Neuromyelitis Optica Autoantibody Research Articles

Syndrome of inappropriate antidiuretic hormone secretion associated with seronegative neuromyelitis optica spectrum disorder.

The syndrome of inappropriate antidiuretic hormone secretion (SIADH) is a potential cause of hyponatremia of the central nervous system (CNS). Although SIADH has been reported to be associated with many other central nervous disorders, its association with neuromyelitis optica (NMO) or NMO spectrum disorders are rare. NMO is a demyelinating disorder characterized by optic neuritis and transverse myelitis. Aquaporin-4 (AQP4), which is the target antigen for a NMO autoantibody, is the predominant CNS water channel. However, some NMO patients show seronegative AQP4 antibody results. The spectrum of NMO has been changed, and new findings about the disease have been reported. Here, we report a case of seronegative NMO spectrum disorder associated with SIADH.

High avidity chimeric monoclonal antibodies against the extracellular domains of human aquaporin-4 competing with the neuromyelitis optica autoantibody, NMO-IgG.

Most of the cases of neuromyelitis optica (NMO) are characterized by the presence of an autoantibody, NMO-IgG, which recognizes the extracellular domains of the water channel, aquaporin-4. Binding of NMO-IgG to aquaporin-4 expressed in end-feet of astrocytes leads to complement-dependent disruption of astrocytes followed by demyelination. One therapeutic option for NMO is to prevent the binding of NMO-IgG to aquaporin-4, using high-avidity, non-pathogenic-chimeric, monoclonal antibodies to this water channel. We describe here the development of such antibodies. cDNAs encoding variable regions of heavy and light chains of monoclonal antibodies against the extracellular domains of human aquaporin-4 were cloned from hybridoma total RNA and fused to those encoding constant regions of human IgG1 and Igκ respectively. Then mammalian expression vectors were constructed to establish stable cell lines secreting mature chimeric antibodies. Original monoclonal antibodies showed high avidity binding to human aquaporin-4, as determined by ELISA. Live imaging using Alexa-Fluor-555-labelled antibodies revealed that the antibody D15107 more rapidly bound to cells expressing human aquaporin-4 than others and strongly enhanced endocytosis of this water channel, while D12092 also bound rapidly to human aquaporin-4 but enhanced endocytosis to a lesser degree. Chimeric D15107 prevented complement-dependent cytotoxicity induced by NMO-IgG from patient sera in vitro. We have established non-pathogenic, high-avidity, chimeric antibodies against the extracellular domains of human aquaporin-4, which provide a novel therapeutic option for preventing the progress and recurrence of NMO/NMO spectrum disorders.

Identification of a Point Mutation Impairing the Binding between Aquaporin-4 and Neuromyelitis Optica Autoantibodies

Neuromyelitis optica (NMO) is characterized by the presence of pathogenic autoantibodies (NMO-IgGs) against supra-molecular assemblies of aquaporin-4 (AQP4), known as orthogonal array of particles (OAPs). NMO-IgGs have a polyclonal origin and recognize different conformational epitopes involving extracellular AQP4 loops A, C, and E. Here we hypothesize a pivotal role for AQP4 transmembrane regions (TMs) in epitope assembly. On the basis of multialignment analysis, mutagenesis, NMO-IgG binding, and cytotoxicity assay, we have disclosed the key role of aspartate 69 (Asp(69)) of TM2 for NMO-IgG epitope assembly. Mutation of Asp(69) to histidine severely impairs NMO-IgG binding for 85.7% of the NMO patient sera analyzed here. Although Blue Native-PAGE, total internal reflection fluorescence microscopy, and water transport assays indicate that the OAP Asp(69) mutant is similar in structure and function to the wild type, molecular dynamic simulations have revealed that the D(69)H mutation has the effect of altering the structural rearrangements of extracellular loop A. In conclusion, Asp(69) is crucial for the spatial control of loop A, the particular molecular conformation of which enables the assembly of NMO-IgG epitopes. These findings provide additional clues for new strategies for NMO treatment and a wealth of information to better approach NMO pathogenesis.

