Aquaporin-4 removal from the plasma membrane of human muller cells by AQP4-IgG from patients with neuromyelitis optica induces changes in cell volume homeostasis: the first step of retinal injury?

1 Aquaporin-4 is removed from the plasma membrane of human Müller cells by AQP4-IgG from NMOSD patients: Changes in cell volume homeostasis as the first step of retinal injury? Vanina Netti1, Juan Fernández1, Luciana Melamud2, Pablo Garcia-Miranda3 , Gisela Di Giusto1, Paula Ford1, Miriam Echevarria3 and Claudia Capurro1# 1 Laboratorio de Biomembranas, Instituto de Fisiología y Biofísica “Bernardo Houssay” (IFIBIO ‐ HOUSSAY), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Departamento de Ciencias Fisiológicas, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina. 2 Consultorio de Neuroinmunología, Centro Universitario de Neurología Dr. J.M. Ramos Mejía, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina. 3 Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, 41013 Seville, Spain. #Corresponding author: Prof. Claudia Capurro, Ph.D. Laboratorio Biomembranas, IFIBIO Houssay, CONICET-UBA, Departamento de Ciencias Fisiológicas. Facultad de Medicina, Universidad de Buenos Aires. Paraguay 2155, piso 7 (1121) Buenos Aires, ARGENTINA. TEL: 54-11-59509500 (ext. 2145). e-mail: [email protected] Running Head: AQP4-IgG affects cell volume regulation in Retinal Müller cells Precis: AQP4-IgG targets AQP4 affecting the capacity to regulate cell volume after an osmotic swelling (RVD), a key function of Retinal Müller cells. Key Words: Aquaporin 4; AQP4-IgG; Human Müller Cells; Cell volume regulation 2 ABSTRACT 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 complementinactivated 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. Keywords Aquaporin 4 · AQP4-IgG · Human Müller cells · Cell volume regulation · Cell proliferation 3 1. INTRODUCTION Neuromyelitis Optica spectrum disorders (NMOSD) is a devastating, inflammatory and demyelinating disease of the central nervous system (CNS) that preferentially affects the optic nerves and spinal cord (Pittock & Lucchinetti, 2016). The majority of patients are seropositive for a highly specific serum immunoglobulin G that targets Aquaporin-4 (AQP4), called AQP4-IgG (Lennon et al., 2004; 2005). AQP4 is the major plasma membrane water channel in the CNS and it is highly expressed in the polarized plasma membrane of astrocytic endfeet facing the blood-brain barrier as two major isoforms (M1 and M23), which are organized in structures known as orthogonal arrays of particles (OAPs) (Nagelhus & Ottersen, 2013). Clinical and pathological studies have proposed that AQP4-IgG binding to astrocytic AQP4 produces complementdependent cytotoxicity, which leads to astrocyte-destructive lesions in spinal cord and optic nerves (Bradl et al., 2018; Chang & Chang, 2019). However, new evidence indicates that AQP4-IgG can also induce complement-independent pathologenesis suggesting that different mechanisms of AQP4-IgG may act concurrently (Ratelade & Verkman, 2012; Bennet et al, 2017). In addition to astrocytes, AQP4 is also highly expressed in Müller cells in the inner retina and in glial membranes contacting retinal ganglion cells as well as in nerve fibers (Reichenbach & Bringmann, 2020). Using optical coherence tomography (OCT), several studies demonstrated that damage to optic nerves in NMOSD is also associated with retinal injury (Gelfand et al., 2013; Sotirchos et al., 2013; Bennett et al., 2015; Peng et al., 2017). Other reports demonstrate that NMOSD patients AQP4-IgG-seropositive without a history of optic neuritis, have microstructural changes in the fovea region, a retinal site rich in Müller cells, supporting the proposal of Müller cells as a target of AQP4-IgG (Oertel et al., 2017). However, until a few years ago there was no direct evidence of retinal lesions in patients with NMOSD (Zeka et al., 2016). Intravitreal injection to adult rats retina of a monoclonal AQP4-specific recombinant antibody derived from an NMOSD patient (rAb-53) showed that, in fact, the