Amiodarone (AMD), an anti-arrhythmic agent, has a number of adverse effects, including peripheral neuropathy. AMD and its metabolites are likely to inhibit lysosomal phospholipases and induce accumulation of phospholipids and other substances in the lysosomes of Schwann cells, thereby being a cause of Schwannopathy or myelinopathy. However, the molecular mechanisms of AMD toxicity in Schwann cells are still unknown. We have established a spontaneously immortalized adult rat Schwann cell line IFRS1, which displays distinct Schwann cell phenotypes and fundamental ability to myelinate neurites in coculture with PC12 cells. In this study, we investigated the effects of AMD on the viability and biochemical properties of IFRS1 cells and the myelination in PC12-IFRS1 coculture system.
IFRS1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 1% fetal calf serum and N2 supplement. Treatment of IFRS1 cells with AMD (1, 5, and 10 μM) for 24 h dose-dependently reduced the cell viability, increased the intracellular phospholipids, and upregulated the expression of a lysosomal marker LIMPII, an oxidative stress marker 4-hydroxy-2-nonenal, autophagy markers LC3-II and p62, and sterol regulatory element-binding protein (SREBP)-1. The upregulation of p62 suggests impaired autophagy, whereas SREBP-1 plays a role in lipid metabolism and its upregulation may be correlated with the AMD-induced phospholipid accumulation.
PC12-IFRS1 cocultures were maintained in myelination medium (DMEM with B27 supplement, 50 μg/ml ascorbic acid, 10 ng/ml nerve growth factor and 25 ng/ml neuregulin-1 type III) for 21 days. Treatment of the cocultures with 20 μM of AMD for 48 h induced detachment of IFRS1 cells from the neurite networks as observed under a phase-contrast microscope and downregulated the expression of myelin protein P0 and neuregulin-1 receptor ErbB3 as revealed by western blot analysis.
These results indicate that AMD induces lysosomal phospholipid storage, oxidative stress and impaired autophagy in Schwann cells, which may lead to demyelination in the peripheral nervous system.