Epigenetic regulation of adult neural stem cells: implications for Alzheimer’s disease

DNA methylation and adult NSCs

Recent observations have suggested that epigenetic mechanisms could be sensors of environmental changes and fine modulators of adult hippocampal neurogenesis [60]. Enrichment of the environment, a well-known stimulus of hippocampal neurogenesis, to which exercise contributes the most, could promote neuronal maturation, possibly through increased methylation activity [59, 60]. In addition, alterations in neurogenesis associated with pathological conditions of the brain have been linked to changes in DNA methylation in the brain [60]. The possible mechanisms by which DNA methylation could influence different stages of adult neural stem cells in both the SGZ and the SVZ will be discussed.

In vitro analysis of DNMT function in differentiating NSCs has proven to be a relevant experimental approach to study the role of DNMTs and DNA methylation in neurogenesis [61]. Neurosphere formation and inhibition of differentiation of cultured quiescent NSCs was maintained by application of epidermal growth factor (EGF) and fibroblast growth factor (FGF). Withdrawal from EGF/FGF supplementation induced their differentiation and subsequent immunostaining confirmed DNMT1 and DNMT3a expression and presence of DNA methylation in undifferentiated NSCs. At the start of differentiation, DNMT1 and DNMT3a were increased but subsequently decreased upon migration and their late differentiation. Thus, while high expression was observed in undifferentiated cells, DNMT1 and DNMT3a expression decreases in the differentiating/migrating NSCs. Importantly, Chromatin immunoprecipitation (ChIP) analysis showed that both increases and decreases in methylation occur in differentiating NSCs at different loci [61]. This possibly reflects a combined repression of stem cell maintenance genes and an activation of cell differentiation genes. Nonetheless, a role for DNA methylation in NSC differentiation and migration is further supported by data showing that administration of the methylation blocker 5-azacytidine (AZA) decreases NSC differentiation and migration [61].

The role of DNMT3a in neuronal differentiation has been further confirmed in the mouse postnatal brain. Wu and colleagues [62] observed expression of DNMT3a in both the SVZ and SGZ in the postnatal mouse, while a more detailed immunohistochemical study found two distinct types of DNMT3a-immunoreactive cells in the SGZ. The first type of immunoreactive cells (those with relatively low immunoreactivity) is ubiquitously expressed throughout the hippocampus, while the second type (displaying high levels of immunoreactivity) was particularly found in the neurogenic region of the SGZ [63]. Immunohistochemical analyses 3 weeks after 5-bromo-2′-deoxyuridine (BrdU) administration showed that the high expressing DNMT3a cells in the SGZ were newborn and expressed the mature neuron marker NeuN. In agreement with this observation, knockout of DNMT3a in vivo results in a profound decrease in postnatal neurogenesis in both the SVZ and SGZ [63]. Culturing NSCs from DNMT3a knockout mice confirmed that DNMT3a is necessary for neuronal differentiation. A 10-fold decrease in newborn neurons upon differentiation induction was observed in DNMT3 knockout NSCs, again indicative of impaired neurogenesis [62]. ChIP analysis revealed that the DNMT3a targets were enriched among the differentially expressed genes in NSCs obtained from DNMT3 knockout mice. Moreover, the down-regulated genes in DNMT3a knockout mice were neurogenic genes while the up-regulated genes were genes involved in astroglial and oligodendroglial differentiation [62]. Thus, DNMT3a seems to act in NSCs as a switch that regulates gene expression towards the non-neuronal lineage when downregulated, and towards a neurogenic fate when upregulated.

