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The epigenetic identity of oligodendrocytes is modulated by post-translational modifications of histones. Acetylation of histone H3 results from the balance between the activity of histone-acetyltransferases (HATs) and histone deacetylases (HDACs) and modulates transcriptional activation. We have previously shown that in rodents histone deacetylation favors oligodendrocyte differentiation, while acetylation is associated with increased levels of transcriptional inhibitors of oligodendrocyte differentiation. Here we report in humans brains, a shift towards histone acetylation in the white matter of the frontal lobes of aged subjects and in patients with chronic multiple sclerosis (MS). Increased immunoreactivity for acetylated histone H3 was observed in the nuclei of NogoA+ oligodendrocytes in a subset of MS samples. These changes were associated with high levels of transcriptional inhibitors of oligodendrocyte differentiation (i.e. TCF7L2, ID2 and SOX2) and higher HAT transcript levels (i.e. CBP, P300) in female MS patients compared to non-neurological controls and correlated with disease duration. Chromatin immunoprecipitation from samples of MS patients revealed enrichment of acetyl-histone H3 at the promoter of the increased target genes (i.e. TCF7L2). The data in chronic lesions contrasted with findings in early MS lesions, where a marked oligodendroglial histone deacetylation was observed. Together these data suggest that histone deacetylation is a process that occurs at the early stages of the disease and whose efficiency decreases with disease duration.
It is well established that the degree of compaction of chromatin and the transcriptional state of genes are associated with distinctive post-translational modifications occurring on precise amino acid residues that occur mainly at the N-terminus of the histones (Liu and Casaccia, 2010). These modifications include acetylation, methylation, sumoylation and ubiquitination of lysine residues; methylation and citrullination of arginine residues; phosphorylation of serines and threonines; ADP- ribosylation and isomerization of prolines (Kouzarides, 2007). One of the best characterized histone modifications that modulate oligodendrocyte differentiation is the equilibrium between histone acetylation and deacetylation (Copray et al., 2009). In young animals, we and others have shown that pharmacological inhibitors of histone deacetylases (HDACs) increase histone acetylation and prevent differentiation of oligodendrocyte progenitors in vitro (Hsieh et al., 2004, Marin-Husstege et al., 2002) and in vivo (Liu et al., 2007, Shen et al., 2005). These data were supported by gene silencing experiments in primary cultures of oligodendrocyte progenitors (Shen et al, 2008b), the detection of defective oligodendrogliogenesis in hdac1 zebrafish mutants (Cunliffe and Casaccia-Bonnefil, 2006) and in mice with conditional ablation of Hdac1 and Hdac2 (Ye et al., 2009) or of the HDAC1 binding molecule Yy1 (He et al., 2007) in the oligodendrocyte lineage.
In aged rodent brains the equilibrium was shifted towards histone acetylation (Shen et al., 2008a) and this correlated with heterotopic and heterochronic expression of genes characteristic of the myelinating phenotype (Shen et al., 2008a and Shen et al., 2008b). Intriguingly, several of the genes down-regulated by HDAC activity encoded for transcription factors downstream of the BMP (i.e. Id2 and Id4), Notch (i.e. Hes5) and Wnt (i.e. Tcf7l2/Tcf4) signaling pathways (He et al., 2007, Fancy et al., 2009, Li et al., 2009, Samanta and Kessler, 2004, Wang et al., 1998). Previous studies have shown that these pathways are down-regulated during developmental myelination and re-expressed in progenitor cells after damage to myelin (John et al 2002; Sim et al., 2006; Fancy et al 2009; Shen et al 2008b) and also in mature cells in the aged rodent brain (Shen et al., 2008a).
Increased histone acetylation has also been described in other organs and related to the presence of inflammatory conditions. In human lung, for instance, the pattern of histone acetylation detected in old subjects, resembled that observed in chronic obstructive pulmonary disease and led to the hypothesis that inflammation could mimic a condition of “premature” aging within different tissues (Ito and Barnes, 2009). Based on these premises, we aimed to test the hypothesis that changes of histone acetylation also occur in patients with multiple sclerosis (MS), an inflammatory disease of the central nervous system, and that these changes are associated with widespread aberrant modulation of gene expression.
