The etiology of multiple sclerosis (MS) is considered to involve genetic, environmental, infective, and immunological factors which affect the integrity of a normally assembled myelin sheath, either directly or indirectly resulting in demyelination. In a correlative study involving protein chemical, mass spectrometric, and electron microscopic techniques we have determined that myelin obtained from victims of MS is arrested at the level of the first growth spurt (within the first 6 yr of life) and is therefore developmentally immature. The data supporting this conclusion include (a) the pattern of microheterogeneity of myelin basic protein (MBP); (b) the NH2-terminal acylation of the least cationic component of MBP ("C-8"); (c) the phase transition temperature (TJ) of myelin isolated from victims of MS correlated with the increased proportion of the least cationic component of MBP; and (d) immunogold electron microscopy using an antibody specific for "C-8" showed that the distribution of gold particles in a 2-yr-old infant was similar to the distribution found in a victim of MS. We postulate that this developmentally immature myelin is more susceptible to degradation by one or a combination of factors mentioned above, providing the initial antigenic material to the immune system. (J. Clin. Invest 1994.94:146-154.)

The pathogenesis of MS is unknown. In our studies, we have demonstrated an important role for citrullinated myelin basic protein (MBP). The accompanying loss of positive charge compromises the ability of MBP to interact with the lipid bilayer. The conversion of arginine to citrulline in brain is carried out by an enzyme peptidyl arginine deiminase (PAD) 2. The amount of PAD 2 in brain was increased in MS normal-appearing white matter. The mechanism responsible for this increase involved hypomethylation of the promoter region in the PAD 2 gene in MS, but no change (compared to normal) was found in thymus tissue DNA from the same MS patients. In addition, no change was observed in other neurological diseases, including Alzheimer's, Parkinson's, and Huntington's. We propose that citrullinated MBP, resulting from elevated levels of PAD 2 represents an important biochemical pathway in the pathogenesis of MS.

Modification of arginine residues by citrullination is catalyzed by peptidylarginine deiminases (PADs), of which five are known, generating irreversible protein structural modifications. We have shown previously that enhanced citrullination of myelin basic protein contributed to destabilization of the myelin membrane in the CNS of multiple sclerosis (MS) patients. We now report increased citrullination of nucleosomal histones by PAD4 in normal-appearing white matter (NAWM) of MS patients and in animal models of demyelination. Histone citrullination was attributable to increased levels and activity of nuclear PAD4. PAD4 translocation into the nucleus was attributable to elevated tumor necrosis factor-␣ (TNF-␣) protein. The elevated TNF-␣ in MS NAWM was not associated with CD3 ϩ or CD8 ϩ lymphocytes, nor was it associated with CD68 ϩ microglia/macrophages. GFAP, a measure of astrocytosis, was the only cytological marker that was consistently elevated in the MS NAWM, suggesting that TNF-␣ may have been derived from astrocytes. In cell cultures of mouse and human oligodendroglial cell lines, PAD4 was predominantly cytosolic but TNF-␣ treatment induced its nuclear translocation. To address the involvement of TNF-␣ in targeting PAD4 to the nucleus, we found that transgenic mice overexpressing TNF-␣ also had increased levels of citrullinated histones and elevated nuclear PAD4 before demyelination. In conclusion, high citrullination of histones consequent to PAD4 nuclear translocation is part of the process that leads to irreversible changes in oligodendrocytes and may contribute to apoptosis of oligodendrocytes in MS.

In previous studies, we documented increased citrullinated myelin basic protein (MBP) was present in MBP isolated from multiple sclerosis (MS) normal appearing white matter (NAWM). This increase was due to the myelin enzyme peptidyl argininedeiminase 2 (PAD2). In this study, we show that methylation of cytosine of the PAD2 promoter in DNA from MS NAWM was decreased to one-third of the level of that in DNA from normal white matter. The PAD2 promoter in DNA from thymus obtained from the same MS patients and white matter DNA from Alzheimer's, Huntington's, and Parkinson's was not hypomethylated. DNA demethylase activity in supernatants prepared from NAWM of MS patients was 2-fold higher than the DNA demethylase from normal, Alzheimer's, Huntington's and Parkinson's disease white matter. The amount of PAD2 enzyme and citrullinated MBP was increased in MS NAWM. The decreased methylation of cytosines in the PAD2 promoter may explain the increased synthesis of PAD2 protein that is responsible for the increased amount of citrullinated MBP, which in turn results in loss of myelin stability in MS brain.

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