Indirect regulation of gene expression by DNMTs is mediated through proteins with methyl-CpG-binding domains (MBDs). MBDs bind to methylated gene promoters, thereby inhibiting gene expression through blockage of transcription factor binding or recruitment of other enzymes that induce transcriptional repression [51]. Similar to the DNMT expression changes described in the previous paragraph, expression of MBD1 correlates with neuronal differentiation [61]. Accordingly, low MBD1 expression was found in undifferentiated neurospheres. Although a moderate increase in expression levels was observed at the induction of differentiation, subsequent down-regulation was seen upon the start of the migratory phase. This suggests that MBD1 target genes are highly expressed in the self-renewing NSCs due to low levels of MBD1 expression. Then, increased MBD1 expression leads to repression of these genes, allowing cell differentiation [61]. Since MBD1 expression is predominantly found in neurons of the adult brain, MBD1 seems to have a specific role in inducing or maintaining neuronal differentiation. Indeed, MBD1-deficient mice have reduced neurogenesis in the postnatal but not embryonic brain [64]. BrdU analysis showed that although there were no differences at day 1, the amount of BrdU labeled cells in the MBD1-deficient mice was significantly decreased 4 weeks after the BrdU injection. This was accompanied by impaired neurogenesis and lower cell density in the DG of the hippocampus. Subsequent phenotypical analysis of the surviving newborn (BrdU labeled) cells revealed that in addition to the overall decrease in BrdU-labeled cells, newborn neurons were significantly more affected than other, more immature, phenotypes. Additionally, the percentage of newborn astrocytes was increased [64]. Thus, MBD1 may be important for neuronal differentiation of NSCs and survival of newborn neurons in the postnatal brain.

The role of MBD1 in adult neurogenesis and NSC differentiation was confirmed by Li and colleagues [65] who provided additional information on the molecular mechanism involved. NSCs isolated from adult MBD1 knockout mice showed increased fibroblast growth factor 2 (FGF2) expression. Moreover, overexpression of MBD1 in both MBD1 knockout and wild type NSCs decreased FGF2 expression. In vitro ChIP analysis confirmed the specific binding of MBD1 to the FGF2 promoter while hypomethylation of the FGF2 promoter in MBD1 knockout mice was observed [66]. Importantly, all the events that either led to a decrease in MBD1 expression or an increase in FGF2 expression resulted in reduced neuronal differentiation [65]. This suggests that neuronal differentiation in the postnatal and adult brain is dependent on methylation of, and MBD1 binding to, the FGF2 promoter, which results in its repression.

A second member of the MBD protein family, methyl-CpG-binding protein 2 (MeCP2) regulates gene expression through a similar mechanism as MBD1. It binds to methylated DNA and functions as a transcriptional repressor. Although MeCP2 expression is predominantly found in neurons, immunohistochemistry on MeCP2 knockout mice brains indicated a different additional function [67]. Although no difference in the amount of newborn neurons was observed in MeCP2 knockout mice, dendritic spine formation and spine density were decreased, resulting in delayed and impaired maturation of the newborn neurons. This was accompanied by a decreased expression of genes important for synaptogenesis [67]. Together, it suggests that, in contrast to a role for MBD1 in early neurogenesis, MeCP2 binding to DNA methylation marks is important for regulating the expression of genes involved in maturation of newborn neurons.

MeCP2 may also function to repress non-neuronal lineage genes and maintain neuronal identity, allowing proper neuronal differentiation. Kohyama and colleagues [68] found high expression of MeCP2 in mature hippocampal neurons of the adult mouse brain. Subsequent analysis of the DNA methylation status of different hippocampal cell types revealed high levels of methylation around the transcriptional start region of the GFAP gene. Moreover, MeCP2 expression was absent in oligodendrocytes and astrocytes in the hippocampus [68]. Thus, also repression of GFAP expression by binding of MeCP2 to methylated DNA loci is important for newborn neuron maturation. Further support for a role for MeCP2 in maintaining neuronal cell fate was shown by in vivo transplantation of MeCP2-expressing neural progenitor cells in non-neurogenic regions [69]. MeCP2 expression allows neuronal differentiation in these areas where usually astrocytic differentiation is observed. Moreover, expression of a truncated mutant form of MeCP2, lacking essential domains of the wild type MeCP2, did not allow NSC neuronal differentiation under astrocytic differentiation-inducing conditions, indicating that MeCP2 binding to methylated DNA is a key regulatory factor of this process [69]. Thus, although MeCP2 may not regulate the initiation of NSC differentiation, it may be important for neuronal differentiation and neuronal cell fate. Furthermore, while MeCP2 is not required for the production of immature neurons in the DG, the newly generated neurons, in the absence of MeCP2, exhibit pronounced deficits in neuronal maturation, including a delayed transition into a more mature stage, altered expression of presynaptic proteins and reduced dendritic spine density, suggesting that MeCP2 plays a role in other aspects of neuronal maturation, including dendritic development and synaptogenesis [67].

