The concept that the adult neural stem or precursor cell population might be better harnessed to regenerate neural tissue after neurological disease is the subject of increasing interest and attention within the neurosciences34
. After pathological processes characterized by myelin damage, there can be a highly effective regenerative process, in which a population of adult neural stem or progenitor cells, OPCs, is rapidly activated andmobilized to generate newmyelinating oligodendrocytes3
Although recent studies indicate the presence of extensive remyelination in the CNS of subjects with multiple sclerosis, they also illustrate that remyelination is not an inevitable consequence of demyelination and that axons can persist in a chronically demyelinated state, in which they are vulnerable to atrophy6,7
. Thus, promoting remyelination remains an important therapeutic objective. As a proportion of non-remyelinating lesions in multiple sclerosis contain OPCs that fail to differentiate into myelinating oligodendrocytes, an understanding of the mechanisms which govern OPC differentiation will be crucial to this endeavor. An explanation that has attracted some attention is that such lesions contain remyelination inhibitors such as PSA-NCAM35
and Notch ligands37
, although the significance of the last is unclear38
. An alternative explanation is that the process fails because of defects in inducers of differentiation, both environmental and cell intrinsic. An important clue to understanding failed repair in multiple sclerosis lesions is provided by the observation that remyelination, like all other regenerative processes, declines in efficiency with aging. Age-associated changes in the environmental signals associated with remyelination have been described in experimental models. However, although it is clear that cell-intrinsic changes occur in OPC biology with aging39
, the intrinsic mechanisms regulating OPC differentiation during remyelination remained unexplored.
In this manuscript, we define the age-dependent differences in intrinsic mechanisms of remyelination efficiency by focusing on the epigenome, a critical component at the interface between environmental signals and coordinated transcriptional programs regulating oligodendrocyte differentiation and myelin gene expression. The epigenome defines the entirety of the components affecting gene expression, including nucleosomal histones, DNA methylation and ATP remodeling complexes. The importance of epigenetics during differentiation has been shown in many lineages. In the oligodendroglial lineages, the importance of histone deacetylation for OPC differentiation has been supported by a large series of experiments in vitro40,41
and in vivo24,25,27,42
. Pharmacological inhibitors of HDAC have also been implicated in preventing the downregulation of neural stem cells markers in cultured OPCs26
. The results of this study identify the epigenetic modulation of transcriptional inhibitors as a critical determinant of remyelination efficiency in young and old mice.
Using the cuprizone model of demyelination, we have shown here that spontaneous remyelination is characterized by the execution of a coordinated and integrated transcriptional response defined by the downregulation of the stem cell marker Sox2
and of the oligodendrocyte differentiation inhibitors Hes5, Hes1, Id2
before the upregulation of the transcription factor Olig1 and subsequent increase in myelin gene expression. Hes5 is highly expressed in proliferating OPCs but gradually decreases as the cells mature into oligodendrocytes29
. Several direct and indirect mechanisms have been characterized to explain the role of Hes5 as inhibitor of myelin gene expression43
. It has also been shown that Hes5 and its upstream Notch receptor are expressed in immature oligodendrocytes around active multiple sclerosis plaques but not within the remyelinated lesions37
. However, genetic manipulation of Notch signaling pathway does not affect remyelination efficiency38
, indicating either that Notch signaling is critical but not rate limiting during the remyelination process or that the remyelination process may be affected by multiple pathways and other transcription factors. A potential candidate gene for affecting repair is Sox2
, a transcription factor involved in stem cell pluripotency26
. We show here that Sox2
is also regulated by age-dependent mechanisms of histone acetylation and HDAC1 and HDAC2 recruitment to the promoter region during the remyelination process. Although the role of Sox2 as inhibitor of myelin gene expression has not been characterized, it is tempting to speculate that its function in the oligodendrocyte lineage might be equivalent to the one described in the peripheral nervous system, where it acts as inhibitor of Schwann cell differentiation and myelination44
The coordinated decrease of multiple inhibitory transcripts is detected only in young mice, not in old, and is characterized by the recruitment of several HDAC isoforms to the promoter with consequent deacetylation of lysine residues in histone H3 and release of RNA Pol II. With aging, this intrinsic ability of the cells to recruit HDAC to the promoter of transcriptional inhibitors progressively decreases. As a consequence, the intracellular environment of aged OPC is poised against differentiation because of the persistence of high levels of transcriptional inhibitors.
The identification of several nuclear HDAC isoforms at the promoter region of Hes5
suggested that multiple HDAC isoforms are involved in repressing differentiation inhibitors and ensure the initiation of the repair process. To define the role of specific HDAC isoforms, we pursued a silencing approach and individually targeted the distinct isoforms of class I HDAC, which we previously showed to be the prominent nuclear HDACs in oligodendrocyte-lineage cells27
. It is noteworthy that silencing of HDAC1 or HDAC2, but not HDAC3 or HDAC8, impaired the timely differentiation of oligodendrocyte progenitor. Cells with decreased HDAC1 or HDAC2 retained the morphological and antigenic characteristics of early progenitors and showed high Sox2 levels. Thus, we conclude that the levels of HDAC1 and HDAC2 isoforms are critical for the execution of a timely program of oligodendrocyte differentiation. We also addressed the role of HDAC activity in repressing differentiation inhibitors, using a broad-spectrum pharmacological approach in vitro
and in vivo
. The effect of HDAC inhibitors (HDACi) in cultured cells was notably cell specific, as it increased the levels of Sox2
in primary cultures of OPCs but not astrocytes or microglia. This effect was accompanied by a significant reduction of myelin gene expression in treated OPCs, whereas we observed no reduction in microglial or astrocytic genes. Inhibition of HDAC activity in vivo
significantly hampered remyelination in the brains of young mice, resulting in substantially fewer myelinated fibers and higher levels of inhibitors, thereby recreating an environment that mimicked the inefficient repression detected in the old brains. Together, these data define the epigenetic control of transcription as a critical determinant of remyelination efficiency in young mice and of remyelination inefficiency in old mice.
The use of HDACi for therapeutic purposes is the subject of an intensive debate. HDACi are at present used as anti-cancer agents because the are able to increase genes involved in growth arrest and promote apoptosis of cancer cells45
. The possibility of using HDACi in demyelinating disorders is more controversial. Some studies have reported a beneficial effect of compounds increasing histone acetylation in mice with experimental allergic encephalomyelitis46
, whereas others have reported a beneficial effect of compounds increasing histone deacetylation in the same inflammatory model of demyelination47
. The clinical improvement in mice treated with compounds increasing histone acetylation was ascribed to increased expression of protective genes46
and possibly to the apoptosis of inflammatory cells48
. The protective effect of increased histone deacetylation, in contrast, was proposed to be due to decreased expression of proinflammatory genes in T cells46
Our study contributes to the debate on the therapeutic potential of HDACi by analyzing a model of demyelination in the absence of the adaptive immune response and suggests that inhibition of HDAC might bear negative consequences for the repair process regardless of its effect on the immune cells. Using the cuprizone model of demyelination consequent to oligodendrogliopathy, we show here that retaining high levels of acetylated histones during remyelination significantly impairs the repair process.
In conclusion, this study defines the age-dependent decline in spontaneous remyelination as the result of the progressive loss of the epigenetic events modulating a coordinated program of gene expression. In addition, it defines the limitations of a pharmacological approach using broad inhibitors and proposes the identification of more specific molecular targets that require further exploration for the possible development of cell-specific therapies aimed at ageappropriate brain repair.