Julien Ratelade

Complement-dependent Cytotoxicity in Neuromyelitis Optica Requires Aquaporin-4 Protein Assembly in Orthogonal Arrays

Background: Complement-dependent cytotoxicity (CDC) plays a central role in neuromyelitis optica (NMO), in which NMO autoantibodies (NMO-IgG) bind to AQP4 on astrocytes. Results: NMO-IgG produced CDC only when AQP4 was assembled in orthogonal arrays of particles (OAPs). Conclusion: AQP4 assembly in OAPs is required for CDC by a mechanism involving multivalent C1q binding. Significance: Our results establish a new mechanism of OAP-dependent pathogenesis in NMO and suggest a novel therapeutic strategy. 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.

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.

Enzymatic deglycosylation converts pathogenic neuromyelitis optica anti-aquaporin-4 immunoglobulin G into therapeutic antibody.

Neuromyelitis optica (NMO) is caused by binding of pathogenic autoantibodies (NMO‐immunoglobulin G [IgG]) to aquaporin‐4 (AQP4) on astrocytes, which initiates complement‐dependent cytotoxicity (CDC) and inflammation. We recently introduced mutated antibody (aquaporumab) and small‐molecule blocker strategies for therapy of NMO, based on prevention of NMO‐IgG binding to AQP4. Here, we investigated an alternative strategy involving neutralization of NMO‐IgG effector function by selective IgG heavy‐chain deglycosylation with bacteria‐derived endoglycosidase S (EndoS).

Neuromyelitis optica IgG and natural killer cells produce NMO lesions in mice without myelin loss

The pathogenesis of neuromyelitis optica (NMO) involves targeting of NMO-immunoglobulin G (NMO-IgG) to aquaporin-4 (AQP4) on astrocytes in the central nervous system. Prior work provided evidence for complement-dependent cytotoxicity (CDC) in NMO lesion development. Here, we show that antibody-dependent cellular cytotoxicity (ADCC), in the absence of complement, can also produce NMO-like lesions. Antibody-dependent cellular cytotoxicity was produced in vitro by incubation of mouse astrocyte cultures with human recombinant monoclonal NMO-IgG and human natural killer cells (NK-cells). Injection of NMO-IgG and NK-cells in mouse brain caused loss of AQP4 and GFAP, two characteristic features of NMO lesions, but little myelin loss. Lesions were minimal or absent following injection of: (1) control (non-NMO) IgG with NK-cells; (2) NMO-IgG and NK-cells in AQP4-deficient mice; or (3) NMO-IgG and NK-cells in wild-type mice together with an excess of mutated NMO-IgG lacking ADCC effector function. NK-cells greatly exacerbated NMO lesions produced by NMO-IgG and complement in an ex vivo spinal cord slice model of NMO, causing marked myelin loss. NMO-IgG can thus produce astrocyte injury by ADCC in a complement-independent and dependent manner, suggesting the potential involvement of ADCC in NMO pathogenesis.

Evidence against cellular internalization in vivo of NMO-IgG, aquaporin-4, and excitatory amino acid transporter 2 in neuromyelitis optica.

Background: Binding of the neuromyelitis optica (NMO) autoantibody (NMO-IgG) to aquaporin-4 (AQP4) is pathogenic in NMO. Results: NMO-IgG binding does not cause endocytosis of itself, AQP4, or glutamate transporter EAAT2 in astrocyte cultures or in vivo. Conclusion: Internalization of NMO-IgG, AQP4, and EAAT2 is not a significant feature of NMO. Significance: Our results challenge a generally accepted paradigm on NMO pathogenesis. 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.