antibody efficiently bound to AQP4 on retinal Müller cells and produced complement-independent retinal injury (Felix et al., 2016). However, as AQP4-IgG is 4 a polyclonal autoantibody that binds multiple epitopes with variable avidity for them (Pittock & Lucchinetti, 2016), the effects of NMOSD patients’ sera on Müller cells warrants further study. One of the major functional roles of Müller cells is to maintain extracellular fluid and ionic homeostasis within the retina, since neural activity alters osmotic gradients leading to cell swelling, which is followed by a regulatory volume decrease (RVD) response, in which AQP4-mediated water transport would play an active role (Pannicke et al., 2010). We have previously reported that RVD in Müller cells is a complex response that involves Transient Receptor Potential Vanilloid 4 (TRPV4) calcium channel-dependent changes in membrane potential (Vm) as well as the activation of K+ and Clchannels and the release of amino acids such as Taurine and Glutamate via the Volume Regulated Anion Channel (VRAC) (Fernández et al., 2013; Netti et al, 2017; Netti et al., 2018). Even more, Jo et al. (2015) showed that TRPV4 -AQP4 interactions constitute a molecular system that finetune astroglial volume regulation by integrating osmosensing, calcium signaling and water transport, and when over-activated, they can trigger pathological swelling. However, the consequences of AQP4-IgG binding to AQP4 on RVD machinery was not investigated, in particular the putative involvement of TRPV4 in NMOSD physiopathology. Another important role of Müller cells is to maintain retinal integrity responding to injury by a mechanism known as reactive gliosis, which involves the activation of the cell cycle and subsequent dedifferentiation to other cell types, having a role of stem cells in the adult retina (Fischer and Reh, 2001; Ooto et al., 2004; Giaume et al., 2007). Interestingly, stem cell transplantation has been considered to be a potential treatment method for neurological disorders including NMOSD (Greco et al., 2014). Since it was proved that AQP4 affects cell proliferation in different cell types (Kong et al., 2008; Li et al., 2016) and several AQPs have a role in cell proliferation by allowing fast cell volume regulation during cell division (Di Giusto et al., 2012; Galán-Cobo et al., 2016), the aim of this study was to investigate whether binding of AQP4-IgG to AQP4 in Müller cells will threat cell volume regulatory capacity and consequently cell proliferation. We used a human retinal Müller cell line (MIO-M1), a good model that expresses endogenous AQP4 and maintains important functional characteristics of Müller cells (Limb et al., 2002) exposed 5 to AQP4-IgG positive sera from different NMOSD patients. Our results propose that retinal injury may be driven by the dysfunction of Müller cells in patients with AQP4-IgG. Understanding these mechanisms will pave the way for therapeutic interventions in NMOSD. 2. MATERIAL AND METHODS 2.1 Cell Cultures The MIO-M1 cell line (kindly provided by Dr. Astrid Limb, University College London, London, UK) is a spontaneously immortalized retinal Müller glial cell line, originated from human retina, that retained many characteristics of Müller cells (Limb et al., 2002). Cells were grown as monolayers in the presence of Dulbecco’s Modified Eagle Medium (DMEM) / glutamax supplemented with 10% fetal calf serum (FCS), with 5 µg/ml streptomycin and 5 U/ml penicillin at 37°C in a humidified atmosphere containing 5% CO2. Cells were routinely subcultured every week. For immunofluorescence and functional studies, MIO-M1 cells were seeded on glass coverslips (diameter 1.2 cm) at 5-10 x 103 cells/ml densities, for 48 hours and then subjected to the different experimental conditions. 