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

Abstract

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.

Keywords: experimental autoimmune encephalitis; ganaxolone; multiple sclerosis; neuroinflammation; γ-amino butyric acid; γ-amino butyric acid transporter-2.