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

Abstract

1.1 Aim Obstructive sleep apnea increases in diabetes and morbid obesity. We tested a hypothesis that circulating autoantibodies in adult type 2 diabetes which increase in association with morbid obesity are capable of causing long-lasting neuronal depolarization and altered calcium release in mouse atrial cardiomyocytes. 1.2 Methods Protein-A eluates from plasma of 14 diabetic obstructive 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 patients were compared for enhancement of inhibitory effects on endothelial cell survival. Size exclusion chromatography performed (in the presence or absence of a specific membrane type 1-matrix metalloproteinase inhibitor) was used to characterize the IgG autoantibody subunit(s) or fragments associated with peak neurotoxicity in diabetic obstructive sleep apnea. 1.3 Results Diabetic obstructive sleep apnea (n = 14) autoantibodies caused a significant increase (P = 0.01) in membrane depolarization in N2a mouse neuroblastoma cells compared to control diabetic patients (n = 15) not suffering with obstructive sleep apnea. Process extension in N2A mouse neuroblastoma cells was significantly inhibited (P = 0.01) by diabetic obstructive sleep apnea (n = 9) autoantibodies compared to effects from identical 10 μg/mL concentrations of control diabetic autoantibodies in patients without obstructive sleep apnea. Ten micromolar concentrations of SCH-202676, a G-protein coupled receptor antagonist (n = 5) or ten micromolar concentration of Y27632, a selective Rho kinase inhibitor (n = 6), each significantly prevented (P < 0.001) neurite outgrowth inhibition by diabetic obstructive 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 Ca2+ in HL-1 mouse atrial cardiomyocytes. The magnitude of intracellular Ca2+ 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. 1.4 Conclusions These results suggest that diabetic obstructive sleep apnea autoantibodies may induce strong depolarization in neuronal cells and alter Ca2+ signaling in atrial cardiomyocytes c...

Diabetes Autoantibodies Mediate Neural- and Endothelial Cell- Inhibitory Effects Via 5-Hydroxytryptamine- 2 Receptor Coupled to Phospholipase C/Inositol Triphosphate/Ca2+ Pathway

“…IP3 causes release of Ca2+ from intracellular stores, the latter plays a role in the activation of protein kinase C. In the current study, specific inhibitors of phospholipase C (PLC-γ), IP3R and an intracellular calcium chelator each nearly or completely prevented acute autoantibody-induced neurite retraction. The selective RhoA/ ROCK inhibitor Y27632 (10 µM) also completed prevented acute neurite retraction consistent with results in previous studies [5,6,7,8,9]. The precise mechanism linking PLC/IP3R/ Ca2+ activation with RhoA/ROCK-signaling in N2A cells is not clear, however, evidence from other laboratories indicates that ROCK activation can occur through the coordinated action(s) of activated Gαq/11 and beta (β) -arrestin-1 [19].…”

“…Dimeric or oligomeric forms of IgG autoantibodies might preferentially target 5-HT-2A receptor heterodimers in the membrane which (in some cases) are comprised by highly stable non-covalent interactions between oppositely-charged amino acid residues in the intracellular domains of physically neighboring GPCRs [23]. Stable heteromers involving GPCRs positively coupled to Gq/11 heterotrimer G proteins may contribute to sustained activation of PLC/IP3R/Ca2+ pathway causing long-lasting Ca2+ release (>10 minutes) similar to that which was observed in different subsets of diabetic autoantibodies [8].…”

“…Moderate-severely obese patients were selected for this mechanistic study, since in prior reports, obesity was significantly associated with pathologies (e.g. atrial fibrillation, obstructive sleep apnea) in which neurotoxic plasma autoantibodies were present at increased levels compared to control diabetic patients [8]. [9].…”