(BBB) into the central nervous system (CNS). Integrins, which are heterodimeric alpha/beta transmembrane proteins, facilitate this process by mediating cell–cell or cell–extracellular matrix protein interactions. Specifically, the beta1 integrin family has been associ- ated with migration of PB lymphocytes across the BBB, as supported by the clinical efficacy of Natalizumab, a monoclonal antibody targeting the alpha4 subunit of integrin alpha4beta1. However, due to widespread expression of alpha4 on most leukocyte subsets, Natalizumab treatment is associated with impaired immune surveil- lance and enhanced susceptibility to lethal viral infections. Therefore, the goal of this study is to characterize the role of novel, subset specific beta1 integrin partners in the migration of pathogenic T lymphocytes across the BBB. Whole cell lysate proteomic analysis revealed that T H 17 cells express integrin alpha8, which hetero- dimerizes exclusively with the beta1 subunit. Western blot, RT-PCR, qPCR and flow cytometric analyses demonstrate that alpha8 is specifically expressed on activated memory CD4 + T lymphocytes and is upregulated in T H 1 and T H 17 polarizing conditions. Immunofluo- rescence studies reveal that alpha8 localizes to the cell surface and is expressed on CD4 + T cell infiltrates in MS and mouse experimental autoimmune encephalomyelitis brains. Moreover, the expression of nephronectin (NPNT), a ligand of alpha8, is constitutively expressed on in vitro cultured BBB endothelial cells (ECs) in resting and inflam- matory conditions. Blockade of the alpha8-NPNT interaction using an RGD-peptide in a static in vitro migration assay decreases T H 1 and T H 17 lymphocyte migration across the EC monolayer. These data highlight a potentially important role of alpha8 in mediating pathogenic T lymphocyte migration across the BBB in MS. Further studies are aimed at demonstrating the therapeutic benefit of alpha8 blocking agents, without promoting systemic immune suppression, in vivo. doi:10.1016/j.jneuroim.2014.08.267 305 PLP1 mutations in patients with multiple sclerosis: Identification of a new mutation and analysis of in vitro effects of PLP1 mutations Nancy Cloake a , Jun Yan a , Michael Pender b , Atefeh Aminian a , Judith Greer a a UQ Centre for Clinical Research, The University of Queensland, Brisbane, Australia; b School of Medicine, The University of Queensland, Brisbane, Australia PLP1 is located on the X-chromosome and encodes myelin proteolipid protein (PLP), which is produced by oligodendrocytes and is the most abundant protein of central nervous system (CNS) myelin. Generally, mutations in PLP1 result in the X-linked dysmyelinating disorders Pelizaeus–Merzbacher disease (PMD) or spastic paraplegia type 2. However, 2 case studies (Warshawsky et al., 2005; Gorman et al., 2007) identified 2 patients with clinical symptoms and signs indistinguishable from multiple sclerosis (MS) who had missense point mutations in PLP1 (resulting in changes Leu30Arg and Arg136Trp in the protein). To investigate whether PLP1 mutations might occur relatively frequently in MS, we sequenced the coding regions of PLP1 in 22 female MS patients who had developed MS after the age of 40 and in 51 healthy females, and identified a missense mutation in exon 2 of PLP1 (resulting in a Leu30Val mutation in the protein) in one of the MS patients. mCherry-tagged plasmids containing wild type or mutant PLP1 sequences encoding the 7 exons of PLP (including two known PMD- related mutations as positive controls) were constructed and transfected into COS-7 cells. In the cells transfected with wild type PLP1, PLP was expressed throughout the cells and at the cell surface by 12 h post-transfection. In contrast, the two PMD-related muta- tions and one of the 3 MS-related PLP1 mutations (Leu30Arg) caused accumulation of PLP in the endoplasmic reticulum of the cells and induction of the unfolded protein response, leading to apoptosis of the cells. While the Leu30Val mutation identified in the current study did not adversely affect the distribution of PLP in the COS-7 cells or induce the unfolded protein response, in silico analysis of HLA binding for the HLA molecules carried by the patient harbouring this mutation suggested that the mutation would produce a novel immunogenic PLP epitope in this patient. These results suggest that PLP1 mutations can occur in patients who meet clinical criteria for MS, and that some of these mutations have the potential to induce oligodendrocyte apoptosis. Other PLP1 muta- tions may produce novel epitopes of PLP that could become targets of autoreactivity in MS. The results support the idea that mutations in myelin-related genes could underlie development of MS in some patients. doi:10.1016/j.jneuroim.2014.08.268 542 Imaging of pathological effects of aquaporin-4 specific antibodies ex vivo and in vivo Marina Herwerth a,b , Rajneesh Srivastava a , Sudhakar Reddy Kalluri a , Thomas Misgeld b , Bernhard Hemmer a a Neurologische Klinik und Poliklinik Rechts der Isar, Technische Universität, München, Germany; b Institute of Neuronal Cell Biology, Technische Universität, München, Germany Neuromyelitis optica (NMO) is an autoimmune disease of the central nervous system (CNS). In the serum of most NMO-patients a specific autoantibody against the water channel, aquaporin 4 (AQP4), can be found. In several experimental studies injection of AQP4-antibodies with complement was shown to cause astrocytic loss in mice and rats. However, the dynamics of AQP4-antibody induced pathological effects remain unknown. Here, we investigat- ed acute effects of sera from NMO patients on brain slices and in the spinal cord in vivo of mice with GFP-labeled astrocytes using two- photon microscopy. Application of sera from AQP4-positive NMO patients induced astrocyte-specific cytotoxicity in a dose-depen- dent manner within 2 h. During this process, astrocytic processes were fragmented and astrocyte somata swelled and lost fluores- cence. This effect was absent after AQP4-antibody depletion or complement inactivation. No astrocytic pathology could be ob- served after application of healthy or AQP4-negative NMO control sera. The early effect appeared to be cell type-specific, as no significant changes were observed in fluorescently labeled oligo- dendrocytes. Thus, we show that human NMO-AQP4-antibodies induce acute astrocyte-specific toxicity ex vivo and in vivo. In the future this approach will be valuable in studying the cytotoxic mechanisms causing astrocyte loss in NMO and in devising new strategies for intervention. doi:10.1016/j.jneuroim.2014.08.269 Abstracts 100