Hypochlorite modification of sphingomyelin generates chlorinated lipid species that induce apoptosis and proteome alterations in dopaminergic PC12 neurons in vitro

Recent observations link myeloperoxidase (MPO) activation to neurodegeneration. In multiple sclerosis MPO is present in areas of active demyelination where the potent oxidant hypochlorous acid (HOCI), formed by MPO from H2O2 and chloride ions, could oxidatively damage myelin-associated lipids. The purpose of this study was (i) to characterize reaction products of sphingomyelin (SM) formed in response to modification by HOCI, (ii) to define the impact of exogenously added SM and HOCI-modified SM (HOCI-SM) on viability parameters of a neuronal cell line (PC12), and (iii) to study alterations in the PC12 cell proteome in response to SM and HOCI-SM. MALDI-TOF-MS analyses revealed that HOCI, added as reagent or generated enzymatically, transforms SM into chlorinated species. On the cellular level HOCI-SM but not SM induced the formation of reactive oxygen species. HOCI-SM induced severely impaired cell viability, dissipation of the mitochondrial membrane potential, and activation of caspase-3 and DNA damage. Proteome analyses identified differential expression of specific subsets of proteins in response to SM and HOCI-SM. Our results demonstrate that HOCI modification of SM results in the generation of chlorinated lipid species with potent neurotoxic properties. Given the emerging connections between the MPO-H2O2-chloride axis and neurodegeneration, this chlorinating pathway might be implicated in neuropathogenesis. (C) 2010 Elsevier Inc. All rights reserved.