We studied a total of 47 MS patients and 69 controls (Table 1 and Table 2). ChIP and qPCR were performed on frozen autoptic tissue specimens from the NAWM samples of the frontal lobes of 23 MS patients with secondary progressive or chronic progressive type of the disease (12 females, 11 males; average age=62.3 +/- 7 years old for males and 61.4 +/- 9.2 years old for females) and 19 non-neurological controls (7 females, 12 males; average age=66.3 +/- 10.3 years old for males and 75.5 +/- 3.8 years old for females). These samples were obtained from the Human Brain and Spinal Fluid Resource Center (VA West Los Angeles Healthcare Center 11301 Wilshire Boulevard, Los Angeles, CA 90073) which is sponsored by NINDS/NIMH, National Multiple Sclerosis Society and Department of Veterans Affairs also from the German Brain Net. Quantitative ChIP for acetyl-H3 was conducted in a subset of 4 MS patients (average age=69.5 +/- 7.8 years old for males and 67.5 +/- 9.2 years old for females) and 4 controls (average age=73 +/- 2.8 years old for males and 76.8 +/- 4.2 years old for females). Samples containing actively demyelinating or chronic lesions were not included in this study. RNA and chromatin quality was checked on agarose gel and only samples that did not show signs of degradation were pursued for further analysis.
For immunohistochemistry, paraffin-embedded frontal tissue samples were obtained from 49 patients who died due to non-neurological diseases, age 21-30 years old (5 females, 4 males), 40-70 years old (10 females, 11 males; mean age 59.5 years old) and 71- 80 years old (11 females, 8 males). Additionally, we included 8 autopsy samples derived from frontal lobes of six patients with MS (3 females, 3 males, mean age 59.5 years old, disease duration between 9 and 41 years) and 19 biopsy samples (6 males, 13 females, the disease duration; only known in 5 patients was between 19 and 92 days). Biopsies were performed for diagnostic reasons to exclude neoplastic or infectious disease. Demyelinating activity in biopsy tissue samples was determined as described earlier (Brück et al 1995). Actively demyelinating lesion areas (n=6) were frequently located at the plaque border, partially demyelinated and infiltrated by T cells and numerous macrophages containing myelin degradation products within the cytoplasm. In demyelinated lesion areas (n=6) no myelin sheaths could be detected by either Luxol-fast blue (LFB) staining or immunohistochemistry for different myelin proteins (e.g. MBP, PLP, CNP, MOG, MAG). These lesion areas were infiltrated by numerous macrophages; however they did not contain myelin degradation products. Remyelinating lesion areas (n=12) were characterized by thin, irregularly formed myelin sheaths; the adjacent peri-plaque white matter (PPWM) (n=10) showed no signs of demyelination; however numerous reactive microglial cells and few T cells were present. Paraffin embedded MS autopsy tissues blocks were from the Institute of Neurology, McGill University, Montreal, Canada; while MS biopsies and the non-diseased brain tissue samples were derived from the Department of Neuropathology, University Medical Center Göttingen, Germany. The study was approved by the Ethics Committee of the University of Göttingen.
Tissue samples were cut in 4μm thick sections that were stained with haematoxylin and eosin (HE) (Non-MS tissue samples) or LFB (MS tissue samples). Immunohistochemical staining was performed with an avidin-biotin technique. After de-paraffinization intrinsic peroxidase activity was blocked by incubation with 0.5% H2O2 in PBS for 20 minutes. Non-specific antibody binding was inhibited with 10% FCS in PBS for 25 minutes. Microwave pre-treatment for better antigen retrieval was performed prior to incubation with rabbit polyclonal anti-acetyl-H3 antibody (1:300) (cat. #06599 Abcam, Cambridge, MA). As secondary antibody anti-rabbit biotinylated Ig (1:400) (Amersham Biosciences, Freiburg, Germany) was used. For double staining we combined anti-acetyl-H3 immunohistochemistry with mouse anti-NogoA immunohistochemistry (1:15,000) (11c7, generous gift from M.E. Schwab, Brain Research Institute, University of Zürich and Department of Biology, Swiss Federal Institute of Technology Zürich, Switzerland), mouse monoclonal anti-GFAP (1: 50) (Dako) or mouse monoclonal anti-KiM1P (1: 5000) (a generous gift by H.-J. Radzun, Department of Pathology, University of Göttingen, Germany. Secondary antibodies were either biotinylated or conjugated to alkaline phosphatase. The number of acetyl-H3-positive cells was determined in at least 10 standardized microscopic fields of 10 000 μm2 each defined by an ocular morphometric grid and expressed as the percentage of total number of nuclei or as percentage of NogoA positive mature oligodendrocytes. In the text and figures, the mean number of cells/mm2 ± SEM is given.
