Neuromyelitis optica: aquaporin-4 based pathogenesis mechanisms and new therapies

Neuromyelitis optica (NMO) is an autoimmune 'aquaporinopathy' of the central nervous system that causes inflammatory demyelinating lesions primarily in spinal cord and optic nerve, leading to paralysis and blindness. NMO lesions show loss of aquaporin-4 (AQP4), GFAP and myelin, infiltration of granulocytes and macrophages, and perivascular deposition of activated complement. Most patients with NMO are seropositive for immunoglobulin autoantibodies (AQP4-IgG) against AQP4, the principal water channel of astrocytes. There is strong evidence that AQP4-IgG is pathogenic in NMO, probably by a mechanism involving complement-dependent astrocyte cytotoxicity, causing leukocyte infiltration, cytokine release and blood-brain barrier disruption, which leads to oligodendrocyte death, myelin loss and neuron death. Here, we review the evidence for this and alternative proposed NMO pathogenesis mechanisms, such as AQP4-IgG-induced internalization of AQP4 and glutamate transporters, complement-independent cell-mediated cytotoxicity, and AQP4-IgG inhibition of AQP4 water transport function. Based on the initiating pathogenic role of AQP4-IgG binding to astrocyte AQP4 in NMO, selective blocker therapies are under development in which AQP4-targeted monoclonal antibodies or small molecules block binding of AQP4-IgG to astrocytes and consequent downstream pathology.     

Aquaporin-4 Autoantibodies in Neuromyelitis Optica: AQP4 Isoform-Dependent Sensitivity and Specificity

Neuromyelitis Optica (NMO) is an autoimmune demyelinating disease, characterized by the presence of autoantibody (NMO-IgG) to Aquaporin-4 (AQP4). NMO-IgG identification supports NMO diagnosis and several diagnostic tests have been developed, but their sensitivity is too variable, and some assay show low sensitivity. This impairs correct diagnosis of NMO. By cell based assay (CBA) we here evaluate the efficacy of different strategies to express AQP4 in mammalian cells in terms of: a) AQP4 translation initiation signals; b) AQP4 isoforms (M1 and M23) and fluorescent tag position; c) NMO serum concentration and AQP4 degradation. Our results demonstrate that when using AQP4-M1, the nucleotide in position −3 of the AUG greatly affects the AQP4-M1/M23 protein ratio, NMO-IgG binding, and consequently test sensitivity. Test sensitivity was highest with M23 expressing cells (97.5%) and only 27.5% with AQP4-M1. The fluorescent tag added to the N-terminus of AQP4-M23 considerably affected the NMO-IgG binding, and test sensitivity, due to disruption of AQP4 suprastructures. Furthermore, sera used at high concentration resulted in AQP4 degradation which affected test sensitivity. To further evaluate the reliability of the M23 based CBA test, samples of one NMO patient collected during about 2 years clinical follow-up were tested. The results of serum titer correlated with disease activity and treatment response. In conclusion, we provide a molecular explanation for the contrasting CBA test data reported and suggest the use of M23 with a C-terminus fluorescent tag as the proper test for NMO diagnosis.     

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.     

Complement-dependent cytotoxicity in neuromyelitis optica requires aquaporin-4 protein assembly in orthogonal arrays

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system in which binding of pathogenic autoantibodies (NMO-IgG) to astrocyte aquaporin-4 (AQP4) causes complement-dependent cytotoxicity (CDC) and inflammation. We previously reported a wide range of binding affinities of NMO-IgGs to AQP4 in separate tetramers versus intramembrane aggregates (orthogonal arrays of particles, OAPs). We report here a second, independent mechanism by which CDC is affected by AQP4 assembly. Utilizing lactate dehydrogenase release and live/dead cell cytotoxicity assays, we found in different cell lines, and with different monoclonal and patient-derived NMO-IgGs, that CDC was greatly (>100-fold) reduced in cells expressing M1- versus M23-AQP4. Studies using a M23-AQP4 mutant containing an OAP-disrupting mutation, and in cells expressing AQP4 in different M1/M23 ratios, indicated that NMO-IgG-dependent CDC requires AQP4 OAP assembly. In contrast, antibody-dependent cell-mediated cytotoxicity produced by natural killer cells did not depend on AQP4 OAP assembly. Measurements of C1q binding and complement attack complex (C9neo) supported the conclusion that the greatly enhanced CDC by OAPs is due to efficient, multivalent binding of C1q to clustered NMO-IgG on OAPs. We conclude that AQP4 assembly in OAPs is required for CDC in NMO, establishing a new mechanism of OAP-dependent NMO pathogenesis. Disruption of AQP4 OAPs may greatly reduce NMO-IgG dependent CDC and NMO pathology.     

