B cell clonal expansion and cerebrospinal fluid (CSF) oligoclonal IgG bands are established features of the immune response in multiple sclerosis (MS). Clone-specific recombinant monoclonal IgG1 Abs (rAbs) derived from MS patient CSF plasmablasts bound to conformational proteolipid protein 1 (PLP1) membrane complexes and, when injected into mouse brain with human complement, recapitulated histologic features of MS pathology: oligodendrocyte cell loss, complement deposition, and CD68+ phagocyte infiltration. Conformational PLP1 membrane epitopes were complex and governed by the local cholesterol and glycolipid microenvironment. Abs against conformational PLP1 membrane complexes targeted multiple surface epitopes, were enriched within the CSF compartment, and were detected in most MS patients, but not in inflammatory and noninflammatory neurologic controls. CSF PLP1 complex Abs provide a pathogenic autoantibody biomarker specific for MS.

Objective: Discover mechanisms by which B cells may drive MS pathogenesis. Background: Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system (CNS), featuring demyelination, activated lymphocytes, astrogliosis, and axonal loss. Suppression of disease progression via B cell-targeted therapies indicates important roles for B cells, requiring further studies of B cell mechanisms. Design/Methods: Cerebellar slice cultures were generated from P10 mice and cultured for 12 days. CD19+ B cells were isolated from peripheral blood mononuclear cells of patients and controls, then added to cerebellar slice cultures or astrocyte cell cultures for 24-96 hours. Slices or cells were fixed and immunostained; media was collected for cytokine analysis. In other studies, B cells were isolated from mice and exposed to stimuli before culturing with cortical murine astrocytes, then analyzed for GFAP reactivity and iNOS production. Results: B cells from MS patients, but not controls, induced astrocyte and microglia morphological changes, oligodendrocyte loss, and demyelination. In order to analyze astrocyte responses, we treated primary astrocyte cultures with murine B cells stimulated under different conditions, or human B cells isolated from MS patients. Under specific activating conditions, murine B cells altered astrocyte morphology, upregulated GFAP expression and intracellular iNOS. Evaluating the astrocyte responses to B lymphocytes from untreated MS patients or controls is currently in progress, as well as identifying mechanisms of B lymphocyte mediated CNS pathology. Conclusions: B cells from MS patients mediate CNS pathology in organotypic slices similar to that observed in histological studies of MS tissue. These effects include morphological changes in astrocytes, oligodendrocyte death, and demyelination. This system will allow for the identification of the target cell(s), as well as assist in identifying key unknown biological mechanisms underlying MS. This study was funded by an Independent Medical Grant from EMD Serono and the Rocky Mountain Multiple Sclerosis Center.

April 21, 2015April 6, 2015Free AccessThe role of fractalkine (CX3CL1) in regulation of CD4+ cell migration to the central nervous system in patients with relapsing-remitting multiple sclerosis (S12.003)Silva Markovic Plese, Kevin Blauth, Xin Zhang, and Manisha ChopraAuthors Info & AffiliationsApril 6, 2015 issue84 (14_supplement)https://doi.org/10.1212/WNL.84.14_supplement.S12.003 Letters to the Editor

B cells are implicated in the etiology of multiple sclerosis (MS). Intrathecal IgG synthesis, cerebrospinal fluid (CSF) oligoclonal bands and lesional IgG deposition suggest a role for antibody-mediated pathology. We examined the binding of IgG1 monoclonal recombinant antibodies (rAbs) derived from MS patient CSF expanded B cell clones to central nervous system (CNS) tissue. MS rAbs displaying CNS binding to mouse and human CNS tissue were further tested for their ability to induce complement-mediated tissue injury in ex vivo spinal cord explant cultures. The staining of CNS tissue, primary human astrocytes and human neurons revealed a measurable bias in MS rAb binding to antigens preferentially expressed on astrocytes and neurons. MS rAbs that recognize myelin-enriched antigens were rarely detected. Both myelin-specific and some astrocyte/neuronal-specific MS rAbs caused significant myelin loss and astrocyte activation when applied to spinal cord explant cultures in the presence of complement. Overall, the intrathecal B cell response in multiple sclerosis binds to both glial and neuronal targets and produces demyelination in spinal cord explant cultures implicating intrathecal IgG in MS pathogenesis.