For RNA isolation, 50mg of tissue was homogenized with 250μL Trizol using mortar and pestle and adjusting the volume to 1 ml. The mixture was transferred into phase-Lock gel heavy 2ml tubes (5 Prime # 2302830, Hamburg, Germany) and centrifuged at 14000 rpm for 15min at 4°C. The aqueous phase containing the RNA was cleaned with 200μL chloroform and the RNA was precipitated and washed with ethanol. The RNA samples were resuspended in water and further purified with RNeasy (Qiagen #74104, Valencia, CA) following the manufacturer's instructions. The quality of RNA was verified by agarose gel electrophoresis and measurement of the A260/A280 ratio and only those samples showing clear ribosomal RNA bands and A260/A280 ratio around 2.0 were further processed for quantitative PCR analysis. For real time PCR, 2 μg of total RNA were reverse transcribed using Super Script RTM First-Strand Synthesis System (Invitrogen #11904018) and oligo-dT as primers for PCR. Quantitative real time PCR was performed using Applied Biosystem SYBR green master mix in a 7900HT Fast RT PCR System (Applied Biosystem, Foster City, CA). The melting curve of each sample was measured to ensure that a single amplicon was obtained. The data were normalized using ribosomal protein 60S as internal control, and analyzed by the Pfaffl ΔΔCt method (Pfaffl, 2001), using the equation ratio= E(target)ΔCt(target) (control-sample)/E(60S)ΔCt (60S)(control-sample), where the control is determined by the average of the normal samples from the same gender as the patients and the efficiency was determined using a standard curve for each primer pair and the simplified Pfaffl equation for the efficiency =10-1/slope. Primers used for qPCR are listed in Table 3.
For chromatin isolation 400mg of frozen brain samples were pulverized in dry ice and cross-linked with 1% formaldehyde for 7min. After stopping the reaction by adding glycine to a final concentration of 125mM, cells were lysed and the isolated nuclear fraction was lysed and sonicated using a Bioruptor sonicator (Bioruptor, Diagenode). Typically 4-10 cycles of sonication of 5 minutes each (15s ON/45s OFF) at 4 degrees were used to reach chromatin fragments of around 500bp in size. Aliquots of isolated cross-linked chromatin (corresponding to ~15μg DNA or 0.6Units/Ab 1Unit=OD at 260nm) were immunoprecipitated using 2μg of antibody (anti-acetyl-H3, Upstate #06-599) purified with protein A-Dynabeads (Invitrogen, Carlsbad, CA) and then reverse cross-linked by incubating at 65°C in a water bath in the presence of RNase A (final concentration 40μl/ml). The proteins of the immunocomplexes were digested with proteinase K (100μg/ml final concentration), the recovered DNA was isolated by phenol-chloroform extraction followed by QIAquick PCR purification kit (Qiagen) and analyzed by quantitative PCR amplification as previously described (Shen et al., 2008b). Primers used for ChIP are listed below:
|Primers used for ChIP|
|Gene||Sequence sense||Sequence antisense||Gene accession Nr.|
|TCF7L2 Prom (-10/+125)||TCGCCCTGTCAATAATCTCC||CGCTCGGATTTGAGTGAGTT||NM_030756|
DNA recovered from chromatin that was not immunoprecipitated (input) and chromatin that underwent the same procedures in the absence of antibodies, were both used as controls. The calculations were done using the standard curve method. The standard curve was performed using 0.5Units/μl of input chromatin diluted 4 fold with each dilution. The signal measured in the immunoprecipitated samples was referred to a standard curve of the signal from the input and thereby as percentage of input.