Eosinophil pathogenicity mechanisms and therapeutics in neuromyelitis optica

Eosinophils are abundant in inflammatory demyelinating lesions in neuromyelitis optica (NMO). We used cell culture, ex vivo spinal cord slices, and in vivo mouse models of NMO to investigate the role of eosinophils in NMO pathogenesis and the therapeutic potential of eosinophil inhibitors. Eosinophils cultured from mouse bone marrow produced antibody-dependent cell-mediated cytotoxicity (ADCC) in cell cultures expressing aquaporin-4 in the presence of NMO autoantibody (NMO-IgG). In the presence of complement, eosinophils greatly increased cell killing by a complement-dependent cell-mediated cytotoxicity (CDCC) mechanism. NMO pathology was produced in NMO-IgG-treated spinal cord slice cultures by inclusion of eosinophils or their granule toxins. The second-generation antihistamines cetirizine and ketotifen, which have eosinophil-stabilizing actions, greatly reduced NMO-IgG/eosinophil-dependent cytotoxicity and NMO pathology. In live mice, demyelinating NMO lesions produced by continuous intracerebral injection of NMO-IgG and complement showed marked eosinophil infiltration. Lesion severity was increased in transgenic hypereosinophilic mice. Lesion severity was reduced in mice made hypoeosinophilic by anti-IL-5 antibody or by gene deletion, and in normal mice receiving cetirizine orally. Our results implicate the involvement of eosinophils in NMO pathogenesis by ADCC and CDCC mechanisms and suggest the therapeutic utility of approved eosinophil-stabilizing drugs.

Light inactivation of water transport and protein–protein interactions of aquaporin–Killer Red chimeras

Aquaporins (AQPs) have a broad range of cellular and organ functions; however, nontoxic inhibitors of AQP water transport are not available. Here, we applied chromophore-assisted light inactivation (CALI) to inhibit the water permeability of AQP1, and of two AQP4 isoforms (M1 and M23), one of which (M23) forms aggregates at the cell plasma membrane. Chimeras containing Killer Red (KR) and AQPs were generated with linkers of different lengths. Osmotic water permeability of cells expressing KR/AQP chimeras was measured from osmotic swelling–induced dilution of cytoplasmic chloride, which was detected using a genetically encoded chloride-sensing fluorescent protein. KR-AQP1 red fluorescence was bleached rapidly (∼10% per second) by wide-field epifluorescence microscopy. After KR bleaching, KR-AQP1 water permeability was reduced by up to 80% for the chimera with the shortest linker. Remarkably, CALI-induced reduction in AQP4-KR water permeability was approximately twice as efficient for the aggregate-forming M23 isoform; this suggests intermolecular CALI, which was confirmed by native gel electrophoresis on cells coexpressing M23-AQP4-KR and myc-tagged M23-AQP4. CALI also disrupted the interaction of AQP4 with a neuromyelitis optica autoantibody directed against an extracellular epitope on AQP4. CALI thus permits rapid, spatially targeted and irreversible reduction in AQP water permeability and interactions in live cells. Our data also support the utility of CALI to study protein–protein interactions as well as other membrane transporters and receptors.

Evidence against Cellular Internalization in Vivo of NMO-IgG, Aquaporin-4, and Excitatory Amino Acid Transporter 2 in Neuromyelitis Optica

Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are thought to be pathogenic in neuromyelitis optica (NMO). Prior work has suggested that a key component of NMO autoantibody (NMO-IgG) pathogenesis is internalization of AQP4 and the associated glutamate transporter EAAT2, leading to glutamate excitotoxicity. Here, we show selective endocytosis of NMO-IgG and AQP4 in transfected cell cultures, but little internalization in brain in vivo. AQP4-dependent endocytosis of NMO-IgG occurred rapidly in various AQP4-transfected cell lines, with efficient transport from early endosomes to lysosomes. Cell surface AQP4 was also reduced following NMO-IgG exposure. However, little or no internalization of NMO-IgG, AQP4, or EAAT2 was found in primary astrocyte cultures, nor was glutamate uptake affected by NMO-IgG exposure. Following injection of NMO-IgG into mouse brain, NMO-IgG binding and AQP4 expression showed a perivascular astrocyte distribution, without detectable cellular internalization over 24 h. We conclude that astrocyte endocytosis of NMO-IgG, AQP4, and EAAT2 is not a significant consequence of AQP4 autoantibody in vivo, challenging generally accepted views about NMO pathogenesis.

Intravenous neuromyelitis optica autoantibody in mice targets aquaporin-4 in peripheral organs and area postrema.

