Shannon R. Hinson

IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel.

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease that selectively affects optic nerves and spinal cord. It is considered a severe variant of multiple sclerosis (MS), and frequently is misdiagnosed as MS, but prognosis and optimal treatments differ. A serum immunoglobulin G autoantibody (NMO-IgG) serves as a specific marker for NMO. Here we show that NMO-IgG binds selectively to the aquaporin-4 water channel, a component of the dystroglycan protein complex located in astrocytic foot processes at the blood-brain barrier. NMO may represent the first example of a novel class of autoimmune channelopathy.

Pathogenic potential of IgG binding to water channel extracellular domain in neuromyelitis optica

Background: Autoantibody specific for the aquaporin-4 astrocytic water channel is restricted to serum and CSF of patients with neuromyelitis optica (NMO) and related CNS inflammatory demyelinating disorders (relapsing optic neuritis and longitudinally extensive transverse myelitis). NMO-typical lesions are distinct from MS-typical lesions. Aquaporin-4 is lost selectively at vasculocentric sites of edema/inflammation coinciding with focal deposits of immunoglobulins (Ig) G, M, and terminal complement products, with and without myelin loss. Evidence for antigen-specific autoantibody pathogenicity is lacking. Methods: We used confocal microscopy and flow cytometry to evaluate the selectivity and immunopathological consequences of Ig binding to surface epitopes of living target cells expressing aquaporin-4 fused at its cytoplasmic N-terminus with GFP. We tested serum, IgG-enriched and IgG-depleted serum fractions, and CSF from patients with NMO, neurologic control patients, and healthy subjects. We also analyzed aquaporin-4 immunoreactivity in myelinated adult mouse optic nerves and spinal cord, and plasma cell Ig isotypes in archived brain tissue from an NMO patient. Results: Serum IgG from patients with NMO binds to the extracellular domain of aquaporin-4; it is predominantly IgG1, and it initiates two potentially competing outcomes, aquaporin-4 endocytosis/degradation and complement activation. Serum and CSF lack aquaporin-4-specific IgM, and plasma cells in CNS lesions of NMO contain only IgG. Paranodal astrocytic endfeet highly express aquaporin-4. Conclusions: NMO patients’ serum IgG has a selective pathologic effect on cell membranes expressing aquaporin-4. IgG targeting astrocytic processes around nodes of Ranvier could initiate demyelination.

Aquaporin-4-binding autoantibodies in patients with neuromyelitis optica impair glutamate transport by down-regulating EAAT2.

Neuromyelitis optica (NMO)-immunoglobulin G (IgG) is a clinically validated serum biomarker that distinguishes relapsing central nervous system (CNS) inflammatory demyelinating disorders related to NMO from multiple sclerosis. This autoantibody targets astrocytic aquaporin-4 (AQP4) water channels. Clinical, radiological, and immunopathological data suggest that NMO-IgG might be pathogenic. Characteristic CNS lesions exhibit selective depletion of AQP4, with and without associated myelin loss; focal vasculocentric deposits of IgG, IgM, and complement; prominent edema; and inflammation. The effect of NMO-IgG on astrocytes has not been studied. In this study, we demonstrate that exposure to NMO patient serum and active complement compromises the membrane integrity of CNS-derived astrocytes. Without complement, astrocytic membranes remain intact, but AQP4 is endocytosed with concomitant loss of Na+-dependent glutamate transport and loss of the excitatory amino acid transporter 2 (EAAT2) . Our data suggest that EAAT2 and AQP4 exist in astrocytic membranes as a macromolecular complex. Transport-competent EAAT2 protein is up-regulated in differentiating astrocyte progenitors and in nonneural cells expressing AQP4 transgenically. Marked reduction of EAAT2 in AQP4-deficient regions of NMO patient spinal cord lesions supports our immunocytochemical and immunoprecipitation data. Thus, binding of NMO-IgG to astrocytic AQP4 initiates several potentially neuropathogenic mechanisms: complement activation, AQP4 and EAAT2 down-regulation, and disruption of glutamate homeostasis.

