We have previously shown that the generation of oligodendrocytes requires an increase in myelin gene expression and a decrease in the levels of transcriptional inhibitors that correlates with the acquisition an of “epigenetic memory”, consisting of histone deacetylation followed by histone methylation34
. To test the hypothesis that aging affects the molecular mechanism responsible for the maintenance of this “memory” in oligodendrocyte lineage cells, we assessed the occurrence of global repressive histone methylation in the nuclei of CC1+ oligodendrocytes in the largest white matter tract of the brain, the corpus callosum (). In the initial experiments, a total of 18 C57Bl/6J mice age 3 weeks, 8 weeks and 8 months and of either gender (n= 3 per gender and age) were sacrificed and the brains cryopreserved. Coronal brain sections were processed for immunohistochemistry, using antibodies against CC1 to identify oligodendrocytes and against dimethylated K9 on histone H3 to identify the secondary modifications that affect specific repressive lysine residues in nucleosomal histone H3 (). While more than half of the nuclei of CC1+ cells (59.5±5.5%) in 3 week young mice were characterized by intense nuclear staining for methylated K9 histone H3 (), only few CC1+ cells (22.77±1.57%) showed dimethylated histone H3 in the nuclei of 8 month old mice (). Besides the decreased percentage of CC1+ oligodendrocytes expressing methylated histone H3 in the older mice, also the expression levels of methylated histone H3 were significantly lower in 8 month old brain () compared to younger mice (1B). Thus, repressive global histone methylation decreases with age.
Age-dependent loss of repressive histone methylation in oligodendrocyte lineage cells
Since during developmental myelination the repressive methylation of lysine 9 in histone H3 is preceded by the removal of acetyl groups34
, we asked whether the age-dependent decrease of global histone methylation was associated with defective histone deacetylation and corresponding increased acetylation. For this reason, brain sections from C57Bl/6 mice of increasing age and of both genders were processed for immunohistochemistry using antibodies specific for the acetylated form of histone H3 (supplementary Fig. 1A-F
) or for acetylated lysine residues (supplementary Fig. 1G-L
). In agreement with the model proposed for developmental myelination, our results showed an inverse correlation between decreased lysine methylation on histone H3 and increased lysine acetylation. A minimum of three mice per gender was used in each age group and the number of acetylated nuclei was counted in white matter tracts analyzed at the corresponding anatomical levels in at least 3 sections per animal (supplementary Fig. 1M and 1N
). The results were then normalized by total number of DAPI+ nuclei in the same region. This quantification revealed a statistically significant increase (p <0.001) in the percentage of acetylated nuclei in the corpus callosum of old male C57Bl/6 mice (x= 67.67±3.01%, average ± SD of total DAPI+ cells) compared to young mice of the same gender (x= 24±2.65% average ± SD of total DAPI+ cells). A similar trend was observed in old females (x= 74.67±4.51% average ± SD of total DAPI+ cells) compared to younger ones (x= 23±2.65% average ± SD of total DAPI+ cells). To further confirm that the increased number of nuclei immunoreactive for acetyl lysine reflected changes occurring in the oligodendrocyte lineage cells, we further analyzed sections by double immunohistochemistry, using the CC1 antibody to identify differentiating and mature oligodendrocytes (). Although a small portion of Aclys+/CC1+ (double positive) cells were typically detected in young mice (x= 23.97±1.05% average ± SD of total CC1+cells), there was a remarkably greater proportion of Aclys+/CC1+ double positive cells in older mice (x= 73.33±2.08% average ± SD of total CC1+ cells (). From these data we conclude that aging oligodendrocytes undergo a progressive decline of the mechanisms associated with transcriptional repression (i.e. histone methylation and deacetylation) and a corresponding increase of histone modifications associated with active transcription (i.e. histone acetylation).
Acetylation is detected at low level in the nuclei of oligodendrocyte in the brain of young mice, but is a common finding in the brain of aged mice
The enzymatic activity responsible for the removal of acetyl group from lysine residues of nucleosomal histones is provided by a family of proteins called histone deacetylases (HDACs) that can be grouped into two subgroups, depending on their responsiveness to specific pharmacological inhibitors. Class I and II HDACs are specifically inhibited by trichostatin A (TSA), while class III activity is responsive to sirtinol. To begin understanding whether increased level of histone acetylation observed during the aging process was the consequence of decreased HDAC activity, we performed enzymatic assays on brain extracts from 8 week old (n=6) and 8 month old (n=6) C57Bl/6 mice of either gender, using a fluorimetric method (). This revealed a 32.7% decrease of total HDAC activity in old females compared to young ones (), and a 50% decrease in old males compared to young ones (). To gain insight regarding the subgroup of histone deacetylase affected during the aging process, we repeated the experiment by adding to the protein extracts increasing concentrations of the class I and II inhibitor TSA () or of the class III inhibitor sirtinol (). In agreement with the major role of class I and II HDACs in contributing to the total HDAC activity, we observed that TSA inhibited HDAC activity in whole cell lysates in a dose dependent fashion. We detected a 75% reduction of the enzymatic activity at concentrations of TSA below the IC50 of the compound (IC50 = 125−250nM) and more than 95% reduction at 500nM (). In contrast, the enzymatic activity of whole cell lysates from the corpora callosa of young and old mice was only partially decreased by concentrations of sirtinol () above the IC50 of this pharmacological inhibitor (IC50=40 uM). Since class II and class III HDACs are mainly cytosolic34,38
while increased histone acetylation was detected in the nuclei of aging oligodendrocytes, we conclude that the decreased HDAC activity detected in older mice was to be attributed to class I HDACs.