2.2 Patients and serum samples Serum samples were collected from patients that fulfilled the original diagnostic criteria for NMO and were AQP4-IgG positive, as previously described (Wingerchuk et al., 2015), from the Neuroimmunologic Clinic Service of the Ramos Mejia Hospital, Argentina and from the Neurology Department at the Virgen of Rocío University Hospital (HUVR), Spain. As control, we included serum from healthy AQP4-IgG negative volunteers. All subjects signed the informed consent form, and the study was approved by the Institutionals Review Boards and conducted in compliance with the Declaration of Helsinki. Demographic and clinical characteristics of patients included in our study were previously reported (Melamud et al., 2012; García-Miranda et al, 2019). Sera were stored at -80°C and complement was inactivated holding 30 minutes at 56°C. 6 2.3 Cells pretreatment with control or patient Sera MIO-M1 human Müller cells were exposed to control or patients heat-inactivated sera (dilution 1/50 in culture media) for 1 hour at 4°C or 12 hours at 37°C. These conditions were selected to avoid or to facilitate AQP4 down-regulation, respectively, as previously described in HEK-293 cells and astrocytes primary culture (Melamud et al., 2012; Hinson et al, 2012). As a control for AQP4 function in Müller cells, in some experiments, cells were preincubated with 100 nM of the specific AQP4 inhibitor N-1,3,4-Thiadiazol-2-yl-3-pyridinecarboxamide (TGN-020, Kida et al., 2017) (Sigma-Aldrich) in the isoosmotic extracellular solution and the same concentration was used throughout the experiments. After these treatments, cells were washed with PBS and then immunofluorescence or functional studies were performed. 2.4 Measurement of Cell Volume Changes, Water permeability, RVD Response and intracellular calcium levels MIO-M1 cells were seeded on glass coverslips (12 mm diameter) at 5-10 x 103 cells/mL densities for 48 hours and then subjected to the different experimental conditions. As previously reported, we simultaneously recorded changes in cell volume and intracellular calcium levels ([Ca2+]i) in single cells by using 14 µM of the calcium-sensitive dye Fura-2 AM (Invitrogen, Thermo Fisher Scientific). Cells were incubated for 60 minutes at 37 ºC and then fluorescence was recorded at the calcium-sensitive (380 nm, Rt/R0 Fura-2) and -insensitive (358 nm, isosbestic) wavelengths with a Nikon TE-200 epifluorescence inverted microscope (Nikon Planfluor 40X oil immersion objective lens), coupled to a device camera (Hamamatsu C4742-95), and connected to a computer with the Metafluor software acquisition program (Universal Imaging Corporation, PA) (Netti et al., 2017). Cells were exposed to hypotonic shock and relative fluorescence (Ft/F0) was recorded. F0 represents the pinhole signal when placed in equilibrium with an iso-osmotic medium with an osmolality OsM0 (300 mOsM) and Ft is the fluorescence from the same region at time t, when placed in equilibrium with a solution with an osmolality of OsMt. Changes in cell volume can be calculated as follows: 7 b b t f f F F V V − −         =1 0 0 where V is cell volume at time t; V0 is cell volume at t = 0; and fb is the relative background. This parameter corresponds to the y intercept of a plot of F0/Ft versus OsM0/OsMt, which represents relative fluorescence in the absence of osmolality changes. RVD after cell exposure to a hypo-osmotic medium was calculated by the following equation: 100 1 % max 0 20 0 max 0 20                −                 −         = V V V V V V RVD where (V/V0)max is the maximal value of V/V0 attained during hypo-osmotic swelling (peak), and (V/V0)10 represents the value of V/V0 observed at time 20 min after the osmotic shock. %RVD20 thus denotes the magnitude of volume regulation 20 minutes after the osmotic shock, with 100% RVD indicating complete volume regulation and 0 % RVD indicating no volume regulation. Osmotic water permeability of MIO-M1 cells was estimated from the time course of V/V0 during the first 2 minutes. Curves for each experiment were fitted with a single exponential function with the software GraphPad Prism 5.0 and the osmotic water permeability coefficient (Pf), was calculated from the exponential time constant (τ) by the equation: w fVOsMA V P... 0  = where V0 is the initial cell water volume; A is the cell surface area; OsM is the osmotic gradient and VW is the partial molar volume of water (18 cm3. mol-1). Volume-area relation (V0/A) of MIO-M1 cells was calculated from confocal images using Imaris 7.1.0 software (Bitplane) with a mean value of 20.85 ± 0.70 (.10-5 cm, n = 27). 