Molecular outcomes of neuromyelitis optica (NMO)-IgG binding to aquaporin-4 in astrocytes

The astrocytic aquaporin-4 (AQP4) water channel is the target of pathogenic antibodies in a spectrum of relapsing autoimmune inflammatory central nervous system disorders of varying severity that is unified by detection of the serum biomarker neuromyelitis optica (NMO)-IgG. Neuromyelitis optica is the most severe of these disorders. The two major AQP4 isoforms, M1 and M23, have identical extracellular residues. This report identifies two novel properties of NMO-IgG as determinants of pathogenicity. First, the binding of NMO-IgG to the ectodomain of astrocytic AQP4 has isoform-specific outcomes. M1 is completely internalized, but M23 resists internalization and is aggregated into larger-order orthogonal arrays of particles that activate complement more effectively than M1 when bound by NMO-IgG. Second, NMO-IgG binding to either isoform impairs water flux directly, independently of antigen down-regulation. We identified, in nondestructive central nervous system lesions of two NMO patients, two previously unappreciated histopathological correlates supporting the clinical relevance of our in vitro findings: (i) reactive astrocytes with persistent foci of surface AQP4 and (ii) vacuolation in adjacent myelin consistent with edema. The multiple molecular outcomes identified as a consequence of NMO-IgG interaction with AQP4 plausibly account for the diverse pathological features of NMO: edema, inflammation, demyelination, and necrosis. Differences in the nature and anatomical distribution of NMO lesions, and in the clinical and imaging manifestations of disease documented in pediatric and adult patients, may be influenced by regional and maturational differences in the ratio of M1 to M23 proteins in astrocytic membranes.

Prediction of Neuromyelitis Optica Attack Severity by Quantitation of Complement-Mediated Injury to Aquaporin-4–Expressing Cells

BACKGROUND Recent reports support a pathogenic role in neuromyelitis optica (NMO) for the aquaporin-4 (AQP4)-specific autoantibody (NMO-IgG). Neuromyelitis optica is an inflammatory demyelinating central nervous system disease, usually relapsing, that causes variable degrees of attack-related disability. The NMO-IgG binds in vitro to the extracellular domain of AQP4, activates complement, and causes astrocyte lesioning. OBJECTIVE To compare the prognostic utility of NMO-IgG titer and quantitative measures of complement-mediated injury to AQP4-expressing cells in NMO attacks. DESIGN, SETTING, AND PARTICIPANTS A retrospective clinical-serological correlative study at Mayo Clinics Neuroimmunology Laboratory was undertaken. Over an 18-month period, we identified NMO-IgG-seropositive patients in whom sufficient serum and adequate clinical information pertaining to NMO attacks (6 severe, 6 mild) were available to analyze clinical-serological correlations. Sera from 9 patients with multiple sclerosis and 9 healthy subjects (all NMO-IgG seronegative) served as controls. Complement activation was measured by quantifying the number of green fluorescent protein-AQP4-transfected HEK 293 cells permeable to the viability dye propidium iodide after exposure to patient serum and active complement. MAIN OUTCOME MEASURES Attack severity (mild or severe), percentage of AQP4-transfected cells lesioned, and NMO-IgG titer. RESULTS The median percentage of AQP4-transfected cells lesioned by complement in the presence of serum from patients with NMO was 14% for patients with mild attacks and 54% for patients with severe attacks (P = .005). Median complement activation values for sera from healthy subjects and patients with multiple sclerosis were 8% and 12%, respectively. Patients with mild NMO attacks and patients with severe NMO attacks did not differ significantly with respect to NMO-IgG titer (P = .089). CONCLUSIONS A laboratory measure of complement-mediated cell injury may serve as a prognostic biomarker in NMO. Larger prospective studies are required to validate this observation.

