GABA transport and neuroinflammation are coupled in multiple sclerosis: regulation of the GABA transporter-2 by ganaxolone.

Interactions between neurotransmitters and the immune system represent new prospects for understanding neuroinflammation and associated neurological disease. GABA is the chief inhibitory neurotransmitter but its actions on immune pathways in the brain are unclear. In the present study, we investigated GABAergic transport in conjunction with neuroinflammation in models of multiple sclerosis (MS). Protein and mRNA levels of γ-amino butyric acid transporter 2 (GAT-2) were examined in cerebral white matter from MS and control (Non-MS) patients, in cultured human macrophages, microglia and astrocytes, and in spinal cords from mice with and without experimental autoimmune encephalomyelitis (EAE) using western blotting, immunocytochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). GABA levels were measured by HPLC. The GAT-2's expression was increased in MS patients' (n=6) white matter, particularly in macrophage lineage cells, compared to Non-MS patients (n=6) (p<0.05). Interferon-γ (IFN-γ) stimulation of human macrophage lineage cells induced GAT-2 expression and reduced extracellular GABA levels (p<0.05) but soluble GABA treatment suppressed HLA-DRα, GAT-2 and XBP-1/s expression in stimulated macrophage lineage cells (p<0.05). Similarly, the synthetic allopregnanolone analog, ganaxolone (GNX), repressed GAT-2, JAK-1 and STAT-1 expression in activated macrophage lineage cells (p<0.05). In vivo GNX treatment reduced Gat-2, Cd3ε, MhcII, and Xbp-1/s expression in spinal cords following EAE induction (p<0.05), which was correlated with improved neurobehavioral outcomes and reduced neuroinflammation, demyelination and axonal injury. These findings highlight altered GABAergic transport through GAT-2 induction during neuroinflammation. GABA transport and neuroinflammation are closely coupled but regulated by GNX, pointing to GABAergic pathways as therapeutic targets in neuroinflammatory diseases.

A cholesterol-dependent CD20 epitope detected by the FMC7 antibody.

Accurate diagnosis of lymphoid malignancies is essential for appropriate therapeutic intervention. In conjunction with other diagnostic determinants, immunophenotypic analysis of differentially expressed cell surface markers, such as CD5, CD20, CD23 and FMC7, is useful in the subclassification of lymphomas and leukemias arising from the B-cell lineage. Recent evidence suggesting that CD20 predicts FMC7 expression has prompted reappraisal of the utility of monitoring both markers. Here, we report that the FMC7 monoclonal antibody (mAb) specifically and strongly recognized CD20 ectopically expressed in hematopoietic and nonhematopoietic cell lines. The reactivity of FMC7 was abolished by mutations in the extracellular domain of CD20. These data confirm the CD20 specificity of FMC7. Like other CD20 mAbs, FMC7 binding was temperature dependent and induced detergent insolubility of CD20. Of significant interest, the CD20 epitope recognized by FMC7 was unusual in that it was exceptionally sensitive to membrane cholesterol. Cholesterol depletion profoundly reduced expression of the FMC7 epitope, whereas cholesterol enrichment enhanced its expression. FMC7 mAb binding thus appears to be a sensitive indicator of the level of plasma membrane cholesterol and reveals a conformational state of CD20 that is regulated by cholesterol.

Identification of a cytoplasmic region of CD20 required for its redistribution to a detergent-insoluble membrane compartment.

CD20 is a B lymphocyte integral membrane protein with signal-transducing properties. Abs directed toward extracellular CD20 epitopes activate nonreceptor tyrosine kinases and modulate cell cycle progression of B lymphocytes. Recently, we demonstrated that binding of CD20 Abs to B cells induces the rapid redistribution of up to 95% of CD20 molecules to low density, detergent-insoluble membrane microdomains and induces the appearance of an approximately 50-kDa tyrosine-phosphorylated protein in the same compartment. Active relocalization of CD20 may thus be critical to the initiation of signaling events by CD20. The CD20 cDNA sequence predicts a nonglycosylated protein with four transmembrane-spanning regions and intracellular amino and carboxyl termini. Here we provide verification of the location of both the intracellular and extracellular regions of the CD20 molecule and identify a membrane-proximal sequence in the cytoplasmic carboxyl tail that is required for CD20 to redistribute to detergent-insoluble membrane microdomains.

Rapid redistribution of CD20 to a low density detergent-insoluble membrane compartment.

CD20 is a B cell integral membrane protein capable of initiating growth-modulating signals in human B lymphocytes upon its engagement with monoclonal anti-CD20 antibodies. In this report, we demonstrate that treatment of B cells with CD20 antibodies induces rapid redistribution of CD20 into a detergent-insoluble membrane compartment. Redistribution is detected as early as 15 s, following antibody addition, and involves up to 95% of CD20 molecules, depending on the antibody used. All of the detergent-insoluble CD20 was found in the low density fractions of sucrose density gradients, indicating that CD20 redistributes to glycolipid-rich membrane domains, analogous to caveolae in some cell types. As CD20 has previously been shown to associate with Src family tyrosine kinases, their co-existence in these compartments suggests a link to the role of CD20 in signal transduction. This study provides insight into the mechanism by which CD20 communicates signals to the cell interior and indicates that the search for membrane-proximal intracellular signaling partners should be directed to the Triton-insoluble fraction.