B cells play a central role in multiple sclerosis (MS) pathology. B and plasma cells may contribute to disease activity through multiple mechanisms: antigen presentation, cytokine secretion, or antibody production. Molecular analyses of B cell populations in MS patients have revealed significant overlaps between peripheral lymphoid and clonally expanded central nervous system (CNS) B cell populations, indicating that B cell trafficking may play a critical role in driving MS exacerbations. In this review, we will assess our current knowledge of the mechanisms and pathways governing B cell migration into the CNS and examine evidence for and against a compartmentalized B cell response driving progressive MS pathology.

Fractalkine (CX3CL1) levels are increased in the cerebrospinal fluid (CSF) of patients with clinically isolated syndrome (CIS), as well as in the CSF and serum samples from patients with relapsing-remitting multiple sclerosis (RRMS). A higher percentage of circulating CD4(+) T-cells expressed its surface receptor (CX3CR1) and intracellular adhesion molecule (ICAM-1) in RRMS patients in comparison to healthy controls (HCs). The CX3CR1(+)ICAM-1(+)CD4(+) T-cells are enriched in the CSF of the RRMS patients. In vitro migration studies revealed that CD4(+) T-cells, which migrated toward a CX3CL1 gradient, expressed higher levels of ICAM-1 than non-migrating cells. CX3CL1 significantly increased IFN-γ and TNF-α gene expression and IFN-γ secretion by CD4(+) T-cells derived from the RRMS patients. CX3CL1 upregulated ICAM-1 expression on the surface of RRMS patient-derived but not HC-derived CD4(+) T-cells. Thus, CX3CL1 induces recruitment of CX3CR1(+)ICAM-1(+)CD4(+) T-cells into the central nervous system (CNS) during the early inflammatory response in MS.

Mechanisms driving pathology in multiple sclerosis (MS) remain widely debated. The effectiveness of anti-B cell therapies in the treatment of RRMS has renewed interest in the role(s) B cells play in promoting CNS immune-mediated damage. Increased and persistent intrathecal IgG synthesis and the presence of oligoclonal bands (OCBs) are biochemical hallmarks of disease and may represent a pathologically relevant targeted B cell response. To study the specificity and pathogenicity of the MS intrathecal B cell response, we have previously generated monoclonal recombinant antibodies (rAbs) that reproduce the in vivo specificities of expanded MS CSF plasma cell clones. Herein, we describe three patterns of rAb immunoreactivity in the mouse CNS: pattern 1 is in discrete domains along myelinated axons; pattern 2 localizes to granule cell bodies of the cerebellum and hippocampus, and to neuronal cell bodies throughout the cerebral cortex and spinal cord; and pattern 3 reactivity is along neuronal cell bodies and neurites. To assess putative roles in pathology, we applied MS rAbs and complement to ex vivo spinal cord explant cultures. Three of the four MS CSF rAbs, but not neuroinflammatory control CSF rAbs, caused significant demyelination as measured by loss of MBP immunoreactivity, astrocyte activation, and terminal complement deposition. MS and control rAbs were also microinjected into discrete brain regions leading to many of these same pathologic features that include loss of myelin markers, astrocyte activation as measured by GFAP and AQP4 immunoreactivity, an increase in CD68+ macrophages/microglia, and deposition of terminal complement complexes. In summary, we have identified MS CSF-derived rAbs that bind to myelin- and neuron-associated antigens in the CNS. Several of the rAbs can cause myelin damage in the presence of complement both in vitro and in vivo, and therefore may play a role in MS pathogenesis. Discovery of the targeted antigens and further characterization of injury induced by these rAbs will advance our understanding of MS pathology and identify novel therapeutic targets.

