In this study we analyzed the equilibrium between histone acetylation, mediated by HATs and histone deacetylation, mediated by HDACs, in the NAWM of chronic MS brains. Similar to what reported for the old rodent brain, also in the NAWM of human brains from aged individuals and chronic MS patients we detected a shift towards acetylation. This shift towards acetylation detected in a subset of female patients correlated with the consistent and reproducible increase of several histone acetyl-transferase family members, including CBP, p300, MYST3 and MYST4. It is worth noting that, although we also detected increased levels of HDAC11 in this subpopulation, the increased of the acetyl-transferases was much greater and likely determined the shift in favor of increased acetylation. These differences were most prominent in a subset of female MS patients and were associated with high levels of developmentally regulated genes (i.e. TCF7L2, SOX2, ID2) compared to controls. Several other genes (i.e. TUBA, HDAC1, HDAC3) were not changed and served as internal controls. In contrast to our findings in NAWM, in early MS lesions with ongoing de- and re-myelinating activity the percentage of acetyl-H3-positive mature oligodendrocytes was significantly reduced compared to adjacent peri-plaque white matter, thereby suggesting that the equilibrium between histone acetylation and deacetylation is affected by multiple parameters, including disease duration.
Oligodendrocyte lineage cells within NAWM in the brain of patients with chronic MS were characterized by high levels of histone acetylation, while cells within early MS lesions showed variable patterns of deacetylation or acetylation of histone H3. The global increase of histone acetylation in the NAWM of MS subjects was consistent with the detection of genes in the BMP4 (Sim et al., 2002
), Notch (John et al., 2002
) and Wnt signaling (Fancy et al., 2009
) pathways and with other elegant studies reporting disregulated gene expression in white matter that lacks clear signs of demyelination (Graumann et al., 2003
; Zeis et al.
, 2008). Taken together, the global changes of histone acetylation and the effects on gene expression, can be interpreted in terms of widespread modifications of the chromatin landscape in demyelinating disorders (Hyunh and Casaccia, 2010
) affecting all the cells present in the white matter regions, even in the absence of active signs of demyelination.
Although the levels of histone acetylation were consistently higher in the brain of MS patients compared to controls, it is worth mentioning that the higher transcript levels for some histone acetyltransferases (i.e. P300
) or downstream Wnt effectors (i.e. TCF7L2
) were only detected in a subset of female patients and correlated with long disease duration. One possible explanation for these gender-based differences was that histone acetylation regulates the promoter for these genes only in female patients and that in male patients the increased levels of HDAC8
might be compensatory. However, the results of the ChIP experiments suggested that the chromatin at the TCF7L2
locus in both male and female MS subjects is more acetylated than in controls. An alternative explanation is a gender-based differential regulation of microRNAs and long-non coding RNA, driven by sex steroids. It has been suggested that estrogen binding to estrogen receptors down-regulates the expression of a subset of microRNA in Drosophila and in mammals (Yamagata et al., 2009
). Conversely, androgens have been reported to up-regulate microRNAs in prostate and muscle cells (Narayanan et al., 2010
) and regulate transcription of long non-coding RNA (Louro et al., 2007
). Acetylation modulates the transcriptional competence of chromatin. Our data suggest that MS disease duration modulates the overall chromatin landscape, rendering it more accessible to other modulating factors, such as androgen or estrogen receptors bound to their respective ligands. The overall effect on transcription, however, is determined by the type of complexes that are recruited to specific genes. The differential regulation of microRNAs by estrogens and androgens, indicates that estrogens might recruit activating complexes and androgen repressive complexes, thereby accounting for the observed gender-based differences of TCF7L2
The NAWM in chronic MS is characterized by a diffuse inflammatory environment, e.g. mild microglial activation and astrogliosis. In other organs, such as the lung, the pattern of increased histone acetylation detected in chronic obstructive pulmonary disease follows the one detected in the lungs of older subjects and led to the concept of inflammation associated premature aging (Ito and Barnes, 2009
). Similar to the lung, the inflammatory environment in the NAWM might induce a progressive increase of global acetylation of histone H3 in patients with chronic MS which resemble the changes observed in the aged CNS and positively correlates with disease duration. The overall change in the total number of acetyl-H3+ nuclei in the normal appearing white matter is relatively modest. However it is important to consider that immunohistochemistry provides a global and semi-quantitative assessment of acetylation. Additional quantification of the protein levels by slot blot or chromatin immunoprecipitation has clearly shown that the amount of acetylation is increased of several folds and that these changes are highly localized at the level of specific gene loci. This would explain why the apparently modest change in the number of immunoreactive acetyl-H3+ nuclei results in important changes in gene expression.
In developing MS lesions, in contrast, we detected a marked downregulation of acetylated histone H3 in oligodendrocytes compared to the peri-plaque white matter. This downregulation was obvious in all lesion areas suggesting that the presence of inflammatory cells in acute MS lesions modulates histone acetylation in oligodendrocyte lineage cells. We have previously shown that developmental myelination in the rodent brain, requires a shift towards deacetylation of nucleosomal histones. In remyelinating lesion areas we could detect oligodendrocyte lineage cells with two distinct patterns of histone acetylation. High levels of acetylation of histone H3 were found in oligodendrocytes within the PPWM while reduced histone H3 acetylation was found in remyelinating areas. One possibility is that these two patterns reflect two distinct stages of remyelination. Alternatively, they could represent intrinsic differences in the capability of oligodendrocytes to remyelinate. However, remyelination is a complex process that in addition to a potential modification by histone acetylation is also dependent on the extent of axonal damage (Frischer et al
Brain 2009), the presence of oligodendroglial progenitor cells (Wolswijk, 1998
; Kuhlmann et al., 2008
; Chang et al. 2002
) the composition of the inflammatory infiltrates (Arnett et al., 2003
; Bieber et al., 2003
; Kotter et al
2005), the anatomical localization of the lesions (Goldschmidt et al., 2009
; Patrikios et al., 2006
) as well as the presence or absence of cell signals which inhibit or promote remyelination (Mi et al., 2005
; Charles et al., 2000
; Mi et al., 2007
; Bachelin et al,2010
Future studies are required to dissect the functional relevance of histone modifications for myelin gene expression in healthy controls as well as in patients with MS and to analyze the functional significance of “reactivating” developmental pathways in the chronic MS brain.