8 2.5 Measurement of Membrane Voltage Changes Transmembrane potential was measured using DIBAC4(3), a slow response anionic dye whose emission is independent of cell volume changes. Cells were loaded with 2.5 M DIBAC4(3) for 15 minutes at 20ºC and placed on the stage of the same microscope described in the previous section. Excitation wavelength was 490 nm and emitted light (above 520 nm) was recorded at 10 second intervals. Fluorescence intensity was monitored until it reached a stable value before starting the experiments. Fluorescence intensity changes after interventions were relativized to stationary values (Ft/F0) and data were corrected for background noise and drift. 2.6 Immunofluorescence assays and quantification of AQP4 For AQP4 detection, MIO-M1 cells were fixed in 4% paraformaldehyde for 1 hour and then permeabilized with 1% glycine and 0.1% Triton X-100 at room temperature. Samples were blocked with 5% Fetal Bovine Serum for 2 hours and sequentially incubated with a rabbit polyclonal anti- AQP4 antibody (1/1000) directed against an epitope located at the intracellular C-terminal domain of rat AQP4 to amino acid residues 249-323 (#AQP-004, Alomone Labs) overnight at 4°C and a secondary antibody (rabbit anti-Cy3 conjugate, dilution 1:100, Jackson Immuno 111-165-003) for 2 hours at room temperature. Then, nuclei were stained with Hoechst (5 µg/ml) for 1 min. Coverslips were mounted with Vectashield mounting medium. Images were captured using epifluorescence on an Olympus FluoView FV1000 confocal microscope and digitalized. The intensity of AQP4-Cy3 fluorescence was quantified by densitometry using Image-J software and then analyzed using the formula: ( ) nIF n  Where n stands for the number of optical sections required scanning the entire cell, and IF (n) stands for integrated fluorescence intensity within a given optical section. 9 To quantify AQP4 at the plasma membrane in MIO-M1 cells, immunofluorescence studies were performed using the same technique but at the beginning, cells were incubated for 30 minutes at 4°C with Alexa Fluor® 488-conjugated wheat germ agglutinin (WGA-488; Thermo Fisher Scientific Cat# W11261) that selectively binds to N-acetylglucosamine and N- acetylneuraminic acid residues and can be used to label glycoproteins for imaging the plasma membrane in live cells. In other experiments, to evaluate the presence of intracellular AQP4 in early endosomes, double immunostaining was performed by using anti-early endosome antigen 1 (EEA1) mouse monoclonal antibodies (1/300, BD Biosciences) and anti-AQP4 antibodies, as described above. Confocal images were analyzed by using Image J colocalization tools and estimated using the M2 superposition coefficient of Manders (SCM), which measures the fraction of WGA/EEA1 that overlaps AQP4 signal, and is described by the following equation 22 )( iii iii xGR xGR SCM   = A value of 1 indicates 100% of superposition between signals of colocalized pixels, while a value of 0 indicates absence of colocalization. For TRPV4 immunostaining, the same protocol was applied and a anti-TRPV4 antibody was used (1/1500; Alomone Labs, #ACC-034). Since plasma membrane TRPV4 expression was low, to identify its presence, we created a mask of plasma membrane as previously described (Netti et al., 2017). Briefly, WGA images were binarized so that the signal from WGA was ascribed the value of “1” and the rest of the image was ascribed the value of “0”. The Boolean logical operation “AND” was then performed on the corresponding images, representing signals from TRPV4-Cy3 and from WGA (binary mask). This resulted in generation of a new image (shown in yellow in the figures) in which only the TRPV4-Cy3 fluorescent signal corresponding to the membrane was present. 