Septate junctions (SJs) display a unique ultrastructural morphology with ladder-like electron densities that are conserved through evolution. Genetic and molecular analyses have identified a highly conserved core complex of SJ proteins consisting of three cell adhesion molecules Neurexin IV, Contactin, and Neuroglian, which interact with the cytoskeletal FERM domain protein Coracle. How these individual proteins interact to form the septal arrays that create the paracellular barrier is poorly understood. Here, we show that point mutations that map to specific domains of neurexin IV lead to formation of fewer septae and disorganization of SJs. Consistent with these observations, our in vivo domain deletion analyses identified the first Laminin G-EGF-Laminin G module in the extracellular region of Neurexin IV as necessary for the localization of and association with Contactin. Neurexin IV protein that is devoid of its cytoplasmic region is able to create septae, but fails to form a full complement of SJs. These data provide the first in vivo evidence that specific domains in Neurexin IV are required for protein-protein interactions and organization of SJs. Given the molecular conservation of SJ proteins across species, our studies may provide insights into how vertebrate axo-glial SJs are organized in myelinated axons.

Slit/Roundabout (Robo) signaling controls midline repulsive axon guidance. However, proteins that interact with Slit/Robo at the cell surface remain largely uncharacterized. Here, we report that theDrosophilatransmembrane septate junction-specific protein Neurexin IV (Nrx IV) functions in midline repulsive axon guidance. Nrx IV is expressed in the neurons of the developing ventral nerve cord, andnrx IVmutants show crossing and circling of ipsilateral axons and fused commissures. Interestingly, the axon guidance defects observed innrx IVmutants seem independent of its other binding partners, such as Contactin and Neuroglian and the midline glia protein Wrapper, which interacts intranswith Nrx IV.nrx IVmutants show diffuse Robo localization, and dose-dependent genetic interactions betweennrx IV/roboandnrx IV/slitindicate that they function in a common pathway.In vivobiochemical studies reveal that Nrx IV associates with Robo, Slit, and Syndecan, and interactions between Robo and Slit, or Nrx IV and Slit, are affected innrx IVandrobomutants, respectively. Coexpression of Nrx IV and Robo in mammalian cells confirms that these proteins retain the ability to interact in a heterologous system. Furthermore, we demonstrate that the extracellular region of Nrx IV is sufficient to rescue Robo localization and axon guidance phenotypes innrx IVmutants. Together, our studies establish that Nrx IV is essential for proper Robo localization and identify Nrx IV as a novel interacting partner of the Slit/Robo signaling pathway.

Glial cells are critical players in every major aspect of nervous system development, function, and disease. Other than their traditional supportive role, glial cells perform a variety of important functions such as myelination, synapse formation and plasticity, and establishment of blood-brain and blood-nerve barriers in the nervous system. Recent studies highlight the striking functional similarities between Drosophila and vertebrate glia. In both systems, glial cells play an essential role in neural ensheathment thereby isolating the nervous system and help to create a local ionic microenvironment for conduction of nerve impulses. Here, we review the anatomical aspects and the molecular players that underlie ensheathment during different stages of nervous system development in Drosophila and how these processes lead to the organization of neuroglial junctions. We also discuss some key aspects of the invertebrate axonal ensheathment and junctional organization with that of vertebrate myelination and axon-glial interactions. Finally, we highlight the importance of intercellular junctions in barrier formation in various cellular contexts in Drosophila. We speculate that unraveling the genetic and molecular mechanisms of ensheathment across species might provide key insights into human myelin-related disorders and help in designing therapeutic interventions.

Glia play crucial roles in ensheathing axons, a process that requires an intricate series of glia-neuron interactions. The membrane-anchored protein Wrapper is present in Drosophila midline glia and is required for ensheathment of commissural axons. By contrast, Neurexin IV is present on the membranes of neurons and commissural axons, and is highly concentrated at their interfaces with midline glia. Analysis of Neurexin IV and wrapper mutant embryos revealed identical defects in glial migration, ensheathment and glial subdivision of the commissures. Mutant and misexpression experiments indicated that Neurexin IV membrane localization is dependent on interactions with Wrapper. Cell culture aggregation assays and biochemical experiments demonstrated the ability of Neurexin IV to promote cell adhesion by binding to Wrapper. These results show that neuronal-expressed Neurexin IV and midline glial-expressed Wrapper act as heterophilic adhesion molecules that mediate multiple cellular events involved in glia-neuron interactions.