Model of aquaporin-4 supramolecular assembly in orthogonal arrays based on heterotetrameric association of M1-M23 isoforms

Tetramers of aquaporin-4 (AQP4) water channels form supramolecular assemblies in cell membranes called orthogonal arrays of particles (OAPs). We previously reported evidence that a short (M23) AQP4 isoform produced by alternative splicing forms OAPs by an intermolecular N-terminus interaction, whereas the full-length (M1) AQP4 isoform does not by itself form OAPs but can coassemble with M23 in OAPs as heterotetramers. Here, we developed a model to predict number distributions of OAP size, shape, and composition as a function M23:M1 molar ratio. Model specifications included: random tetrameric assembly of M1 with M23; intertetramer associations between M23 and M23, but not between M1 and M23 or M1; and a free energy constraint limiting OAP size. Model predictions were tested by total internal reflection fluorescence microscopy of AQP4-green-fluorescent protein chimeras and native gel electrophoresis of cells expressing different M23:M1 ratios. Experimentally validated model predictions included: 1), greatly increased OAP size with increasing M23:M1 ratio; 2), marked heterogeneity in OAP size at fixed M23:M1, with increased M23 fraction in larger OAPs; and 3), preferential M1 localization at the periphery of OAPs. The model was also applied to test predictions about binding to AQP4 OAPs of a pathogenic AQP4 autoantibody found in the neuroinflammatory demyelinating disease neuromyelitis optica. Our model of AQP4 OAPs links a molecular-level interaction of AQP4 with its supramolecular assembly in cell membranes.     

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 (Asp⁶⁹) of TM2 for NMO-IgG epitope assembly. Mutation of Asp⁶⁹ 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 Asp⁶⁹ mutant is similar in structure and function to the wild type, molecular dynamic simulations have revealed that the D⁶⁹H mutation has the effect of altering the structural rearrangements of extracellular loop A. In conclusion, Asp⁶⁹ 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.     

Supramolecular aggregation of aquaporin‐4 is different in muscle and brain: correlation with tissue susceptibility in neuromyelitis optica

Neuromyelitis optica (NMO) is an autoimmune demyelinating disease of the central nervous system (CNS) caused by autoantibodies (NMO‐IgG) against the water channel aquaporin‐4 (AQP4). Though AQP4 is also expressed outside the CNS, for example in skeletal muscle, patients with NMO generally do not show clinical/diagnostic evidence of skeletal muscle damage. Here, we have evaluated whether AQP4 supramolecular organization is at the basis of the different tissue susceptibility. Using immunofluorescence we found that while the sera of our cohort of patients with NMO gave typical perivascular staining in the CNS, they were largely negative in the skeletal muscle. This conclusion was obtained using human, rat and mouse skeletal muscle including the AQP4‐KO mouse. A biochemical analysis using a new size exclusion chromatography approach for AQP4 suprastructure fractionation revealed substantial differences in supramolecular AQP4 assemblies and isoform abundance between brain and skeletal muscle matching a lower binding affinity of NMO‐IgG to muscle compared to the brain. Super‐resolution microscopy analysis with g‐STED revealed different AQP4 organization in native tissues, while in the brain perivascular astrocyte endfoot membrane AQP4 was mainly organized in large interconnected and raft‐like clusters, in the sarcolemma of fast‐twitch fibres AQP4 aggregates often appeared as small, relatively isolated linear entities. In conclusion, our results provide evidence that AQP4 supramolecular structure is different in brain and skeletal muscle, which is likely to result in different tissues susceptibility to the NMO disease.     

