Aquaporin-4 Removal from the Plasma Membrane of Human Müller Cells by AQP4-IgG from Patients with Neuromyelitis Optica Induces Changes in Cell Volume Homeostasis: the First Step of Retinal Injury?

Aquaporin-4 (AQP4) is the target of the specific immunoglobulin G autoantibody (AQP4-IgG) produced in patients with neuromyelitis optica spectrum disorders (NMOSD). Previous studies demonstrated that AQP4-IgG binding to astrocytic AQP4 leads to cell-destructive lesions. However, the early physiopathological events in Müller cells in the retina are poorly understood. Here, we investigated the consequences of AQP4-IgG binding to AQP4 of Müller cells, previous to the inflammatory response, on two of AQP4's key functions, cell volume regulation response (RVD) and cell proliferation, a process closely associated with changes in cell volume. Experiments were performed in a human retinal Müller cell line (MIO-M1) exposed to complement-inactivated sera from healthy volunteers or AQP4-IgG positive NMOSD patients. We evaluated AQP4 expression (immunofluorescence and western blot), water permeability coefficient, RVD, intracellular calcium levels and membrane potential changes during hypotonic shock (fluorescence videomicroscopy) and cell proliferation (cell count and BrdU incorporation). Our results showed that AQP4-IgG binding to AQP4 induces its partial internalization, leading to the decrease of the plasma membrane water permeability, a reduction of swelling-induced increase of intracellular calcium levels and the impairment of RVD in Müller cells. The loss of AQP4 from the plasma membrane induced by AQP4-IgG positive sera delayed Müller cells' proliferation rate. We propose that Müller cell dysfunction after AQP4 removal from the plasma membrane by AQP4-IgG binding could be a non-inflammatory mechanism of retinal injury in vivo, altering cell volume homeostasis and cell proliferation and consequently, contributing to the physiopathology of NMOSD.

Neuromyelitis optica: pathogenicity of patient immunoglobulin in vivo.

OBJECTIVE: Severe inflammation and astrocyte loss with profound demyelination in spinal cord and optic nerves are typical pathological features of neuromyelitis optica (NMO). A diagnostic hallmark of this disease is the presence of serum autoantibodies against the water channel aquaporin-4 (AQP-4) on astrocytes. METHODS: We induced acute T-cell-mediated experimental autoimmune encephalomyelitis in Lewis rats and confronted the animals with an additional application of immunoglobulins from AQP-4 antibody-positive and -negative NMO patients, multiple sclerosis patients, and control subjects. RESULTS: The immunoglobulins from AQP-4 antibody-positive NMO patients are pathogenic. When they reach serum titers in experimental animals comparable with those seen in NMO patients, they augment clinical disease and induce lesions in the central nervous system that are similar in structure and distribution to those seen in NMO patients, consisting of AQP-4 and astrocyte loss, granulocytic infiltrates, T cells and activated macrophages/microglia cells, and an extensive immunoglobulin and complement deposition on astrocyte processes of the perivascular and superficial glia limitans. AQP-4 antibody containing NMO immunoglobulin injected into naïve rats, young rats with leaky blood-brain barrier, or after transfer of a nonencephalitogenic T-cell line did not induce disease or neuropathological alterations in the central nervous system. Absorption of NMO immunoglobulins with AQP-4-transfected cells, but not with mock-transfected control cells, reduced the AQP-4 antibody titers and was associated with a reduction of astrocyte pathology after transfer. INTERPRETATION: Human anti-AQP-4 antibodies are not only important in the diagnosis of NMO but also augment disease and induce NMO-like lesions in animals with T-cell-mediated brain inflammation.

Marked central nervous system pathology in CD59 knockout rats following passive transfer of Neuromyelitis optica immunoglobulin G.

Neuromyelitis optica spectrum disorders (herein called NMO) is an inflammatory demyelinating disease of the central nervous system in which pathogenesis involves complement-dependent cytotoxicity (CDC) produced by immunoglobulin G autoantibodies targeting aquaporin-4 (AQP4-IgG) on astrocytes. We reported evidence previously, using CD59-/- mice, that the membrane-associated complement inhibitor CD59 modulates CDC in NMO (Zhang and Verkman, J. Autoimmun. 53:67-77, 2014). Motivated by the observation that rats, unlike mice, have human-like complement activity, here we generated CD59-/- rats to investigate the role of CD59 in NMO and to create NMO pathology by passive transfer of AQP4-IgG under conditions in which minimal pathology is produced in normal rats. CD59-/- rats generated by CRISPR/Cas9 technology showed no overt phenotype at baseline except for mild hemolysis. CDC assays in astrocyte cultures and cerebellar slices from CD59-/- rats showed much greater sensitivity to AQP4-IgG and complement than those from CD59+/+ rats. Intracerebral administration of AQP4-IgG in CD59-/- rats produced marked NMO pathology, with astrocytopathy, inflammation, deposition of activated complement, and demyelination, whereas identically treated CD59+/+ rats showed minimal pathology. A single, intracisternal injection of AQP4-IgG in CD59-/- rats produced hindlimb paralysis by 3 days, with inflammation and deposition of activated complement in spinal cord, optic nerves and brain periventricular and surface matter, with most marked astrocyte injury in cervical spinal cord. These results implicate an important role of CD59 in modulating NMO pathology in rats and demonstrate amplification of AQP4-IgG-induced NMO disease with CD59 knockout.

The intrinsic pathogenic role of autoantibodies to aquaporin 4 mediating spinal cord disease in a rat passive-transfer model.

Neuromyelitis optica (NMO) is causally linked to autoantibodies (ABs) against aquaporin 4 (AQP4). Here, we focused on the pathogenic effects exclusively mediated by human ABs to AQP4 in vivo. We performed cell-free intrathecal (i.th.) passive transfer experiments in Lewis rats using purified patient NMO immunoglobulin G (IgG) and various recombinant human anti-AQP4 IgG-ABs via implanted i.th. catheters. Repetitive application of patient NMO IgG fractions and of recombinant human anti-AQP4 ABs induced signs of spinal cord disease. Magnetic resonance imaging (MRI) revealed longitudinal spinal cord lesions at the site of application of anti-AQP4 IgG. Somatosensory evoked potential amplitudes were reduced in symptomatic animals corroborating the observed functional impairment. Spinal cord histology showed specific IgG deposition in the grey and white matter in the affected areas. We did not find inflammatory cell infiltration nor activation of complement in spinal cord areas of immunoglobulin deposition. Moreover, destructive lesions showing axon or myelin damage and loss of astrocytes and oligodendrocytes were all absent. Immunoreactivity to AQP4 and to the excitatory amino acid transporter 2 (EAAT2) was markedly reduced whereas immunoreactivity to the astrocytic marker glial fibrillary acid protein (GFAP) was preserved. The expression of the NMDA-receptor NR1 subunit was downregulated in areas of IgG deposition possibly induced by sustained glutamatergic overexcitation. Disease signs and histopathology were reversible within weeks after stopping injections. We conclude that in vivo application of ABs directed at AQP 4 can induce a reversible spinal cord disease in recipient rats by inducing distinct histopathological abnormalities. These findings may be the experimental correlate of "penumbra-like" lesions recently reported in NMO patients adjacent to effector-mediated tissue damage.