Greatly improved survival and neuroprotection in aquaporin-4-knockout mice following global cerebral ischemia

Aquaporin‐4 (AQP4), the principal water channel in astrocytes, is involved in brain water movement, inflammation, and neuroexcitation. In this study, there was strong neuroprotection in mice lacking AQP4 in a model of global cerebral ischemia produced by transient, bilateral carotid artery occlusion (BCAO). Survival and neurological outcome were greatly improved in the AQP4‐/‐ vs. AQP4+/+ mice after occlusion, with large and robust differences in both outbred (CD1) and inbred (C57bl/6) mouse strains without or with mechanical ventilation. Improved survival was also seen in mice lacking the scaffold protein α‐syntrophin, which manifest reduced astrocyte water permeability secondary to defective AQP4 plasma membrane targeting. Intracranial pressure elevation and brain water accumulation were much reduced in the AQP4‐/‐ vs. AQP4+/+ mice after carotid artery occlusion, as were blood–brain barrier (BBB) disruption and neuronal loss. Brain slices from AQP4‐/‐ mice showed significantly reduced cell swelling and cytotoxicity in response to oxygen–glucose deprivation, compared with slices from AQP4+/+ mice. Our findings suggest that the neuroprotective effect of AQP4 deletion in global cerebral ischemia involves reduced astrocyte swelling and brain water accumulation, resulting in reduced BBB disruption, inflammation, and neuron death. AQP4 water transport inhibition may improve survival and neurological outcome after cardiac arrest and in other conditions associated with global cerebral ischemia.—Katada, R., Akdemir, G., Asavapanumas, N., Ratelade, J., Zhang, H., Verkman, A. S. Greatly improved survival and neuroprotection in aquaporin‐4 knockout mice following global cerebral ischemia. FASEB J. 28, 705–714 (2014). www.fasebj.org

SUPER-RESOLUTION IMAGING OF AQUAPORIN-4 ORTHOGONAL ARRAYS OF PARTICLES IN CELL MEMBRANES

Summary Aquaporin-4 (AQP4) is a water channel expressed in astrocytes, skeletal muscle and epithelial cells that forms supramolecular aggregates in plasma membranes called orthogonal arrays of particles (OAPs). AQP4 is expressed as a short isoform (M23) that forms large OAPs, and a long isoform (M1) that does not form OAPs by itself but can mingle with M23 to form relatively small OAPs. AQP4 OAPs were imaged with ∼20 nm spatial precision by photoactivation localization microscopy (PALM) in cells expressing chimeras of M1- or M23-AQP4 with photoactivatable fluorescent proteins. Native AQP4 was imaged by direct stochastic optical reconstruction microscopy (dSTORM) using a primary anti-AQP4 antibody and fluorescent secondary antibodies. We found that OAP area increased from 1878±747 to 3647±958 nm2 with decreasing M1:M23 ratio from 1:1 to 1:3, and became elongated. Two-color dSTORM indicated that M1 and M23 co-assemble in OAPs with a M1-enriched periphery surrounding a M23-enriched core. Native AQP4 in astrocytes formed OAPs with an area of 2142±829 nm2, which increased to 5137±1119 nm2 with 2-bromopalmitate. PALM of AQP4 OAPs in live cells showed slow diffusion (average ∼10−12 cm2/s) and reorganization. OAP area was not altered by anti-AQP4 IgG autoantibodies (NMO-IgG) that cause the neurological disease neuromyelitis optica. Super-resolution imaging allowed elucidation of novel nanoscale structural and dynamic features of OAPs.

Neuromyelitis optica IgG does not alter aquaporin-4 water permeability, plasma membrane M1/M23 isoform content, or supramolecular assembly.

Neuromyelitis optica (NMO) is thought to be caused by immunoglobulin G autoantibodies (NMO‐IgG) against astrocyte water channel aquaporin‐4 (AQP4). A recent study (Hinson et al. (2012) Proc Natl Acad Sci USA 109:1245‐1250) reported that NMO‐IgG inhibits AQP4 water permeability directly and causes rapid cellular internalization of the M1 but not M23 isoform of AQP4, resulting in AQP4 clustering, enhanced complement‐dependent cytotoxicity, and tissue swelling. Here, we report evidence challenging this proposed mechanism of NMO‐IgG‐mediated pathology. We measured osmotic water permeability by stopped‐flow light scattering on plasma membrane vesicles isolated from AQP4‐expressing CHO cells, an approach that can detect changes in water permeability as small as 5% and is not confounded by internalization effects. We found similar single‐molecule water permeability for M1‐AQP4 tetramers and M23‐AQP4 clusters (orthogonal arrays of particles, OAPs). Exposure of AQP4 to high concentrations of NMO‐IgG from six seropositive NMO patients, and to high‐affinity recombinant monoclonal NMO antibodies, did not reduce AQP4 water permeability. Also, NMO‐IgG did not reduce water permeability in AQP4‐reconstituted proteoliposomes. In transfected cells expressing M1‐ or M23‐AQP4 individually, NMO‐IgG caused more rapid internalization of M23‐ than M1‐AQP4. In cells coexpressing both isoforms, M1‐ and M23‐AQP4 comingled in OAPs that were internalized together in response to NMO‐IgG. Super‐resolution imaging and native gel electrophoresis showed that the size of AQP4 OAPs was not altered by NMO sera or recombinant NMO antibodies. We conclude that NMO‐IgG does not: (i) inhibit AQP4 water permeability, (ii) cause preferential internalization of M1‐AQP4, or (iii) cause intramembrane AQP4 clustering.