Clinical Relevance of Serum Aquaporin-4 Antibody Levels in Neuromyelitis Optica

Neuromyelitis optica (NMO) is an inflammatory disease that selectively affects the optic nerves and spinal cord. The discovery of NMO-IgG targeting aquaporin-4 (AQP4) in NMO patients suggested that NMO is a distinct entity, with a fundamentally different etiology from that of multiple sclerosis (MS). Although NMO usually leads to grave disability because of the more severe tissue destruction compared with classical MS, there have been several reports describing a benign form of NMO over a long disease term. NMO-IgG/AQP4 antibodies show high specificity but medium sensitivity for NMO, while the clinical relevance of AQP4 antibody titers remains to be determined. We aimed to clarify the clinical relevance of AQP4 antibody levels determined by a bridging enzyme-linked immunosorbent assay in 38 patients with NMO or NMO spectrum disorder. The AQP4 antibody levels were higher in patients with optic neuritis (ON) than in those without ON (p = 0.0164). Among the 12 patients examined in a longitudinal study, four showed an increase in the ELISA values during some relapses, and eight showed no clear correlation between the ELISA values and relapse. Of the four patients who demonstrated a steady rise in the antibody levels over time, two patients had no concomitant relapses, despite elevation of the AQP4 antibody levels. We conclude that high AQP4 antibody levels are associated with the occurrence of ON, but that the antibody levels themselves are not closely correlated with the onset of relapse.     

Histamine treatment induces rearrangements of orthogonal arrays of particles (OAPs) in human AQP4-expressing gastric cells

To test the involvement of the water channel aquaporin (AQP)-4 in gastric acid physiology, the human gastric cell line (HGT)-1 was stably transfected with rat AQP4. AQP4 was immunolocalized to the basolateral membrane of transfected HGT-1 cells, like in native parietal cells. Expression of AQP4 in transfected cells increased the osmotic water permeability coefficient (Pf) from 2.02 +/- 0.3 x 10-4 to 16.37 +/- 0.5 x 10-4 cm/s at 20 degrees C. Freeze-fracture EM showed distinct orthogonal arrays of particles (OAPs), the morphological signature of AQP4, on the plasma membrane of AQP4-expressing cells. Quantitative morphometry showed that the density of OAPs was 2.5 +/- 0.3% under basal condition and decreased by 50% to 1.2 +/- 0.3% after 20 min of histamine stimulation, mainly due to a significant decrease of the OAPs number. Concomitantly, Pf decreased by approximately 35% in 20-min histamine-stimulated cells. Both Pf and OAPs density were not modified after 10 min of histamine exposure, time at which the maximal hormonal response is observed. Cell surface biotinylation experiments confirmed that AQP4 is internalized after 20 min of histamine exposure, which may account for the downregulation of water transport. This is the first evidence for short term rearrangement of OAPs in an established AQP4-expressing cell line.     

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.     

Identification of two major conformational aquaporin-4 epitopes for neuromyelitis optica autoantibody binding

Neuromyelitis optica (NMO) is an autoimmune demyelinating disease characterized by the presence of anti-aquaporin-4 (AQP4) antibodies in the patient sera. We recently reported that these autoantibodies are able to bind AQP4 when organized in the supramolecular structure called the orthogonal array of particles (OAP). To map the antigenic determinants, we produced a series of AQP4 mutants based on multiple alignment sequence analysis between AQP4 and other OAP-forming AQPs. Mutations were introduced in the three extracellular loops (A, C, and E), and the binding capacity of NMO sera was tested on AQP4 mutants. Results indicate that one group of sera was able to recognize a limited portion of loop C containing the amino acid sequence (146)GVT(T/M)V(150). A second group of sera was characterized by a predominant role of loop A. Deletion of four AQP4-specific amino acids ((61)G(S/T)E(N/K)(64)) in loop A substantially affected the binding of this group of sera. However, the binding capacity was further reduced when amino acids in loop A were mutated together with those in loop E or when those in loop C were mutated in combination with loop E. Finally, a series of AQP0 mutants were produced in which the extracellular loops were progressively changed to make them identical to AQP4. Results showed that none of the mutants was able to reproduce in AQP0 the NMO-IgG epitopes, indicating that the extracellular loop sequence by itself was not sufficient to determine the rearrangement required to create the epitopes. Although our data highlight the complexity of the disease, this study identifies key immunodominant epitopes and provides direct evidence that the transition from AQP4 tetramers to AQP4-OAPs involves conformational changes of the extracellular loops.     