Diagnosis of Neuromyelitis Spectrum Disorders: Comparative Sensitivities and Specificities of Immunohistochemical and Immunoprecipitation Assays

OBJECTIVE To compare the sensitivity and specificity of immunofluorescence (IF) and immunoprecipitation (IP) assays using green fluorescent protein-tagged aquaporin-4 (AQP4) in 6335 patients for whom serological evaluation was requested on a service basis. DESIGN Case-control study. SETTING Mayo Clinic Neuroimmunology Laboratory (Rochester, Minnesota) and Departments of Neurology (Rochester, Minnesota; Scottsdale, Arizona; and Jacksonville, Florida). Patients Group 1, 835 Mayo Clinic patients, 100 with a neuromyelitis optica (NMO) spectrum disorder diagnosis and 735 without NMO spectrum disorder; group 2, 5500 non-Mayo Clinic patients. Main Outcome Measure Sensitivity and specificity of each assay for NMO or NMO spectrum disorder, individually and combined. RESULTS In group 1, the sensitivity rates for NMO were IF, 58%; IP, 33%; and combined assays, 63%. The sensitivity rates for relapsing longitudinally extensive transverse myelitis were IF, 29%; IP, 6%; and combined assays, 29%. The specificity rates for NMO and relapsing longitudinally extensive transverse myelitis were IF, 99.6%; IP, 99.3%; and combined assays, 99.2%. In group 2, NMO-IgG was detected by IF in 498 of 5500 patients (9.1%) and by IP in 331 patients (6.0%); 76 of the 331 patients seropositive by IP (23%) were negative by IF. Clinical information was available for 124 patients (including 16 of those seropositive by IP only); 123 had a definite NMO spectrum disorder and 1 was at risk for NMO (monophasic optic neuritis). CONCLUSIONS In this large, clinical practice-based study, NMO-IgG detected by IF or IP was highly specific for NMO spectrum disorders. The IP assay was significantly less sensitive than IF. Combined testing improved sensitivity by 5%.

Neurological autoimmunity targeting aquaporin-4.

Neuromyelitis optica (NMO) is the first inflammatory autoimmune demyelinating disease of the CNS for which a specific tissue target molecule has been identified--the astrocytic water channel aquaporin-4 (AQP4). Immunological insights have propelled significant advances in understanding the clinical, radiologic and immunopathologic characteristics of the disease in the last 5 years. In this review, we describe features distinguishing CNS AQP4 autoimmunity from classical multiple sclerosis (MS). In NMO, disease attacks preferentially involve the optic nerves and spinal cord (hence the name), but neurological signs in the initial attack of AQP4 autoimmunity in children commonly involve the brain. A clinically validated serum biomarker, NMO-IgG, distinguishes relapsing CNS inflammatory demyelinating disorders related to NMO from MS. The NMO-IgG autoantibody is AQP4-specific. Clinical, radiological and immunopathological data support its role in the pathogenesis of NMO spectrum disorders. Lesions characteristic of NMO are distinct from MS: AQP4 and its coupled glutamate transporter, excitatory amino acid transporter 2 (EAAT2), are lost, with and without associated myelin loss, IgG, IgM and complement are deposited in a vasculocentric pattern, edema and inflammation are prominent. In vitro studies demonstrate that binding of NMO-IgG to astrocytic AQP4 initiates multiple potentially neuropathogenic mechanisms: complement activation, AQP4 and EAAT2 downregulation with disruption of water and glutamate homeostasis, enhanced blood-brain barrier permeability, plasma protein and granulocyte influx, and antibody-dependent cell-mediated cytotoxicity. Development of effective, and potentially curative, therapies requires validated models of the disease, in animals and cell culture systems.

Autoimmune Glial Fibrillary Acidic Protein Astrocytopathy: A Novel Meningoencephalomyelitis.

