracted from mammalian brain tissue have been identified in patients with neuromyotonia, Morvan’s syndrome, limbic encephalitis and a few cases of adult-onset epilepsy. These conditions often improve following immunomodulatory therapies. However, the proportions of the different syndromes, the numbers with associated tumours and the relationships with potassium channel subunit antibody specificities have been unclear. We documented the clinical phenotype and tumour associations in 96 potassium channel antibody positive patients (titres >400 pM). Five had thymomas and one had an endometrial adenocarcinoma. To define the antibody specificities, we looked for binding of serum antibodies and their effects on potassium channel currents using human embryonic kidney cells expressing the potassium channel subunits. Surprisingly, only three of the patients had antibodies directed against the potassium channel subunits. By contrast, we found antibodies to three proteins that are complexed with 125I-α-dendrotoxin-labelled potassium channels in brain extracts: (i) contactin-associated protein-2 that is localized at the juxtaparanodes in myelinated axons; (ii) leucine-rich, glioma inactivated 1 protein that is most strongly expressed in the hippocampus; and (iii) Tag-1/contactin-2 that associates with contactin-associated protein-2. Antibodies to Kv1 subunits were found in three sera, to contactin-associated protein-2 in 19 sera, to leucine-rich, glioma inactivated 1 protein in 55 sera and to contactin-2 in five sera, four of which were also positive for the other antibodies. The remaining 18 sera were negative for potassium channel subunits and associated proteins by the methods employed. Of the 19 patients with contactin-associated protein-antibody-2, 10 had neuromyotonia or Morvan’s syndrome, compared with only 3 of the 55 leucine-rich, glioma inactivated 1 protein-antibody positive patients (P < 0.0001), who predominantly had limbic encephalitis. The responses to immunomodulatory therapies, defined by changes in modified Rankin scores, were good except in the patients with tumours, who all had contactin-associated-2 protein antibodies. This study confirms that the majority of patients with high potassium channel antibodies have limbic encephalitis without tumours. The identification of leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 as the major targets of potassium channel antibodies, and their associations with different clinical features, begins to explain the diversity of these syndromes; furthermore, detection of contactin-associated protein-2 antibodies should help identify the risk of an underlying tumour and a poor prognosis in future patients.

KCNQ2 Is a Nodal K+ Channel

Mutations in the gene encoding the K+ channel KCNQ2 cause neonatal epilepsy and myokymia, indicating that KCNQ2 regulates the excitability of CNS neurons and motor axons, respectively. We show here that KCNQ2 channels are functional components of axon initial segments and nodes of Ranvier, colocalizing with ankyrin-G and voltage-dependent Na+ channels throughout the CNS and PNS. Retigabine, which opens KCNQ channels, diminishes axonal excitability. Linopirdine, which blocks KCNQ channels, prolongs the repolarization of the action potential in neonatal nerves. The clustering of KCNQ2 at nodes and initial segments lags that of ankyrin-G during development, and both ankyrin-G and KCNQ2 can be coimmunoprecipitated in the brain. KCNQ3 is also a component of some initial segments and nodes in the brain. The diminished activity of mutant KCNQ2 channels accounts for neonatal epilepsy and myokymia; the cellular locus of these effects may be axonal initial segments and nodes.

Morvan syndrome: clinical and serological observations in 29 cases.

A study was undertaken to describe the clinical spectrum, voltage‐gated potassium channel (VGKC) complex antibody specificities, and central nervous system localization of antibody binding in 29 patients diagnosed with Morvan syndrome (MoS).