To obtain the ratio of acetyl-H3 / H3, nuclear fractions containing 5 μg protein diluted to 100 ng /mL protein were loaded in triplicate onto wetted nitrocellulose membranes in a Biodot (slot format) ultrafiltration unit (Biorad, Hercules, CA) under vacuum, as described in Mastronardi et al., 2006. The ratio of acetyl-H3/ H3 was calculated from immunoslot blots following detection of acetyl-H3 and H3. Samples were loaded onto slots in triplicate.
Results are expressed as mean ± standard deviation (S.D.). A t-test with Welch's correction was performed to evaluate significance in the overall comparison of groups. The Spearman's test was used to analyze the correlation of the transcript levels of different genes among each other and against disease duration. A linear regression was used to test whether slopes and intercepts included in the correlation were significantly different. Differences between individual groups were determined by the Scheffé test or the Mann Whitney U-test. In all cases differences were considered to be significant when *=p < 0.05; **=p<0.01.
The premise of the current study was to characterize histone acetylation and deacetylation in the human aged brain, because our previous studies in old rodent brains had correlated these changes to myelin repair (Shen et al., 2008b). We focused our analysis on the percentage of acetyl-H3 positive nuclei over total cells in the frontal normal appearing white matter (NAWM) samples of 28 patients who died to non-neurological diseases aged 21 – 30 years old (n=9) or 71 – 80 years old (n=19) (Table 2). Analysis of the cellular composition of these samples revealed majority of oligodendroglial lineage cells. Astrocytes, identified by GFAP immunoreactivity, accounted for 1.2% to 5.3% of the cells while microglial cells, identified using the marker KiM1P ranged between 2.5% and 6.5% of the cells in white matter samples. In average, 90% of the astrocytes and 33.2% of the microglial cells expressed acetyl-H3 as demonstrated by double stainings combing acetyl-H3 with astrocytic or microglial markers. Since staining of the autopsy tissue samples with oligodendroglial markers, such as Olig2 or NogoA resulted in insufficient staining quality (most likely due to the sensitivity of these markers to prolonged fixation times), we quantified the percentage of oligodendrocytes in white matter in biopsy samples from patients (n=5) with pharmaco-resistant seizures without any obvious pathology (except for mild gliosis). NogoA immunoreactivity accounted for 92.8% positive nuclei and confirmed that the vast majority of cells in the white matter were oligodendrocytes. A comparison of the cellular composition in young and old NAWM did not reveal any change in the percentage of astrocytes with age, although slightly higher numbers of microglial cells were detected in NAWM from young women and men (5.8 and 5.7% of white matter cells) compared to aged men (3.0%). Double immunoreactivity for NogoA and acetyl-H3 in tissue biopsies suggested that most of the acetyl-H3 positive cells in NAWM are oligodendrocytes (Fig 1B). In control tissue samples the lowest percentage of acetyl-H3 positive nuclei was observed in young females (79 +/- 4.2%) and the highest percentage in older males (93 +/- 1.7%) and females (92 +/- 1.4%) (Fig. 1C).
