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. 2012 Dec;60(12):2027-39.
doi: 10.1002/glia.22417. Epub 2012 Sep 14.

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

Andrea Rossi  1 , Julien Ratelade, Marios C Papadopoulos, Jeffrey L Bennett, A S Verkman
Affiliations

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

Andrea Rossi et al. Glia. 2012 Dec.

Abstract

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.

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Figures

Fig. 1
Fig. 1
Characterization of AQP4-transfected CHO cells. (A) Immunofluorescence of CHO cells stably transfected with M1- or M23-AQP4, stained with anti-C-terminus AQP4 antibody and fluorescent secondary antibody, and imaged by confocal microscopy (top), TIRFM (middle), and dSTORM (bottom). (B) AQP4 immunoblot of cell homogenate following SDS/PAGE (top) and (nondenaturing) BN/PAGE (bottom). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 2
Fig. 2
AQP4 supramolecular assembly in OAPs does not affect its single-molecule water permeability. (A) (left) Membrane fractionation method. (Right) Immunoblot of membrane fractions for AQP4 and indicated plasma membrane, Golgi, and ER markers. (B) AQP4 immunoblot following BN/PAGE of plasma membrane vesicles from M1- and M23-AQP4-transfected CHO cells. (C) Plasma membrane vesicle size determined by quasi-elastic light scattering (top) and by dSTORM of surface-immobilized vesicles immunostained with AQP4 antibody. (D) Stopped-flow light scattering measurement of plasma membrane osmotic water permeability. (left) Plasma membrane vesicles from non-transfected and M1-or M23-AQP4-expressing cells were subjected to a 250-mM inwardly directed osmotic gradient, resulting in vesicle shrinkage (increased scattered light intensity). (Right) Relative single-molecule water permeability (Pf) deduced from light scattering, vesicle size, and AQP4 expression data (SE, n = 5, difference not significant).
Fig. 3
Fig. 3
NMO-IgG does not inhibit AQP4 water permeability. (A) Plasma membrane vesicles from M23-AQP4-expressing CHO cells were immobilized on a coverglass, incubated with NMO serum (left) or recombinant NMO antibody (right), and immunostained green for AQP4 and red for NMO-IgG. (B) (left) Red-to-green fluorescence ratios for experiments as in A as a function of serum/rAb concentration (SEM, n = 4). Data fitted to saturable, single-site binding model. (Right) R/G for six different NMO sera and three rAbs. (C) Plasma membrane osmotic water permeability measured as in Fig. 2D. M23-AQP4-containing vesicles were incubated with sera/rAbs as in panel B. (Left) Representative light scattering curves. (Right) Relative osmotic water permeability (Pf in s−1) deduced from light scattering data (SE, n = 5, differences not significant). (D) Osmotic water permeability in control and AQP4-reconstituted proteoliposomes following NMO-IgG incubations as done in panel B (SE, n = 5, differences not significant). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 4
Fig. 4
NMO-IgG binding causes greater internalization of M23-than M1-AQP4 when expressed separately in transfected cells. (A) CHO cells stably expressing M1- or M23-AQP4 were labeled with 20 µg/mL rAb-58-Cy3 at 4°C, washed, and chased for 1 h at 37°C. (Left) Cy3 fluorescence shown before (t = 0) and at 1 h chase. Fluorescence of rAb-58-Cy3 remaining at the cell surface was quenched with bromocresol green (+quencher). (Right) Percentage of internalized antibody after 0 and 1 h chase (SE, n = 10, *P < 0.01). Representative of two sets of experiments. (B) (Left) CHO cells expressing M1- or M23-AQP4 stained for surface AQP4 (red) and plasma membrane (WGA, green) before (t = 0) and after 1 h incubation at 37°C with 50 µg/mL NMO-IgG (rAb-58) or control-IgG (ctrl-IgG). (Right) Percentage of AQP4 remaining at the cell surface (SE, n = 6, *P < 0.01). Representative of two sets of experiments done on CHO and U87MG cells. (C) TIRFM of U87MG cells transfected with M23-AQP4/GFP chimera (M23-GFP), incubated with 50 µg/mL rAb58 for 1 h at 4°C, and then chased at 37°C for indicated times (full time course in Supp. Info. Movie 1). Arrowheads of the same color in consecutive micrographs show internalized OAPs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 5
Fig. 5
NMO-IgG binding causes simultaneous cointernalization of M1- and M23-AQP4 when coexpressed in transfected cells. (A) CHO cells were transiently transfected with M23-AQP4 only or together with an equal amount of M1-AQP4, and incubated with 50 µg/mL NMO-IgG (rAb-58) or control-IgG (ctrl-IgG) for 2 h at 37°C. (Left) Immunoblot of AQP4 and Na+/K+ ATPase following SDS/PAGE of plasma membrane (PM) and intracellular vesicles (cytoplasmic, C) fractions. (Right) M1- and M23-AQP4 amounts from blots as in A (SE, n = 3). (B) (Left) Cells treated as in A were stained for surface AQP4 (red) and plasma membrane (WGA, green). (Right) Percentage of remaining AQP4 at the cell surface after 2 h incubation with NMO-IgG (SE, n = 10, *P < 0.01). Representative of two sets of experiments. (C) TIRFM of U87MG cells transfected with M1-mCherry (red) and M23-GFP (green), incubated with 50 µg/mL rAb58 for 1 h at 4°C, and then chased at 37°C for indicated times (full time course in Supp. Info. Movie 2). Arrowheads of the same color in consecutive micrographs show internalized OAPs containing both isoforms. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 6
Fig. 6
NMO-IgG does not cause cell surface AQP4 clustering. (A) Live cell PALM of cells cotransfected with M1- and M23-AQP4-PAmCherry. Cells imaged before (0 min) and at 45 min after incubation of NMO serum. (B) Average OAP area (SE, n = 6, difference not significant).
Fig. 7
Fig. 7
N-terminus GFP addition disrupts OAP formation by M23-AQP4. (A) N-terminus chimeras showing schematic (left), TIRFM (center), and BN/PAGE (right). (B) C-terminus chimera. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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