Introduction 1 2 3 4 5 The cause of MS is unknown. Two major theories have emerged: an autoimmune or a neurodegenerative cause. The autoimmune etiology is considered to arise from sensitization of T-cells in the periphery by a mechanism of molecular mimicry, usually involving a viral antigen with peptide sequences similar to those found in myelin proteins, especially MBP, a candidate autoantigen in MS. The neurodegenerative hypothesis involves metabolic changes in the myelin constituents, which destabilize the membrane architecture, resulting in myelin degradation. Since the myelin proteins are primarily responsible for maintaining the structure of the membrane we focused our attention on myelin proteins, in particular, modifications that are found on MBP. 1 Fig. 1 Post-translational modification of MBP human sequence  6 7 9 1 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Since we determined that up-regulation of PAD 2, preceded demyelination in the animal model, we searched for the mechanism responsible. An examination of the nucleotide sequence of the PAD 2 promoter revealed that it was high in CpG sequences. CpG sequences are important, because cytosine is a target for methylation by DNA methyltransferase, which produces 5-methylcytosine. Methylation of the promoter region of a gene silences the gene, whereas demethylation increases transcription. n n 2 Fig. 2 Deimination pathway in the pathogenesis of demyelinating disease. left closed lollipops open lollipops  28 As a result of the work summarized in this overview, we postulate the following sequence of events during the pathogenesis of MS. The citrullinated MBP, because of its decreased positive charge, prevents proper compaction of the bilayer, so that the multilamellar structure is less stable. Since PAD 2 has been shown to be present in myelin by immunogold electron microscopy (unpublished results), the conversion of non-citrullinated to citrullinated MBP can occur locally in myelin. Preliminary results have shown that immunogold labeling of PAD 2 in mouse optic nerve was not random, but was found in clusters in the axon itself and in the myelin sheath. In the latter, the clusters were found mainly at the junction of the myelin sheath and the axon (periaxonal) with some gold particles distributed in the myelin sheath. The presence of the PAD 2 enzyme at these sites suggests that the citrullinated MBP would be formed in these clusters in myelin, eventually resulting in plaques. When we examined the extent of labeling in the optic nerve of the spontaneously demyelinating transgenic mouse, we found a threefold increase in the number of gold particles. This increase in PAD 2 labeling demonstrated that increased amounts of PAD 2 were present in the transgenic mouse, accounting for an increase in the citrullinated MBP. Since citrullinated MBP is present in the normal myelin sheath (20% of the total MBP), citrullinated MBP can be tolerated to this extent. However, increased amounts as found in chronic MS (threefold), and in fulminating MS (six to sevenfold), cannot be accommodated in a compact myelin sheath, resulting in destabilization. 29 4 5 30 31