Autoimmune limbic encephalitis in 39 patients: immunophenotypes and outcomes

Background: About 40% of patients with limbic encephalitis do not have detectable CNS antibodies. Some of these patients have immune-mediated limbic encephalitis, but their frequency is unknown. Aims: (1) To determine the spectrum of limbic encephalitis identified on clinical grounds in a single institution, and compare it with that in patients referred for antibody analysis. (2) To correlate clinical outcomes with the cellular location of the autoantigens. Methods: Prospective clinical case studies. Immunohistochemistry with rat brain, live hippocampal neurones, HeLa cells expressing Kv potassium channels and immunoblot. Results: In 4 years, 17 patients were identified in the Hospital of the University of Pennsylvania, Philadelphia, USA, and the serum or CSF samples of 22 patients diagnosed elsewhere were also studied. 9 of our 17 (53%) patients had antibodies to known neuronal antigens (paraneoplastic or voltage gated potassium channels (VGKCs)) and 5 (29%) to novel cell-membrane antigens (nCMAg) typically expressed in the hippocampus and sometimes in the cerebellum. Considering the entire series, 19 of 39 (49%) patients had antibodies to known antigens, and 17 (44%) to nCMAg. Follow-up (2–48 months, median 19 months) was available for 35 patients. When compared with patients with antibodies to intraneuronal antigens, a significant association with response to treatment was found in those with antibodies to cell-membrane antigens in general (VGKC or nCMAg, p = 0.003) or to nCMAg (p = 0.006). Conclusions: (1) 82% of patients with limbic encephalitis prospectively identified on clinical grounds had CNS antibodies; (2) responsiveness to treatment is not limited to patients with VGKC antibodies; (3) in many patients (29% from a single institution), the autoantigens were unknown but were found to be highly enriched in neuronal cell membranes of the hippocampus; and (4) these antibodies are associated with a favourable outcome.

A comprehensive analysis of the epidemiology and clinical characteristics of anti-LRP4 in myasthenia gravis.

Double-seronegative myasthenia gravis (dSN-MG, without detectable AChR and MuSK antibodies) presents a serious gap in MG diagnosis and understanding. Recently, autoantibodies against the low-density lipoprotein receptor-related protein 4 (LRP4) have been identified in several dSN-MG sera, but with dramatic frequency variation (∼2-50%). We have developed a cell based assay (CBA) based on human LRP4 expressing HEK293 cells, for the reliable and efficient detection of LRP4 antibodies. We have screened about 800 MG patient sera from 10 countries for LRP4 antibodies. The overall frequency of LRP4-MG in the dSN-MG group (635 patients) was 18.7% but with variations among different populations (range 7-32.7%). Interestingly, we also identified double positive sera: 8/107 anti-AChR positive and 10/67 anti-MuSK positive sera also had detectable LRP4 antibodies, predominantly originating from only two of the participating groups. No LRP4 antibodies were identified in sera from 56 healthy controls tested, while 4/110 from patients with other neuroimmune diseases were positive. The clinical data, when available, for the LRP4-MG patients were then studied. At disease onset symptoms were mild (81% had MGFA grade I or II), with some identified thymic changes (32% hyperplasia, none with thymoma). On the other hand, double positive patients (AChR/LRP4-MG and MuSK/LRP4-MG) had more severe symptoms at onset compared with any single positive MG subgroup. Contrary to MuSK-MG, 27% of ocular dSN-MG patients were LRP4 antibody positive. Similarly, contrary to MuSK antibodies, which are predominantly of the IgG4 subtype, LRP4 antibodies were predominantly of the IgG1 and IgG2 subtypes. The prevalence was higher in women than in men (female/male ratio 2.5/1), with an average disease onset at ages 33.4 for females and 41.9 for males. Overall, the response of LRP4-MG patients to treatment was similar to published responses of AChR-MG rather than to MuSK-MG patients.

Cellular mechanisms of connexin32 mutations associated with CNS manifestations

Both oligodendrocytes and myelinating Schwann cells express the gap junction protein connexin32 (Cx32). Mutations in the gene encoding Cx32 (GJB1) cause the X‐linked form of Charcot‐Marie‐Tooth disease (CMTX). Although most CMTX patients do not have clinical central nervous system (CNS) manifestations, subclinical evidence of CNS dysfunction is common. We investigated the cellular effects of a subgroup of GJB1/Cx32 mutations that have been reported to cause clinical CNS dysfunction. We hypothesized that these mutants have dominant‐negative effects on other connexins expressed by oligodendrocytes, specifically Cx45. We expressed these and other Cx32 mutants in communication‐incompetent as well as Cx45‐expressing HeLa cells, and analyzed the transfected cells by immunocytochemistry and immunoblotting. In communication‐incompetent cells, the mutants associated with CNS phenotypes failed to reach the cell membrane and were instead retained in the endoplasmic reticulum (A39V, T55I) or Golgi apparatus (M93V, R164Q, R183H), although rare gap junction plaques were found in cells expressing M93V or R183H. In HeLa cells stably expressing Cx45, these Cx32 mutants showed a similar expression pattern, and did not alter the pattern of Cx45 expression. These results indicate that Cx32 mutants that are associated with a CNS phenotype do not interact with Cx45, but may instead have other toxic effects in oligodendrocytes.