Evidences for a Leaky Scanning Mechanism for the Synthesis of the Shorter M23 Protein Isoform of Aquaporin-4: IMPLICATION IN ORTHOGONAL ARRAY FORMATION AND NEUROMYELITIS OPTICA ANTIBODY INTERACTION*

Aquaporin-4 (AQP4) exists as two major isoforms that differ in the length of the N terminus, the shorter AQP4-M23 and the longer AQP4-M1. Both isoforms form tetramers, which can further aggregate in the plasma membrane to form typical orthogonal arrays of particles (OAPs) whose dimension depends on the ratio of the M1 and M23. In this study, we tested the hypothesis that the M23 isoform can be produced directly by the M1 mRNA. In cells transiently transfected with AQP4-M1 coding sequence we observed besides AQP4-M1 the additional presence of the AQP4-M23 isoform associated with the formation of typical OAPs observable by two-dimensional blue native/SDS-PAGE and total internal reflection microscopy. The mutation of the second in-frame methionine M23 in AQP4-M1 (AQP4-M1^M23I) prevented the expression of the M23 isoform and the formation of OAPs. We propose “leaky scanning” as a translational mechanism for the expression of AQP4-M23 protein isoform and that the formation of OAPs may occur even in the absence of AQP4-M23 mRNA. This mechanism can have important pathophysiological implications for the cell regulation of the M1/M23 ratio and thus OAP size. In this study we also provide evidence that AQP4-M1 is mobile in the plasma membrane, that it is inserted and not excluded into immobile OAPs, and that it is an important determinant of OAP structure and size.     

Post-Golgi supramolecular assembly of aquaporin-4 in orthogonal arrays

The supramolecular assembly of aquaporin-4 (AQP4) in orthogonal arrays of particles (OAPs) involves N-terminus interactions of the M23-AQP4 isoform. We found AQP4 OAPs in cell plasma membranes but not in endoplasmic reticulum (ER) or Golgi, as shown by: (i) native gel electrophoresis of brain and AQP4-transfected cells, (ii) photobleaching recovery of green fluorescent protein-AQP4 chimeras in live cells and (iii) freeze-fracture electron microscopy (FFEM). We found that AQP4 OAP formation in plasma membranes, but not in the Golgi, was not related to AQP4 density, pH, membrane lipid composition, C-terminal PDZ domain interactions or α-syntrophin expression. Remarkably, however, fusion of AQP4-containing Golgi vesicles with (AQP4-free) plasma membrane vesicles produced OAPs, suggesting the involvement of plasma membrane factor(s) in AQP4 OAP formation. In investigating additional possible determinants of OAP assembly we discovered membrane curvature-dependent OAP assembly, in which OAPs were disrupted by extrusion of plasma membrane vesicles to ～110 nm diameter, but not to ～220 nm diameter. We conclude that AQP4 supramolecular assembly in OAPs is a post-Golgi phenomenon involving plasma membrane-specific factor(s). Post-Golgi and membrane curvature-dependent OAP assembly may be important for vesicle transport of AQP4 in the secretory pathway and AQP4-facilitated astrocyte migration, and suggests a novel therapeutic approach for neuromyelitis optica.     

Aquaporin-4 dynamics in orthogonal arrays in live cells visualized by quantum dot single particle tracking

