Increased Neuronal Depolarization Evoked by Autoantibodies in Diabetic Obstructive Sleep Apnea: Role for Inflammatory Protease(s) in Generation of Neurotoxic Immunoglobulin Fragment

Abstract

1 Abstract 1.1 Aim Obstructive sleep apnea increases in diabetes and mor-bid obesity. We tested a hypothesis that circulating au-toantibodies in adult type 2 diabetes which increase in association with morbid obesity are capable of caus-ing long-lasting neuronal depolarization and altered cal-cium release in mouse atrial cardiomyocytes. 1.2 Methods Protein-A eluates from plasma of 14 diabetic obstruc-tive sleep apnea patients and 17 age-matched diabetic patients without sleep apnea were tested for effects on depolarization and neurite out growth in N2a mouse neuroblastoma cells. The mechanism of autoantibody-mediated neurite outgrowth inhibition was investigated in co-incubation experiments of diabetic obstructive sleep apnea autoantibodies with specific antagonists of G-protein coupled receptors or the RhoA/ Rho kinase signaling pathway. Following long-term storage of the protein-A eluates (to allow spontaneous proteolysis and IgG subunit dissociation), plasma autoantibodies from diabetic obstructive sleep apnea, cancer or control pa-tients were compared for enhancement of inhibitory ef-fects on endothelial cell survival. Size exclusion chro-matography performed (in the presence or absence of a specific membrane type 1-matrix metalloproteinase in-hibitor) was used to characterize the IgG autoantibody subunit(s) or fragments associated with peak neurotox-icity in diabetic obstructive sleep apnea. 1.3 Results Diabetic obstructive sleep apnea (n = 14) autoantibod-ies caused a significant increase (P = 0.01) in mem-brane depolarization in N2a mouse neuroblastoma cells compared to control diabetic patients (n = 15) not suf-fering with obstructive sleep apnea. Process extension in N2A mouse neuroblastoma cells was significantly in-hibited (P = 0.01) by diabetic obstructive sleep apnea (n = 9) autoantibodies compared to effects from iden-tical 10 µg/ mL concentrations of control diabetic au-toantibodies in patients without obstructive sleep ap-nea. Ten micromolar concentrations of SCH-202676, a G-protein coupled receptor antagonist (n = 5) or ten micromolar concentration of Y27632, a selective Rho ki-nase inhibitor (n = 6), each significantly prevented (P < 0.001) neurite outgrowth inhibition by diabetic ob-structive sleep apnea autoantibodies. Autoantibodies in representative patients with obstructive sleep apnea and either atrial fibrillation or left ventricular hypertrophy evoked acute large increases in intracellular Ca 2+ in HL-1 mouse atrial cardiomyocytes. The magnitude of intra-cellular Ca 2+ release was dose-dependently significantly correlated to the electrocardiographic Cornell voltage-duration product. Gel filtration of diabetic obstructive sleep apnea autoantibodies revealed peak neurotoxicity associated with MWs corresponding to IgG light chain dimer(s), monomers or half-light chains as well as a novel ∼ 5.5 kD putative light chain fragment.