Identification of major S-nitrosylated proteins in murine experimental autoimmune encephalomyelitis

Nitrosative stress has been implicated in the pathophysiology of several CNS disorders, including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). We have recently shown that protein nitrosothiols (PrSNOs) accumulate in the brain of MS patients, and there is indirect evidence that PrSNO levels are also increased in EAE. In this study we sought to identify the major PrSNOs in the spinal cord of EAE animals prepared by active immunization of C57/BL6 mice with MOG(35-55) peptide. For this purpose, PrSNOs from control and EAE mice at various disease stages were derivatized with HPDP-biotin, and the biotinylated proteins were isolated with streptavidin-agarose. Proteins from total and streptavidin-bound fractions were then analyzed by Western blotting using antibodies against the major S-nitrosylated substrates of CNS tissue. With this approach we found that the proportion of S-nitrosylated neurofilament proteins, NMDA receptors, alpha/beta-tubulin, beta-actin, and GAPDH is increased in EAE. Other potential substrates either were not S-nitrosylated in vivo (HCN3, HSP-72, CRMP-2, gamma-actin, calbindin) or their S-nitrosylation levels were unaltered in EAE (Na/K ATPase, hexokinase, glycogen phosphorylase). We also discovered that neuronal specific enolase is the major S-nitrosylated protein in acute EAE. Given that S-nitrosylation affects protein function, it is likely that the observed changes are significant to the pathophysiology of inflammatory demyelination.