Freeze-fracture electron microscopy (FFEM) indicates that aquaporin-4 (AQP4) water channels can assemble in cell plasma membranes in orthogonal arrays of particles (OAPs). We investigated the determinants and dynamics of AQP4 assembly in OAPs by tracking single AQP4 molecules labeled with quantum dots at an engineered external epitope. In several transfected cell types, including primary astrocyte cultures, the long N-terminal "M1" form of AQP4 diffused freely, with diffusion coefficient approximately 5 x 10(-10) cm(2)/s, covering approximately 5 microm in 5 min. The short N-terminal "M23" form of AQP4, which by FFEM was found to form OAPs, was relatively immobile, moving only approximately 0.4 microm in 5 min. Actin modulation by latrunculin or jasplakinolide did not affect AQP4-M23 diffusion, but deletion of its C-terminal postsynaptic density 95/disc-large/zona occludens (PDZ) binding domain increased its range by approximately twofold over minutes. Biophysical analysis of short-range AQP4-M23 diffusion within OAPs indicated a spring-like potential, with a restoring force of approximately 6.5 pN/microm. These and additional experiments indicated that 1) AQP4-M1 and AQP4-M23 isoforms do not coassociate in OAPs; 2) OAPs can be imaged directly by total internal reflection fluorescence microscopy; and 3) OAPs are relatively fixed, noninterconvertible assemblies that do not require cytoskeletal or PDZ-mediated interactions for formation. Our measurements are the first to visualize OAPs in live cells.     

A Small-molecule Screen Yields Idiotype-specific Blockers of Neuromyelitis Optica Immunoglobulin G Binding to Aquaporin-4

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system caused by binding of anti-aquaporin-4 (AQP4) autoantibodies (NMO-IgG) to AQP4 on astrocytes. A screen was developed to identify inhibitors of NMO-IgG-dependent, complement-dependent cytotoxicity. Screening of 50,000 synthetic small molecules was done using CHO cells expressing human AQP4 and a human NMO recombinant monoclonal antibody (rAb-53). The screen yielded pyrano[2,3-c]pyrazoles that blocked rAb-53 binding to AQP4 and prevented cytotoxicity in cell culture and spinal cord slice models of NMO. Structure-activity analysis of 82 analogs yielded a blocker with IC₅₀ ∼ 6 μm. Analysis of the blocker mechanism indicated idiotype specificity, as (i) pyrano[2,3-c]pyrazoles did not prevent AQP4 binding or cytotoxicity of other NMO-IgGs, and (ii) surface plasmon resonance showed specific rAb-53 binding. Antibody structure modeling and docking suggested a putative binding site near the complementarity-determining regions. Small molecules with idiotype-specific antibody targeting may be useful as research tools and therapeutics.     

Immunobiology of neuromyelitis optica spectrum disorders

Neuromyelitis optica spectrum disorder (NMOSD) is a rare autoimmune inflammatory disease of the central nervous system. Most of the cases are positive for autoantibodies targeting the water channel aquaporin-4 (AQP4-IgG). Activated B and T cells, innate immunity cells, pro-inflammatory cytokines, and activated complement contribute to the formation of the NMOSD lesions. Optic neuritis, longitudinally extensive myelitis, and area postrema syndrome are core clinical manifestations. NMOSD diagnosis is based on clinical manifestations, magnetic resonance imaging findings, and AQP4-IgG positivity. Cell-based assays are the preferred method for the detection of AQP4-IgG. Acute relapses are treated with IV methylprednisolone or plasma exchange. Recent advances on the NMOSD immunobiology led to approved treatments such as eculizumab, satralizumab, and inebilizumab.     

An Optimized Immunohistochemistry Technique Improves NMO-IgG Detection: Study Comparison with Cell-Based Assays

Cell-based assays (CBA) have increased the sensitivity of the neuromyelitis optica (NMO)-IgG/aquaporin-4-antibody detection compared to classical tissue-based indirect assays. We describe the sensitivity of an optimized immunohistochemistry (IHC-o) to detect NMO-IgG/aquaporin-4-antibody in comparison with that of two CBA: an in-house (CBA-ih) and a commercial (CBA-c) assay (Euroimmun, Germany). Coded serum from 103 patients with definite NMO and 122 inflammatory controls were studied by IHC-o, CBA-ih, and CBA-c. IHC-o used the same protocol described to detect antibodies against cell surface antigens. CBA-ih used live cells transfected with the aquaporin-4-M23-isoform. The sensitivity of the IHC-o was 74.8% (95% confidence interval [CI] 65-83) and was similar to that of the CBA-ih 75.7% (95% CI 66-84) and the CBA-c 73.8% (95% CI 64-82). The specificity of the three assays was 100% (95% CI 97-100). Interassay concordance was high, 100 of 103 samples were coincident in all techniques. The optimized immunohistochemistry proves to be as sensitive and specific as the cell-based assays. This assay extends the available tools for NMO-IgG/aquaporin-4-antibody detection.     

