5-hydroxymethylcytosine (5hmC) is a newly discovered modification of DNA that originates from the oxidation of 5-methylcytosine (5mC) by the Ten-eleven translocation 1-3 (Tet1-3) proteins. This modification is essential for several complex processes requiring dynamic changes in DNA methylation patterns. In fact, 5hmC represents both a new epigenetic state in genomic DNA and an intermediate in the process of DNA demethylation. More importantly, this exciting discovery has begun to shed light on the dynamic nature of 5mC in many disorders, including neurodegenerative diseases. Consistently, deregulation of the 5hmC metabolisms and alteration of 5hmC genomic patterns have been linked to neurodegeneration in Huntington's disease and Alzheimer's disease.
Our group has recently proposed that defective 5hmC regulation is also involved in Multiple Sclerosis (MS). We showed that global levels of 5hmC are decreased in MS PBMCs, probably as a result of the diminished expression of the methylcytosine dioxygenase TET2. Significantly, further recent data, which form the preliminary results background of this proposal, indicate that the very same alterations are actually observed in the post-mortem NAWM of MS brains. Overall, these data indicate that MS may have an epigenetic component that manifests itself as a loss of 5hmC most likely due to the defective expression of the TET2 enzyme. Based on this background, we propose to perform further experimental strategies in order to better investigate the origin of the 5hmC loss in the MS Brain. We will determine if the origin of the altered expression of TET2 and other proteins that co-operate in its function, can be attributed to epigenetic modifications of their promoters, as is the case in other pathologies. Results would be fruitful not only to understand the molecular features of MS, but also to set the stage for the future development of strategies to counteract the disease via epigenetic drugs.
It has been hypothesised that epigenetic alteration might be an underlying factor in MS. A significant clue is that MS is a complex disease so that epigenetic mechanisms might explain its various non-Mendelian irregularities and susceptibility to environmental stressors. Consistently, epigenetic abnormalities have been reported in the MS brain and blood in various studies. In the brain, 5hmC has various functions which are linked to neuronal plasticity and oligodendrocytes differentiation and survival [14]. It is therefore plausible that a reduced level of 5hmC might be involved in MS pathogenesis. Significantly, data obtained in the murine model of human MS actually indicate that TET2 deficiency favours the progression of this type of autoimmune disease. This is consistent with the observation that TET enzymes participate in complex mechanisms of gene regulation by modulating the methylation level of gene expression regulatory sequences. Significantly, this action is required for the correct modulation of inflammatory response through pro-inflammatory factors' repression [15]. In addition, genetic evidence suggests that TET2 gene variants are actually associated with the risk of MS [16]. It is therefore plausible that the TET2 decline we observe in NAWM can represent a further manifestation of a process that affects TET2 in MS, which can translate into global epigenetic alterations and exacerbate the inflammatory state of MS individuals. The investigation of a connection between 5hmC, TET2 enzyme activity and MS will open new pathways towards a molecular knowledge of its pathogenesis. The reduction of TET2 and 5hmC may indicate a more complex phenomenon of global epigenetic pattern alteration, whose significance for MS pathogenesis could be a relevant topic for future research based on the results we believe can be achieved by this project. It is significant to note that similar approaches have been successful in discovering a similar 5hmC deficiency in the brains of patients with other neurodegenerative pathologies, such as Alzheimer's and Huntington's diseases [17, 18]. High-throughput sequencing data indicate that disease-associated DhMRs showed a good correlation with genes and signalling pathways involved in the pathogenesis of these diseases [18]. The involvement of 5hmC in MS is a brand new result from this group. This project aims to further investigate this link. However, being an observational study, it cannot directly detect a cause-effect relationship between TET2 deficiency, 5hmC alterations, and MS pathogenesis. It will, however, be able to establish relevant associations that can be a starting point for future investigations to define their mechanisms. It is implied that the subsequent definition of a possible causative role of 5hmC in MS pathogenesis will be pivotal because it can open up new possibilities for new treatments based on epigenetic manipulation, for example by using epigenetic drugs.