Aquaporin-4: orthogonal array assembly, CNS functions, and role in neuromyelitis optica

Aquaporin-4 (AQP4) is a water-selective transporter expressed in astrocytes throughout the central nervous system, as well as in kidney, lung, stomach and skeletal muscle. The two AQP4 isoforms produced by alternative spicing, M1 and M23 AQP4, form heterotetramers that assemble in cell plasma membranes in supramolecular structures called orthogonal arrays of particles (OAPs). Phenotype analysis of AQP4-null mice indicates the involvement of AQP4 in brain and spinal cord water balance, astrocyte migration, neural signal transduction and neuroinflammation. AQP4-null mice manifest reduced brain swelling in cytotoxic cerebral edema, but increased brain swelling in vasogenic edema and hydrocephalus. AQP4 deficiency also increases seizure duration, impairs glial scarring, and reduces the severity of autoimmune neuroinflammation. Each of these phenotypes is likely explicable on the basis of reduced astrocyte water permeability in AQP4 deficiency. AQP4 is also involved in the neuroinflammatory demyelinating disease neuromyelitis optica (NMO), where autoantibodies (NMO-IgG) targeting AQP4 produce astrocyte damage and inflammation. Mice administered NMO-IgG and human complement by intracerebral injection develop characteristic NMO lesions with neuroinflammation, demyelination, perivascular complement deposition and loss of glial fibrillary acidic protein and AQP4 immunoreactivity. Our findings suggest the potential utility of AQP4-based therapeutics, including small-molecule modulators of AQP4 water transport function for therapy of brain swelling, injury and epilepsy, as well as small-molecule or monoclonal antibody blockers of NMO-IgG binding to AQP4 for therapy of NMO.

Involvement of antibody-dependent cell-mediated cytotoxicity in inflammatory demyelination in a mouse model of neuromyelitis optica

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system that can cause paralysis and blindness. The pathogenesis of NMO involves binding of immunoglobulin G autoantibodies to aquaporin-4 (AQP4) on astrocytes, which is thought to cause complement-dependent cytotoxicity (CDC) and a secondary inflammatory response leading to oligodendrocyte and neuronal damage. Here, we investigate in vivo the role of antibody-dependent cell-mediated cytotoxicity (ADCC) triggered by AQP4 autoantibodies (AQP4-IgG) in the development of NMO pathology. A high-affinity, human recombinant monoclonal AQP4-IgG was mutated in its Fc region to produce ‘NMO superantibodies’ with enhanced CDC and/or ADCC effector functions, without altered AQP4 binding. Pathological effects of these antibodies were studied in a mouse model of NMO produced by intracerebral injection of AQP4-IgG and human complement. The original (non-mutated) antibody produced large NMO lesions in this model, with loss of AQP4 and GFAP immunoreactivity, inflammation and demyelination, as did a mutated antibody with enhanced CDC and ADCC effector functions. As anticipated, a mutated AQP4-IgG lacking CDC, but having tenfold enhanced ADCC, produced little pathology. However, unexpectedly, a mutated antibody with ninefold enhanced CDC, but lacking ADCC, produced much less pathology than the original AQP4-IgG. Also, pathology was greatly reduced following administration of AQP4-IgG and complement to mice lacking the FcγIII receptor involved in effector cell activation during ADCC, and to normal mice injected with an Fcγ receptor blocking antibody. Our results provide evidence for the central involvement of ADCC in NMO pathology and suggest ADCC as a new therapeutic target in NMO.