Unique distributions of the gap junction proteins connexin29, connexin32, and connexin47 in oligodendrocytes

Oligodendrocytes of adult rodents express three different connexins: connexin29 (Cx29), Cx32, and Cx47. In this study, we show that Cx29 is localized to the inner membrane of small myelin sheaths, whereas Cx32 is localized on the outer membrane of large myelin sheaths; Cx29 does not colocalize with Cx32 in gap junction plaques. All oligodendrocytes appear to express Cx47, which is largely restricted to their perikarya. Cx32 and Cx47 are colocalized in many gap junction plaques on oligodendrocyte somata, particularly in gray matter. Cx45 is detected in the cerebral vasculature, but not in oligodendrocytes or myelin sheaths. This diversity of connexins in oligodendrocytes (in different populations of cells and in different subcellular compartments) likely reflects functional differences between these connexins and perhaps the oligodendrocytes themselves.

Autoimmune channelopathies and related neurological disorders.

Ion channels are crucial elements in neuronal signaling and synaptic transmission, and defects in their function are known to underlie rare genetic disorders, including some forms of epilepsy. A second class of channelopathies, characterized by autoantibodies against ligand- and voltage-gated ion channels, cause a variety of defects in peripheral neuromuscular and ganglionic transmission. There is also emerging evidence for autoantibody-mediated mechanisms in subgroups of patients with central nervous system disorders, particularly those involving defects in cognition or sleep and often associated with epilepsy. In all autoimmune channelopathies, the relationship between autoantibody specificity and clinical phenotype is complex. But with this new information, autoimmune channelopathies are detected and treated with increasing success, and future research promises new insights into the mechanisms of dysfunction at neuronal synapses and the determinants of clinical phenotype.

Molecular genetics of X-linked Charcot-Marie-Tooth disease

The X-linked form of Charcot-Marie-Tooth disease (CMT1X) is the second most common molecularly designated form of hereditary motor and sensory neuropathy. The clinical phenotype is characterized by progressive distal muscle atrophy and weakness, areflexia, and variable sensory abnormalities. Affected males have moderate-to-severe symptoms, whereas heterozygous females are usually mildly affected or even asymptomatic. Several patients also have manifestations of central nervous system involvement or hearing impairment. Electrophysiological and pathological studies of peripheral nerves show evidence of demyelinating neuropathy with prominent axonal degeneration. A large number of mutations in the GJB1 gene encoding the gap junction (GJ) protein connexin32 (C×32) cause CMT1X. C×32 is expressed by Schwann cells and oligodendrocytes, as well as by other tissues, and the GJ formed by C×32 play an important role in the homeostasis of myelinated axons. The reported CMT1X mutations are diverse and affect both the promoter region as well as the coding region of GJB1. Many C×32 mutants fail to form functional GJ, or form GJ with abnormal biophysical properties. Furthermore, C×32 mutants are often retained intracellularly either in the endoplasmic reticulum or Golgi in which they could potentially have additional dominant-negative effects. Animal models of CMT1X demonstrate that loss of C×32 in myelinating Schwann cells causes a demyelinating neuropathy. No definite phenotype-genotype correlation has yet been established for CMT1X and effective molecular based therapeutics for this disease, remain to be developed.

Connexin32 Mutations Cause Loss of Function in Schwann Cells and Oligodendrocytes Leading to PNS and CNS Myelination Defects

The gap junction (GJ) protein connexin32 (Cx32) is expressed by myelinating Schwann cells and oligodendrocytes and is mutated in X-linked Charcot-Marie-Tooth disease. In addition to a demyelinating peripheral neuropathy, some Cx32 mutants are associated with transient or chronic CNS phenotypes. To investigate the molecular basis of these phenotypes, we generated transgenic mice expressing the T55I or the R75W mutation and an IRES-EGFP, driven by the mouse Cnp promoter. The transgene was expressed in oligodendrocytes throughout the CNS and in Schwann cells. Both the T55I and the R75W mutants were localized in the perinuclear cytoplasm, did not form GJ plaques, and did not alter the expression or localization of two other oligodendrocytic GJ proteins, Cx47 and Cx29, or the expression of Cx29 in Schwann cells. On wild type background, the expression of endogenous mCx32 was unaffected by the T55I mutant, but was partly impaired by R75W. Transgenic mice with the R75W mutation and all mutant animals with Gjb1-null background developed a progressive demyelinating peripheral neuropathy along with CNS myelination defects. These findings suggest that Cx32 mutations result in loss of function in myelinated cells without trans-dominant effects on other GJ proteins. Loss of Cx32 function alone in the CNS causes myelination defects.