2.7 Western blotting 16 due to cell death since cell viability, assessed by Trypan Blue exclusion method, was not affected by the presence of AQP4-IgG as compared to the control sera or in the presence of TGN-020 (data not shown). We further evaluated MIO-M1 cell proliferation in the presence of control or AQP4-IgG+ sera by measuring the incorporation of the thymidine analog 5-Bromodeoxyuridin (BrdU) as a marker of DNA synthesis and calculated the percentage of proliferating cells. Results showed that BrdU positive cells (BrdU+) are reduced in the presence of AQP4-IgG, as compared to control serum (Figure 6C). Quantification of the total number of BrdU+ cells effectively demonstrated that the percentage of proliferative cells is significantly reduced with the AQP4-IgG+ sera from the three patients (Figure 6D). As expected, pretreatement of MIO-M1 cells with TGN-020 showed a similar reduction of BrdU incorporation. These results demonstrate that before complement activation, AQP4 loss from the plasma membrane altered the capacity of Müller cells to proliferate, probably having an impact on retinal tissue repair in vivo. 4. DISCUSSION In the present work, we confirm that AQP4 expressed in human retinal Müller cells represents a pathogenic ocular target of AQP4-IgG, as previously suggested by clinical observations and in vitro animal models (Gelfand et al., 2012; Sotirchos et al., 2012; Green et al., 2009; Levin et al., 2013). One of the main findings of this study is that the auto-antibody binding to AQP4, before complement activation, does not directly inhibit AQP4 water permeability by blocking the water pore, but induces its partial internalization. In fact, we showed that, under conditions where membrane fluidity and metabolic processes are restricted (4°C for 1 hour), AQP4 remains in the plasma membrane and its water channel activity is not affected. This was further confirmed by comparing these results with those obtained by the use of TGN-020 inhibitor, which specifically blocks the AQP4 pore as shown by molecular dynamics studies (Toft-Bertelsen et al. 2020). The long-term exposure to AQP4-IgG+ sera for 12 h at 37°C, a condition that does not prevent 17 metabolic processes, reduced AQP4 expression and induced its partial removal of the plasma membrane to the endosomal compartment. AQP4 down-regulation induced by AQP4-IgG+ sera in Müller cells is in line with the report of Felix et al. (2016) using a monoclonal antibody, as well as with our and other previous reports in astrocytes (Melamud et al., 2012; Hindson et al., 2012). In fact, these studies showed that while AQP4-IgG+ sera induced the endocytosis of M1 isoform in astrocytes, M23 still remain in the plasma membrane, rearranging to larger OAPs. However, several other AQP4 isoforms were later described, produced by alternative splicing (AQP4b, d, e and f) or by translational readthrough that contains a C-terminal extension (AQP4ex) (De Bellis et al., 2017; Lisjak et al., 2020). It was proposed that AQP4ex could have an important regulatory role in OAP assembly and stability, being critical for the binding of pathogenic human AQP4-IgG autoantibodies in astrocytes (Palazzo et al., 2019). In the present study, Western blot analysis for AQP4 revealed that human Müller cells express several isoforms, but the major band observed at the plasma membrane, which was partially removed after AQP4-IgG binding, is that of ~40 kDa, which may correspond to AQP4-ex. However, we cannot discard that the AQP4e isoform, a protein of also ~40 kDa present in the plasma membrane and participating in the cell volume regulation of astrocytes (Lisjak et al., 2017), could be also involved. The present study does not attempt to clarify this point; however, our observations support the idea that, in human Müller cells, AQP4-IgG would not bind to the canonical M1 and M23 isoforms. Future studies are needed to address this novel issue since, as previously reported, the expression of particular AQP4 isoforms depend mainly on post-transcriptional regulation, according to the