Challenging AQP4 druggability for NMO-IgG antibody binding using molecular dynamics and molecular interaction fields

Neuromyelitis optica (NMO) is a multiple sclerosis-like immunopathology disease affecting optic nerves and the spinal cord. Its pathological hallmark is the deposition of a typical immunoglobulin, called NMO-IgG, against the water channel Aquaporin-4 (AQP4). Preventing NMO-IgG binding would represent a valuable molecular strategy for a focused NMO therapy. The recent observation that aspartate in position 69 (D69) is determinant for the formation of NMO-IgG epitopes prompted us to carry out intensive Molecular Dynamics (MD) studies on a number of single-point AQP4 mutants. Here, we report a domino effect originating from the point mutation at position 69: we find that the side chain of T62 is reoriented far from its expected position leaning on the lumen of the pore. More importantly, the strength of the H-bond interaction between L53 and T56, at the basis of the loop A, is substantially weakened. These events represent important pieces of a clear-cut mechanistic rationale behind the failure of the NMO-IgG binding, while the water channel function as well as the propensity to aggregate into OAPs remains unaltered. The molecular interaction fields (MIF)-based analysis of cavities complemented MD findings indicating a putative binding site comprising the same residues determining epitope reorganization. In this respect, docking studies unveiled an intriguing perspective to address the future design of small drug-like compounds against NMO. In agreement with recent experimental observations, the present study is the first computational attempt to elucidate NMO-IgG binding at the molecular level, as well as a first effort toward a less elusive AQP4 druggability.     

Neuromyelitis optica: Passive transfer to rats by human immunoglobulin

Recurrent attacks of optic neuritis and myelitis are the hallmarks of both neuromyelitis optica (NMO) and multiple sclerosis (MS). NMO immunoglobulin G (NMO-IgG), which recognizes astrocytic aquaporin-4 (AQP4) water channels, is a specific serum autoantibody that distinguishes NMO from MS. The pathogenic role of the anti-AQP4 antibody (AQP4-Ab, NMO-IgG) in NMO has been speculated based on several studies in vitro. The aim of this study was to demonstrate the pathogenicity of AQP4-Ab in vivo. We obtained IgG from patients who underwent therapeutic plasmapheresis, and developed an animal model by passive transfer of IgG to rats. The active lesions of the rats exhibited pathological characteristics strikingly similar to those of NMO, marked by astrocytic loss and perivascular deposition of immunoglobulin and complements. These findings provide the first evidence of the pathogenicity of AQP4-Ab in vivo and support the therapeutic efficacy of eliminating the antibodies by plasmapheresis.     

Reversible, Temperature-Dependent Supramolecular Assembly of Aquaporin-4 Orthogonal Arrays in Live Cell Membranes

The shorter “M23” isoform of the glial cell water channel aquaporin-4 (AQP4) assembles into orthogonal arrays of particles (OAPs) in cell plasma membranes, whereas the full-length “M1” isoform does not. N-terminal residues are responsible for OAP formation by AQP4-M23 and for blocking of OAP formation in AQP4-M1. In investigating differences in OAP formation by certain N-terminus mutants of AQP4, as measured by freeze-fracture electron microscopy versus live-cell imaging, we discovered reversible, temperature-dependent OAP assembly of certain weakly associating AQP4 mutants. Single-particle tracking of quantum-dot-labeled AQP4 in live cells and total internal reflection fluorescence microscopy showed >80% of M23 in OAPs at 10-50°C compared to <10% of M1. However, OAP formation by N-terminus cysteine-substitution mutants of M1, which probe palmitoylation-regulated OAP assembly, was strongly temperature-dependent, increasing from <10% at 37°C to >70% at 10°C for the double mutant M1-C13A/C17A. OAP assembly by this mutant, but not by native M23, could also be modulated by reducing its membrane density. Exposure of native M1 and single cysteine mutants to 2-bromopalmitate confirmed the presence of regulated OAP assembly by S-palmitoylation. Kinetic studies showed rapid and reversible OAP formation during cooling and OAP disassembly during heating. Our results provide what to our knowledge is the first information on the energetics of AQP4 OAP assembly in plasma membranes.