A similar immunohistochemical analysis was then conducted with tissue samples from the NAWM of patients with chronic MS. We examined eight tissue blocks with NAWM of the frontal lobes from six MS patients (3 females, 3 males, mean age 59.5 years). As expected and described earlier (Kutzelnigg et al. 2005) all tissue samples showed a mild to moderate activation of microglial cells, astrogliosis and minimal acute axonal damage (Table 4). On average, 7.9% of the cells were KiM1P-positive microglial cells and 7.2% were GFAP-positive astrocytes in MS tissue, whereas in controls 4.7% of the cells were microglia and 3.2% astrocytes. Immunoreactivity for acetyl-H3 was detected in 92.9% +/- 1.4% of the nuclei in the NAWM of patients with chronic MS, thereby suggesting that the vast majority of oligodendroglial lineage cells were immunoreactive for acetyl-H3. Tissue samples from non-neurological patients (mean age 59.5 years, 11 males, and 10 females) had a slight, but significantly lower percentage of acetyl-H3-positive nuclei (85.0% +/- 2.4%) (Fig 1 D). To further quantify potential differences in the levels of histone acetylation between MS and control groups, we isolated proteins from frozen brain homogenates from 23 patients with chronic MS and 20 non-neurological controls. The relative levels of acetylation of histone H3 were quantified by slot-blot analysis and normalized by dividing the signal intensity of acetyl-H3 by the total levels of histone H3. Similar to our immunohistochemical findings, the relative levels of histone H3 acetylation was significantly higher in MS patients compared to controls (Fig. 1E). Importantly, a subgroup of patients showed two to five times higher levels of acetyl-H3 than controls. This marked increase could not be explained by the relatively mild increase in the numbers of astrocytes and microglial cells in the NAWM of MS patients and therefore could be considered as an indirect measure of the global acetyl-H3 levels in cells.
Previous studies have reported the importance of histone acetylation in modulating the levels of downstream effectors of the Notch, BMP4 and Wnt signaling pathway and negatively impact oligodendrocyte differentiation (Li et al., 2009). For this reason we obtained RNA from the NAWM of the frontal lobes of 21 chronic MS patients and 18 age and sex-matched non-neurological controls (Table 1), and analyzed the transcript levels of downstream signaling components of the BMP4 (i.e. ID2) and Wnt (i.e. TCF7L2) pathway. Additional transcript levels were assessed for myelin genes (i.e. MAL; MOG) and other transcription regulators (i.e. SOX2). Tubulin-alpha (i.e. TUBA) was used as a control (Fig. 2). Despite the inter-individual variability characteristic of post-mortem human material, we noticed that 8/10 female and 0/6 male patients consistently showed statistically significant higher levels of TCF7L2 transcripts compared to sex-matched controls (Fig. 2). The subgroup of female MS patients with increased levels of TCF7L2 also showed higher levels of SOX2 and ID2 transcripts compared to controls. The age of these female patients ranged between 50 and 60 years and the disease duration was longer than 20 years. To begin understanding the potential implications of increased levels of TCF7L2 in female patients, we assessed the existence of a correlation between the levels of TCF7L2 and those of the myelin gene MOG (Fig. 3A). Also in this case, two subgroups of patients could be identified within the female MS population: one with a positive, although statistically not significant correlation between TCF7L2 and MOG, the other one with a negative correlation (Fig. 3A). None of these correlations were detected in male patients (Fig. 3A). The subgroup of female patients with higher transcript levels for TCF7L2 was also the one showing a statistically significant positive correlation with disease duration (Fig. 3B). Together, these data suggest the existence of gender-related mechanisms that impact the functional output of increased histone acetylation and modulate the transcriptional network, so that only female MS patients with a long history of the disease show increased levels of downstream signaling effectors such as Wnt and BMP4 pathways.
We and others have previously shown that the expression of Tcf7l2, Sox2 and Ids in rodent oligodendrocyte progenitors is modulated by histone acetylation, which depends on the equilibrium between HDACs and HATs (Shen et al., 2008b; Ye et al., 2009). Therefore, we asked whether the transcript levels for these two major classes of enzymes were altered in the NAWM of chronic MS patients (Fig. 4). Of the several HDAC family members we opted to focus on members of class I, due to their predominant nuclear localization in oligodendrocyte lineage cells (Shen et al., 2005) and on Hdac11, due to its enrichment in oligodendrocytes (Liu et al., 2009). Despite the increased levels of histone acetyl-H3 in the oligodendrocytes of the NAWM of MS patients, we did not detect a significant decrease in the transcript levels of HDAC1 and HDAC2, two isoforms previously linked to successful oligodendrogliogenesis (Ye et al., 2009; Shen et al., 2008b). However, we observed a tendency towards increased HDAC8 (in males) and HDAC11 (in females) transcript levels in the NAWM of the frontal lobes of MS patients compared to controls (Fig. 4).
