Aquaporin-4 facilitates cell proliferation in retinal müller cells: implications in neuromyelitis optica

Abstract

Müller cells are involved in controlling extracellular homeostasis in the retina, regulating cell swelling by a regulatory volume decrease (RVD) mechanism that depends on the efflux of solutes and water through Aquaporin-4 (AQP4). Müller cells are also important for retinal integrity, as they respond to injury by re-entering the cell cycle for tissue repair. Since AQP4 was reported to modulate cell volume during cell cycle progression and facilitate proliferation in astrocytes, the aim of this study was to evaluate, using the novel inhibitor TGN-020, if AQP4 was involved in human Müller cells? proliferation in physiological conditions. Considering that AQP4 is the target of autoantibody IgG-NMO present in the sera of patients with Neuromyelitis Optica (NMO), we also evaluated if cell proliferation was altered in the presence of IgG-NMO. MIO-M1 human Müller cells were exposed to 100 nM TNG-020 or vehicle or to 1/50 dilution of IgG-NMO positive or control sera. Cell volume (videomicroscopy) and cell proliferation (cell count, cell cycle analysis by flow cytometry and BrdU incorporation by immunofluorescence) were measured. AQP4 inhibition with TGN-020 reduced osmotic water permeability (Pf, µm/s) from 20.3±1.2 to 12.2±0.4 (n=5, p<0.001) and %RVD 15min from 54±4 to 17±3 (n=5, p<0.001). MIO-M1 cell proliferation was decreased by TGN-020 (doubling time in hours, control vs. TGN-020: 31±1 vs. 40±3, n=4, p<0.05) without affecting cell viability. TGN-020 also increased the % of cells in G1/G0 phase, decreased the S phase of cell cycle and reduced BrdU incorporation by 20%. IgG-NMO positive sera decreased AQP4 plasma membrane expression in MIO-M1 cells, reducing Pf from 22.4±1.5 to 15.9±0.6 µm/s (n=6, p<0.001) and %RVD 15min from 66±5 to 48±4 (n=6, p<0.005), as well as cell proliferation (doubling time in hours, control vs. IgG-NMO: 59±5 vs. 86±4, n=3, p<0.05) in comparison to control sera. We propose that inhibition or removal of AQP4 from the plasma membrane reduces AQP4-mediated water permeability altering cell proliferation. This is of particular importance in NMO, as the decreased ability of Müller cells to proliferate may affect retinal tissue repair.