specific homeostatic needs of each tissue (Palazzo et al, 2019). The here observed endocytosis of AQP4 by AQP4-IgG is functionally relevant, since it leads to the decrease of plasma membrane water permeability and the impairement of one of the major roles of Müller cells, the control of the RVD response. In fact, after the partial removal of AQP4 by AQP4-IgG, the changes of intracellular calcium levels, which determine the efficiency of RVD mechanisms (Netti et al., 2017), were also significantly reduced. These results propose an association between the presence of AQP4 and the osmotically-induced calcium signals in Müller 18 cells, as previously shown in astrocytes (Thrane et al., 2011), which is probably altered in the presence of AQP4-IgG, affecting cell volume regulation response. Interestingly, AQP4-IgG does not affect neither TRPV4 expression/activity nor Vm changes occurring during cell swelling and RVD in Müller cells, indicating that AQP4 is the main contributor to the RVD response. This was further confirmed with the specific blocker of the pore of AQP4 by TGN-020. We propose that the activation of RVD effectors would be sensitive to the dynamics of cell swelling and/or its consequences (changes in membrane tension, dilution of intracellular mediators, etc), as previously reported for some AQPs (Galizia et al., 2008; Di Giusto et al., 2012). Therefore, the rapid influx of water in the presence of AQP4 will lead to a faster regulation of cell volume changes by activating AQP4-dependent cell volume regulation machinery, as previously shown in mousecultured glial cells (Mola et al., 2016). Previous reports in mouse Müller cells described that AQP4 deletion is associated with reactive gliosis and gliovascular modifications which involves the activation of the cell cycle and cell differentiation (Nicchia et al., 2016). We here provide experimental evidence demonstrating, for the first time, that before complement activation, the loss of AQP4 from the plasma membrane induced by AQP4-IgG+ sera delayed the ability of Müller cells to proliferate. We also show that AQP4 inhibition with TGN-020 effectivelly reduced the proliferation of Müller cells without inducing cell death, directly demonstrating the contribution of AQP4 to this process. These results are in agreement with our and other studies in different cell types demonstrating that AQPs play a key role in cell volume control, an important prerequisite for the regulation of various cell properties, including proliferation, migration and differentiation (Galán-Cobo et al., 2016; Di Giusto et al., 2012). Even more, it was previously reported that AQP4 has a crucial role in regulating proliferation and differentiation of adult neural stem cells by its action on intracellular calcium dynamics (Kong et al., 2008) . We here show that the blockage or endocytosis of AQP4, with TGN-020 and AQP4- IgG respectively, not only altered intracellular calcium levels during the osmotic swelling and RVD but also delayed cell proliferation. Therefore, we propose that AQP4-mediated rapid changes in cell volume and in calcium signals facilitate cell proliferation in physiological conditions. Shedding 19 light on how AQP4 is able to regulate Müller cells proliferation can increase our knowledge of their biological behaviours and improve their application in regenerative and reparative medicine for NMOSD patients. Interenstingly, recent reports revealed that bone marrow mesenchymal stem cells derived from patients with NMOSD exhibited a decreased proliferation rate, together with a decreased expression of several cell cycle promoting and proliferation-associated genes, as compared to control healthy donors (Yang et al., 2019). The key role of AQP4 on cell proliferation might also explain the in vivo reported structural and functional Müller cells’ dysfunction observed, by full-field electroretinography and spectral-domain OCT, only in NMOSD patients who were AQP4-IgG positive (You et al. 2019). Further studies are warranted to investigate these mechanisms and its potential implications for the prognosis and treatment of optic neuritis in NMOSD. In conclusion, we here propose that AQP4-IgG present in the sera of NMOSD patients causes the removal of endogenously expressed AQP4 from the plasma membrane of retinal Müller cells, affecting osmotic water permeability, the consequent activation of the cell volume regulation machinery and cell proliferation. 