The pathogenesis of neuromyelitis optica (NMO) involves binding of IgG autoantibodies (NMO-IgG) to aquaporin-4 (AQP4) on astrocytes in the central nervous system (CNS). We studied the in vivo processing in mice of a recombinant monoclonal human NMO-IgG that binds strongly to mouse AQP4. Following intravenous administration, serum [NMO-IgG] decreased with t1/2 ∼18 hours in wildtype mice and ∼41 hours in AQP4 knockout mice. NMO-IgG was localized to AQP4-expressing cell membranes in kidney (collecting duct), skeletal muscle, trachea (epithelial cells) and stomach (parietal cells). NMO-IgG was seen on astrocytes in the area postrema in brain, but not elsewhere in brain, spinal cord, optic nerve or retina. Intravenously administered NMO-IgG was also seen in brain following mechanical disruption of the blood-brain barrier. Selective cellular localization was not found for control (non-NMO) IgG, or for NMO-IgG in AQP4 knockout mice. NMO-IgG injected directly into brain parenchyma diffused over an area of ∼5 mm2 over 24 hours and targeted astrocyte foot-processes. Our data establish NMO-IgG pharmacokinetics and tissue distribution in mice. The rapid access of serum NMO-IgG to AQP4 in peripheral organs but not the CNS indicates that restricted antibody access cannot account for the absence of NMO pathology in peripheral organs.

Aquaporins at a glance

With a concentration of 55,000 mM, water is by far the most prevalent molecule in biological systems. For many years it was assumed that biological membranes are freely water permeable, so that the existence of a family of water channels would not have been predicted. However, biophysical studies in

Orthogonal array formation by human aquaporin-4: Examination of neuromyelitis optica-associated aquaporin-4 polymorphisms

Pathogenic autoantibodies target aquaporin-4 (AQP4) water channels in individuals with neuromyelitis optica (NMO). Recently, allelic mutations were reported at residue 19 of AQP4 in three cases of NMO, and it was suggested that polymorphisms may influence disease by altering AQP4 supramolecular assembly into orthogonal arrays of particles (OAPs). We analyzed the determinants of OAP formation by human AQP4 to investigate the possible role of polymorphisms in NMO pathogenesis. NMO-associated mutations R19I and R19T in AQP4 did not affect OAP assembly, palmitoylation-dependent regulation of assembly, or NMO autoantibody binding. Residue-19 polymorphisms in AQP4 are thus unlikely to be disease relevant.

Binding Affinity and Specificity of Neuromyelitis Optica Autoantibodies to Aquaporin-4 M1/M23 Isoforms and Orthogonal Arrays

Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are highly specific for the neuroinflammatory disease neuromyelitis optica (NMO). We measured the binding of NMO autoantibodies to AQP4 in human astrocyte-derived U87MG cells expressing M1 and/or M23 AQP4, or M23 mutants that do not form orthogonal array of particles (OAPs). Binding affinity was quantified by two-color fluorescence ratio imaging of cells stained with NMO serum or a recombinant monoclonal NMO autoantibody (NMO-rAb), together with a C terminus anti-AQP4 antibody. NMO-rAb titrations showed binding with dissociation constants down to 44 ± 7 nm. Different NMO-rAbs and NMO patient sera showed a wide variation in NMO-IgG binding to M1 versus M23 AQP4. Differences in binding affinity rather than stoichiometry accounted for M1 versus M23 binding specificity, with consistently greater affinity of NMO-IgG binding to M23 than M1 AQP4. Binding and OAP measurements in cells expressing different M1:M23 ratios or AQP4 mutants indicated that the differential binding of NMO-IgG to M1 versus M23 was due to OAP assembly rather than to differences in the M1 versus M23 N termini. Purified Fab fragments of NMO-IgG showed similar patterns of AQP4 isoform binding, indicating that structural changes in the AQP4 epitope upon array assembly, and not bivalent cross-linking of whole IgG, result in the greater binding affinity to OAPs. Our study establishes a quantitative assay of NMO-IgG binding to AQP4 and indicates remarkable, OAP-dependent heterogeneity in NMO autoantibody binding specificity.