Because acetylation results from the equilibrium between HATs and HDACs, we also measured the transcript levels of several HAT isoforms in the NAWM of female and male MS patients. We detected statistically significant higher levels of CBP and P300, in white matter samples from the same subgroup of female MS patients that also showed increased TCF7L2 compared to controls (Fig. 5A-B). Interestingly, the levels of these HATs showed a positive correlation with TCF7L2 levels only in female, but not in male MS patients (Fig. 5C). Thus gender plays an important role in modulating the transcriptional output of increased histone acetylation.
To prove further that acetylation of histones was directly related to the increased transcript levels of transcriptional inhibitors, we performed chromatin immunoprecipitation (ChIP). Briefly chromatin samples were isolated from pulverized NAWM of the frontal lobes of frozen samples from non-neurological and chronic MS patient brains. Samples were cross-linked and then immunoprecipitated with antibodies that bind to acetyl-H3. Following digestion of the chromatin associated proteins, the DNA was used as a template for quantitative PCR with primers specific for the region surrounding the start sites of the genes encoding TCF7L2. TUBA, a constitutively expressed cytoskeletal component, was used as control (Fig. 6). The amount of DNA immunoprecipitated with acetyl-H3 antibody was expressed as percentage of input control. The levels of acetylation of histone H3 in regions corresponding to the TCF7L2 gene were statistically significantly (p=0.0034) higher in MS patient (x=7.9089 +/- 3.964945, n=9) compared to the levels detected in non-neurological controls (x=2.080 +/- 2.522142, n=7). No significant changes were detected in constitutively regulated genes, such as TUBA.
The higher levels of histone acetylation at the TCF7L2 locus were more prominent in samples from female MS patients than corresponding samples from male patients (Fig.6B). Thus, additional factors in male MS patients (i.e. hormone receptors or hormone receptor effect on chromatin or microRNAs, as discussed) might dampen the effect of histone acetylation on the activation of the TCF7L2 promoter.
Molecular studies in rodent models had suggested that histone acetylation is associated with the maintenance of an undifferentiated state of oligodendrocyte progenitor cells and that differentiation is associated with decreased levels of histone acetylation. Our studies in chronic MS samples suggested that deacetylation is impaired with disease duration, but did not reveal any information regarding the initial stages of the disease. To further analyze the acetylation/deacetylation of histone H3 in oligodendrocytes in early MS lesions, we determined the percentage of acetyl-H3-positive oligodendrocytes in newly developed MS lesions with ongoing de- and/or remyelination. Whereas in the periplaque white matter the vast majority of NogoA-positive oligodendrocytes expressed acetyl-H3 (83% +/-5%) this percentage was significantly decreased in actively demyelinating, demyelinated and remyelinating lesion areas (between 47 +/- 4% and 50 +/- 11%) (Fig 7A - C). In seven biopsies remyelinating lesion areas and adjacent peri-plaque white matter were available for quantitative analysis; whereas four biopsies showed a markedly reduced percentage of acetyl-H3 and NogoA-positive oligodendrocytes in remyelinating areas, the other three biopsies revealed a similar fraction of acetyl-H3 expressing mature oligodendrocytes compared to the peri-plaque white matter. These data suggest that deacetylation of histone H3 is a process that can be detected in early MS lesions and that it is less efficient in patients with long disease duration.
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 and CBP) 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 transcript levels.
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.
This work was supported by grants NS042925-08, ARRA-NS042925-08S1 and NMSS RG-4134 to P. Casaccia; grants CSD2006-00049 and PR2008-0243 Ministerio de Ciencia y Tecnología to G. López-Rodas, and grant DFG 120-377 to T. Kuhlmann. The authors wish to thank Dr. Jack Antel (Neuroimmunology Unit, Montreal Neurological Institute, McGill Montreal), the Brain and Spinal Fluid Resource Center, VA West Los Angeles Healthcare Center 11301 Wilshire Blv. Los Angeles, CA 90073 and the Brain-Net Germany supported by the Federal Ministry of Health (01GI0505) for providing MS tissue and control samples.