Importance A novel astrocytic autoantibody has been identified as a biomarker of a relapsing autoimmune meningoencephalomyelitis that is immunotherapy responsive. Seropositivity distinguishes autoimmune glial fibrillary acidic protein (GFAP) meningoencephalomyelitis from disorders commonly considered in the differential diagnosis. Objective To describe a novel IgG autoantibody found in serum or cerebrospinal fluid that is specific for a cytosolic intermediate filament protein of astrocytes. Design, Setting, and Participants Retrospective review of the medical records of seropositive patients identified in the Mayo Clinic Neuroimmunology Laboratory from October 15, 1998, to April 1, 2016, in blinded comprehensive serologic evaluation for autoantibody profiles to aid the diagnosis of neurologic autoimmunity (and predict cancer likelihood). Main Outcomes and Measures Frequency and definition of novel autoantibody, the autoantigens immunochemical identification, clinical and magnetic resonance imaging correlations of the autoantibody, and immunotherapy responsiveness. Results Of 103 patients whose medical records were available for review, the 16 initial patients identified as seropositive were the subject of this study. Median age at neurologic symptom onset was 42 years (range, 21-73 years); there was no sex predominance. The novel neural autoantibody, which we discovered to be GFAP-specific, is disease spectrum restricted but not rare (frequency equivalent to Purkinje cell antibody type 1 [anti-Yo]). Its filamentous pial, subventricular, and perivascular immunostaining pattern on mouse tissue resembles the characteristic magnetic resonance imaging findings of linear perivascular enhancement in patients. Prominent clinical manifestations are headache, subacute encephalopathy, optic papillitis, inflammatory myelitis, postural tremor, and cerebellar ataxia. Cerebrospinal fluid was inflammatory in 13 of 14 patients (93%) with data available. Neoplasia was diagnosed within 3 years of neurologic onset in 6 of 16 patients (38%): prostate and gastroesophageal adenocarcinomas, myeloma, melanoma, colonic carcinoid, parotid pleomorphic adenoma, and teratoma. Neurologic improvement followed treatment with high-dose corticosteroids, with a tendency of patients to relapse without long-term immunosuppression. Conclusions and Relevance Glial fibrillary acidic protein-specific IgG identifies a distinctive, corticosteroid-responsive, sometimes paraneoplastic autoimmune meningoencephalomyelitis. It has a lethal canine equivalent: necrotizing meningoencephalitis. Expression of GFAP has been reported in some of the tumor types identified in paraneoplastic cases. Glial fibrillary acidic protein peptide-specific cytotoxic CD8+ T cells are implicated as effectors in a transgenic mouse model of autoimmune GFAP meningoencephalitis.

Syndrome of inappropriate antidiuresis may herald or accompany neuromyelitis optica

Aquaporin-4 (AQP4) is the target autoantigen of an immunoglobulin G (IgG) autoantibody that distinguishes a spectrum of inflammatory demyelinating CNS disorders (the neuromyelitis optica [NMO] spectrum) from multiple sclerosis (MS) and other CNS demyelinating disorders.1 Compelling evidence supports this IgG having a central role in the pathogenesis of NMO. AQP4 is concentrated in astrocytic foot processes at interfaces between CNS parenchyma and fluid compartments, both CSF and blood,1 and in areas involved in osmosensitivity and osmoregulation, including supraoptic and paraventricular nuclei of the hypothalamus and sensory circumventricular organs, the subfornical organ, the organum vasculosum of the lamina terminalis, and the area postrema.2 A single case of the syndrome of inappropriate antidiuresis (SIAD) has been described in NMO.3 Here we report the frequency of SIAD in NMO.

Glial fibrillary acidic protein immunoglobulin G as biomarker of autoimmune astrocytopathy: Analysis of 102 patients

A novel autoimmune central nervous system (CNS) disorder with glial fibrillary acidic protein (GFAP)‐IgG as biomarker was recently characterized. Here, 102 patients with GFAP‐IgG positivity are described.