The age-dependent increase of histone acetylation correlates with the progressive decrease of HDAC activity
The decreased HDAC enzymatic activity observed in protein extracts from aged brains could be attributed to a functional block or decreased protein expression. To distinguish between these two possibilities we asked whether the expression of class I and class II HDACs in oligodendrocytes were affected by the aging process. For this purpose, coronal brain sections from aging C57Bl/6 mice were processed for immunohistochemistry using antibodies against specific HDAC isoforms of class I () and of class II (). Although we observed a generalized age-dependent decrease of HDAC expression (), only class I HDACs showed a nuclear localization (), while class II HDACs were either absent from oligodendrocytes or primarily cytosolic (). Since defective histone deacetylation was observed in the nuclei of CC1+ cells in older mice, we conclude that the decreased expression of class I HDACs is primarily responsible for the defective removal of acetyl groups from lysine residues on histone H3 observed in oligodendrocyte lineage cells in the aging brain.
Progressive decrease of class I HDAC expression in the medial corpus callosum of aged mice
Progressive decrease of class II HDAC expression, in the medial corpus callosum of aged mice
Together all these data indicate that the aging process is associated with the decreased ability of mature oligodendrocytes to retain repressive changes of nucleosomal histones that have been shown to affect the regulation of gene expression. To determine whether the age-dependent decline in histone methylation and deacetylation was also associated with aberrant gene expression, we performed a semi-quantitative RT-PCR of RNA samples isolated from white matter tracts of younger and older mice, using conditions that allowed the detection of transcripts within the linear range (). A prominent finding of our study was the age-dependent decrease of oligodendrocyte-specific transcription factors, including Sox10 and Olig2, but not Mash1, Sox11 and Olig1 (), in older mice compared to younger ones. This decrease in the expression levels of these transcriptional activators was paralleled by the detection of statistically higher levels of transcriptional inhibitors (i.e. Hes5, Id4 and Nkx2.2) and precursor markers (i.e. Sox2) in aging mice compared to younger controls (). Since the expression levels of these inhibitors during oligodendrocyte progenitor differentiation is dependent on histone deacetylase activity (Shen et al., 2005), we hypothesized that that aging would allow the re-expression of inhibitors in mature oligodendrocytes. To test this hypothesis we performed immunohistochemistry of white matter tracts of young and old mice with antibodies specific for the precursor markers (Sox2), a molecule that is epigenetically down-regulated during the differentiation of progenitors into mature oligodendrocytes (). In agreement with the prediction that loss of epigenetic memory results in the re-expression of progenitor molecules in mature cells, we detected greater number of Sox2+/CC1+ cells in aged mice, a finding that was rarely observed in young animals (). To further confirm that the increased levels of Hes5 and Id4 detected in older mice resulted from defective epigenetic memory, rather than accumulation of oligodendrocyte progenitors, we performed additional immunohistochemical experiments using antibodies specific for progenitor markers, such as PDGFR alpha () and NG2 (). Although fewer progenitors were detected in all the white matter tracts of older mice compared to the young ones, the decline in progenitor number was more evident in specific regions (i.e. the corpus callosum) than others (i.e. cerebellar peduncles) (). Together, these results suggested that the epigenetic memory loss detected in oligodendrocytes of older mice resulted in changes of gene expression, including the up-regulation of transcriptional inhibitors.
Increased expressions of transcriptional inhibitors in the aging corpus callosum
Aberrant expression of Sox2 in CC1 positive cells
To further test this hypothesis in an in vitro system, we used primary cultures of oligodendrocyte progenitors. When maintained in the presence of mitogens, such as PDGF and bFGF, these cells actively proliferate and are characterized by high levels of expression of transcriptional inhibitors of the bHLH family, including Hes5, Id2 and Id4 (). In addition, these cells express the transcriptional activator Sox10 and the homeodomain protein NKx2.2 (). Upon removal of mitogens from the medium, these cells promptly exit from the cell cycle and down-regulate the levels of Hes5, Id2 and Id4, while the levels of Sox10 and Nkx2.2 do not change significantly (). In the presence of the histone deacetylase inhibitor TSA, the cells are still able to exit from the cell cycle, but the levels of Hes5, Id2 and Id4 remain elevated and this translates into greater protein levels of these inhibitors (). Thus, treatment of differentiating oligodendrocytes with HDAC inhibitors mimics the results obtained from our studies of transcription factor expression in white matter tracts of aged mice.
The pattern of gene expression detected in the corpus callosum of older mice can be recapitulated in vitro by treating differentiating oligodendrocytes with pharmacological inhibitors of HDAC
Together our findings identify modification of chromatin components occurring in oligodendrocytes that indicate a progressive loss of the epigenetic memory. The resulting changes in gene expression detected in older oligodendrocytes compared to young ones may provide a mechanistic explanation for the changes of the functional properties of oligodendrocytes in the aged brain.