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A: Cells were treated for 1 hour at 4°C or 12 hours at 37°C with control or AQP4-IgG+ sera and then immunofluorescence were performed using a polyclonal anti-AQP4 antibody (red). Top panel: Confocal images illustrating similar staining pattern of plasma membrane AQP4 after exposure to control or AQP4-IgG+ sera for 1 hour at 4°C. Lower panel: Confocal images illustrating changes in the staining pattern after exposure for 12 hours at 37°C to AQP4-IgG+ serum, as compared to control serum. Images are representative of three independent experiments. B: Detailed magnification of cells exposed to control or AQP4-IgG+ sera for 12 hours at 37°C (Scale bars: 10 µm). C: Total fluorescence quantification of positively-stained cells after incubation for 12 hours at 37°C with control or 3 different AQP4-IgG+ sera exposure. Immunofluorescence values per cell (AU: arbitrary units) are expressed as mean ± SEM for 23 - 52 cells from 3 experiments for each condition **p<0.01 and *p<0.05 control vs. AQP4-IgG+ sera. 0 5 10 15 20 Control Serum AQP4-IgG+ Sera P1 P2 P3 AQP4 Fluorescence intensity/cell (AU .104) ** * * 12 h 37ºC Control Serum AQP4-IgG+ Serum 12 h 37ºC C Figure 2 A AQP4-IgG + Serum AQP4 WGA merge Control Serum C B 0 0.2 0.4 0.6 0.8 1 Control Serum AQP4-IgG+ Serum OD Plasma membrane AQP4/ β-actin (AU) ** β-actin 43 KDa AQP4 20 25 37 50 75 37 Plasma membrane AQP4 β-actin 43 KDa 37 50 37 AQP4 EEA1 merge merge* AQP4-IgG + Serum Control Serum Figure 2: AQP4 expression and localization in plasma membrane of MIO-M1 cells after exposure to control or AQP4-IgG positive sera. A: Immunoflurescence confocal images of MIO-M1 cells (40X) stained with a polyclonal anti-AQP4 antibody (red), WGA plasma membrane staining (green) and merge of both images (yellow) after treatment for 12 hours at 37°C with control (left) or AQP4-IgG sera (right). Images are representative of three independent experiments (Scale bars: 20 µm). B: Western blot showing the expression of several AQP4 isoforms in whole cell lysates and in enriched plasma membrane fractions (left) and AQP4 plasma membrane expression after treatment for 12 hours at 37°C with control or AQP4-IgG sera (right). Values are expressed as mean ± SEM, **p<0.005 control vs. AQP4- IgG positive sera. C: Immunoflurescence confocal images of MIO-M1 cells (60X) stained with a polyclonal anti-AQP4 antibody (red), EEA1 (green) and merge of both images (yellow) after treatment for 12 hours at 37°C with control (left) or AQP4-IgG sera (right). Merge* show enlarged boxed areas from merged image. Figure 7 Figure 7: Proposed model to explain the complement-independent disfunction of Müller cells after AQP4-IgG binding to AQP4. A: In physiological conditions, neural activity induces water and K+ uptake and cell swelling, which is followed by RVD response. RVD is a complex mechanism that involves AQP4 and TRPV4-dependent changes in membrane potential (Vm) as well as the activation of K+ and Cl− channels and the release of Taurine (Tau) and Glutamate (Glu) via VRAC (left upper panel). RVD capacity is also actively modulated during G0/G1 phase of cell cycle and plays an important role in cell cycle progress (1) to promote cell proliferation (2) and maintain retinal homeostasis (3). B: In the presence of AQP4-IgG in NMOSD, AQP4 is partially removed from the plasma membrane and the activation of the AQP4-dependent RVD response is affected, resulting in Müller cells’ disfunction (right upper panel). Consequently, cell cycle is delayed (1) and the rate of cell proliferation decreases (2), probably contributing to the physiopathology of NMOSD in the retina (3)