Neurotoxic Species of Misfolded SOD1 Recognized by Antibodies Against the P2X4 Subunit of the ATP Receptor Accumulate in Damaged Neurons of Transgenic Animal Models of Amyotrophic Lateral Sclerosis

We recently reported that degenerating motor neurons of superoxide dismutase mutant 1 (SOD1) rodents exhibit immunoreactivity to P2X4 antibodies. Neurons with strong P2X4-like immunoreactivity (P2X4-LIR) do not show an apoptotic phenotype and are often associated with microglial cells that display neuronophagic activity. Western blot analysis showed that P2X4 antibodies recognize not only the P2X4 adenosine triphosphate receptor protein but also a hitherto unidentified lowYmolecular weight band. Here, we identify the molecular counterpart of the strong P2X4-LIR observed in association with neuronal degeneration in SOD1 animals. After matrix-assisted laser desorption/ionization time-of-flight, we found that the lowYmolecular weight P2X4-immunoreactive protein was SOD1. Further analysis demonstrated that the P2X4 antibody recognizes a form of misfolded mutant SOD1 that is expressed in neuronal cells undergoing degeneration but not in glial cells. Cross-reactivity could have been caused by the abnormal exposure of an epitope in the inner hydrophobic region of SOD1 that shared structural homology with the P2X4-immunizing peptide used for raising the antibody. No positive P2X4 immunostaining was detected in mice overexpressing human wild-type SOD1. Intracerebral injections of affinity chromatographyYisolated P2X4-immunoreactive SOD1 species promote microglial and astroglial activation. We conclude that neuronal SOD1 conformers with P2X4-LIR may have pathogenetic relevance in the promotion of neuroinflammation.