Aquaporin‐4 Mz isoform: Brain expression, supramolecular assembly and neuromyelitis optica antibody binding

Water channel aquaporin-4 (AQP4) is expressed in astrocytes throughout brain and spinal cord. Two major AQP4 isoforms are expressed, M1 and M23, having different translation initiation sites. A longer isoform (Mz) has been reported in rat with translation initiation 126-bp upstream from that of M1. By immunoblot analysis of SDS and native gels probed with a C-terminus anti-AQP4 antibody, Mz was detected in rat brain as a distinct band of size ∼39 kDa. Mz was absent in human and mouse brain because of in-frame stop codons. The ability of rat Mz to form orthogonal arrays of particles (OAPs) was investigated by single particle tracking and native gel electrophoresis. We found that Mz, like M1, diffused rapidly in the cell plasma membrane and did not form OAPs. However, when co-expressed with M23, Mz associated in OAPs by forming heterotetramers with M23. Unexpectedly, Mz-expressing cells bound neuromyelitis optica autoantibodies (NMO-IgG) poorly, <5-fold compared with M1-expressing cells. Truncation analysis suggested that the poor NMO-IgG binding to Mz involves residues 31-41 upstream of Met-1. We conclude that Mz AQP4 is (a) present at low level in rat but not human or mouse brain, (b) unable to form OAPs on its own but able to associate with M23 AQP4 in heterotetramers, and (c) largely unable to bind NMO-IgG because of N-terminus effects on the structure of the AQP4/NMO-IgG binding site.

Higher order structure of aquaporin-4

Unlike other mammalian AQPs, multiple tetramers of AQP4 associate in the plasma membrane to form peculiar structures called Orthogonal Arrays of Particles (OAPs), that are observable by freeze-fracture electron microscopy (FFEM). However, FFEM cannot give information about the composition of OAPs of different sizes, and due to its technical complexity is not easily applicable as a routine technique. Recently, we employed the 2D gel electrophoresis BN–SDS/PAGE that for the first time enabled the biochemical isolation of AQP4-OAPs from several tissues. We found that AQP4 protein is present in several higher-order complexes (membrane pools of supra-structures) which contain different ratios of M1/M23 isoforms corresponding to AQP4-OAPs of different size. In this paper, we illustrate in detail the potentiality of 2D BN/SDS-PAGE for analyzing AQP4 supra-structures, their relationship with the dystrophin glycoprotein complex and other membrane proteins, and their role as a specific target of Neuromyelitis Optica autoantibodies.

Live Cell Analysis of Aquaporin-4 M1/M23 Interactions and Regulated Orthogonal Array Assembly in Glial Cells

Aquaporin-4 (AQP4) can assemble into supramolecular aggregates called orthogonal arrays of particles (OAPs). In cells expressing single AQP4 isoforms, we found previously that OAP formation by AQP4-M23 requires N terminus interactions just downstream of Met-23 and that the inability of AQP4-M1 to form OAPs involves blocking by residues upstream of Met-23. Here, we studied M1/M23 interactions and regulated OAP assembly by nanometer-resolution tracking of quantum dot-labeled AQP4 in live cells expressing differentially tagged AQP4 isoforms and in primary glial cell cultures in which native AQP4 was labeled with a monoclonal recombinant neuromyelitis optica autoantibody. OAP assembly was assessed independently by Blue Native gel electrophoresis. We found that OAPs in native glial cells could be reproduced in transfected cells expressing equal amounts of AQP4-M1 and -M23. Mutants of M23 that do not themselves form OAPs, including M23-F26Q and M23-G28P, were able to fully co-associate with native M23 to form large immobile OAPs. Analysis of a palmitoylation-null M1 mutant (C13A/C17A) indicated palmitoylation-dependent OAP assembly only in the presence of M23, with increased M1 palmitoylation causing progressive OAP disruption. Differential regulation of OAP assembly by palmitoylation, calcium elevation, and protein kinase C activation was found in primary glial cell cultures. We conclude that M1 and M23 co-assemble in AQP4 OAPs and that specific signaling events can regulate OAP assembly in glial cells.

Neuromyelitis Optica Immunoglobulin G in a Child

Neuromyelitis optica or Devic's syndrome is an uncommon demyelinating disorder that preferentially attacks the spinal cord and optic nerves. Although it is well described in adults, childhood neuromyelitis optica has rarely been reported in the literature and is frequently misdiagnosed as severe multiple sclerosis. Recently, a serum immunoglobulin G test for neuromyelitis optica has become available which may clarify and accelerate the diagnosis. This report describes a child with recurrent myelitis and an elongated spinal cord lesion who was found to have positive neuromyelitis optica autoantibody. We believe that neuromyelitis optica autoantibody testing should be performed in cases of pediatric transverse myelitis with multiple vertical segments or recurrence.