Anti-Aquaporin-1 Autoantibodies in Patients with Neuromyelitis Optica Spectrum Disorders John S. Tzartos 1,2 *, Christos Stergiou 1 , Konstantinos Kilidireas 3 , Paraskevi Zisimopoulou 1 , Thomas Thomaidis 2 , Socrates J. Tzartos 1,4 * 1 Department of Biochemistry, Hellenic Pasteur Institute, Athens, Greece, 2 Department of Neurology, General Hospital ‘‘Red Cross", Athens, Greece, 3 Department of Neurology, Aeginition Hospital, School of Medicine, National and Kapodistrian University, Athens, Greece, 4 Department of Pharmacy, University of Patras, Patras, Greece Abstract Autoantibodies against aquaporin-4 (AQP4), a water channel in CNS astrocytes, are detected in ,50–80% of patients with neuromyelitis optica spectrum disorders (NMOsd), characterized by longitudinally extensive transverse myelitis (LETM) and/ or optic neuritis. Although these autoantibodies present an invaluable biomarker for NMOsd and for the differential diagnosis of multiple sclerosis (MS), diagnosis of anti-AQP4-seronegative NMOsd remains challenging. We hypothesized that seronegative NMOsd patients might have autoantibodies against aquaporin-1 (AQP1), another water channel in CNS astrocytes. We initially developed a radioimmunoprecipitation assay to search for anti-AQP1 antibodies in sera from 632 individuals. Anti-AQP1 or anti-AQP4 autoantibodies were detected in 16.7% and 12%, respectively, of 348 patients with suspected NMOsd. Anti-AQP1 specificity was confirmed by competition, protein immunoblotting and ELISA assays, whereas epitope localization was studied by immunoadsorption on intact cells expressing AQP1 and peptide mapping experiments. Most anti-AQP1 autoantibodies were of the complement-activating IgG1 subclass and the majority bound to the extracellular domain of AQP1, suggesting a possible pathogenic role. Five out of 42 MS patients had anti-AQP1 antibodies, but 2 of them also had spinal cord lesions, while the anti-AQP1 antibodies in the other 3 bound to the cytoplasmic domain of AQP1. Anti-AQP1 antibodies were not detected in 100 healthy individuals or 142 patients with non-demyelinating neuroimmune diseases. Analysis of 17 anti-AQP1+/anti-AQP4- patients with suspected NMOsd showed that 5 had NMO and 11 had LETM. 12/17 of these sera bound predominantly to the extracellular AQP1 loop-A. Overall, we found that anti-AQP1 autoantibodies are present in a subgroup of patients with chronic demyelination in the CNS and similarities with anti-AQP4- seronegative NMOsd, offering a novel potential biomarker for CNS demyelination disorders. Citation: Tzartos JS, Stergiou C, Kilidireas K, Zisimopoulou P, Thomaidis T, et al. (2013) Anti-Aquaporin-1 Autoantibodies in Patients with Neuromyelitis Optica Spectrum Disorders. PLoS ONE 8(9): e74773. doi:10.1371/journal.pone.0074773 Editor: Jun-ichi Kira, Kyushu University, Japan Received February 20, 2013; Accepted August 7, 2013; Published September 23, 2013 Copyright: ß 2013 Tzartos et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by overheads of the Department of Biochemistry of the Hellenic Pasteur Institute and co-financed by the EU (European Social Fund) and Greek national funds, through the Program "Education and Lifelong Learning" of the NSRF - Research Funding Programs: ‘‘Thales’’ and ‘‘Aristeia’’. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journal’s policy and have the following conflicts: J. Tzartos, Ch. Stergiou, K. Kilidireas, and S. Tzartos are the inventors in a patent application that relates to the detection of antibodies to aquaporin-1 in demyelinating diseases of the CNS. S. Tzartos is currently in the process of establishing a private diagnostics laboratory. P. Zisimopoulou and Th. Thomaidis report no disclosures. This does not alter their adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: jtzartos@gmail.com (JST); tzartos@pasteur.gr (SJT) Introduction Patients suffering from chronic inflammatory demyelinating diseases of the central nervous system (CNS) present a variable disease course, pathology, and response to treatment [1]. As a consequence, specific biomarkers are needed to classify these patients and adopt the most appropriate treatment. Currently, autoantibodies to aquaporin-4 (AQP4), a water channel abun- dantly expressed in CNS astrocytes, are a widely accepted biomarker in patients suffering from neuromyelitis optica spectrum disorders (NMOsd). The NMOsd group of demyelinating disorders includes the classic neuromyelitis optica (NMO), with major characteristics longitudinally extensive transverse myelitis (LETM) and optic neuritis [2], only LETM, only recurrent optic neuritis and related disorders [3]. In a high proportion of NMOsd patients, brain lesions are also present [4,5], thus complicating diagnosis, and the detection of anti-AQP4 antibodies is therefore vital for the early differentiation of NMO from MS [6] and for appropriate treatment [7]. Unfortunately, about 20–50% of NMOsd patients are seronegative for anti-AQP4 autoantibodies [8–10]. AQP4 belongs to the large AQP family with 13 members (AQP0-12), which form tetramers in the cell membrane, each monomer acting as a water channel [11]. AQPs have been identified in several mammalian tissues [12]. Several reports have shown that, in addition to AQP4, human CNS astrocytes also abundantly express AQP1 on their surface [13–17]. Specifically, it has been shown that AQP1 is highly expressed in areas prone to develop NMO lesions (spinal cord, optic nerves and brain white matter) [17]. Moreover, both AQP1 and AQP4 are overexpressed in the brain in some neurological diseases, such as MS, compared to ‘‘normal’’ brains [13], possibly for maintenance of water homeostasis. We therefore wondered whether patients with NMOsd, but seronegative for anti-AQP4 antibodies, had antibodies to AQP1. NMO-related autoantibodies (named NMO-IgG) were initially identified using an immunofluorescence assay in which serum from NMO patients was applied to mouse PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e74773