Early studies identified mutations in MeCP2 that cause neurodevelopmental alterations accounting for the majority of Rett syndrome cases and more recent studies suggest that MeCP2 plays an important role in brain development, aging and in neurological disorders [70]. The extreme abundance of MeCP2 expression in the brain, estimated extend to one molecule of MeCP2 for every two nucleosomes in neuronal chromatin [71] suggesting that it may play a key role in neurological disorders associated with aberrant DNA methylation, such as AD. Particularly in the case of Rett Syndrome, the most common genetic cause of severe intellectual disability in females, several studies in animal models of the disease have demonstrated that animal do not develop an irreversible condition and that phenotypic rescue may be possible, highlighting the need to understand the biological role of MeCP2 and particularly its involvement in the regulation of DNA methylation in the brain [72].

DNA de-methylation and adult NSCs

Hydroxymethylated DNA immunoprecipitation (hMeDIP) followed by high-throughput sequencing has recently started to reveal the genome-wide distribution patterns of 5-hmC in many tissues and cells. Using this technique, recent reports have suggested a functional role of 5-hmC during neural differentiation [75, 84, 85]. Specifically, one of these studies revealed dynamic changes in DNA hydroxymethylation during neural differentiation and identified differentially hydroxymethylated regions between ESCs and NPCs [84]. Of interest, 5-hmC is found in most tissues and its levels seem to be highest in brain, and enriched in synaptic genes [86].

As described above, the Gadd45 family of proteins mediates DNA demethylation. This family of proteins responds to changes in the environment by releasing gene repression at specific genes through the promotion of DNA demethylation [16, 54, 87]. Gadd45b is important specifically for the sequential steps of activity-induced neurogenesis in the adult hippocampus. Gadd45b is expressed in mature neurons in the hippocampus and neuronal activity is an important factor in controlling the rate of neurogenesis [81]. Ma and colleagues [81] studied activity-induced neurogenesis in the hippocampus of adult transgenic mice lacking Gadd45b. The increase in NSC proliferation after electroconvulsive therapy (ECT) observed in the hippocampus of control mice was significant decreased in Gadd45b knockout mice. Moreover, deficits in dendritic growth were observed in Gadd45b knockout mice, indicating that Gadd45b is important for neuronal maturation [81]. Methylated DNA immunoprecipitation (MeDIP) analysis revealed that Gadd45b is necessary for demethylation at different genes encoding growth factors involved in neurogenesis, including FGF1 [81], which regulates self-renewal and proliferation of NSCs similar to FGF2 [54]. These results indicate that Gadd45b is an immediate early gene expressed in mature neurons upon neural activity that subsequently regulates growth factor expression through DNA demethylation. Secretion of these growth factors, FGF1 specifically, induces increased neurogenesis in the surrounding neurogenic niche [81]. Therefore, Gadd45b provides a link between environmental signals (neuronal activity) and epigenetic DNA modifications that regulate adult neural stem cells.

Histone acetylation and adult NSCs

Acetylation of histone proteins is a dynamic process and especially the removal of acetylation marks by HDACs is important in neurogenesis [88]. Transcriptional repression through HDAC activity is essential for adult NSC proliferation and self-renewal. For example, the orphan nuclear receptor homologue of the Drosophila tailless gene (Tlx or NR2E1) regulates NSC self-renewal and interacts with different HDAC enzymes to regulate gene expression. Sun and colleagues [89] used ChIP analysis to show a direct interaction between Tlx and HDAC3, HDAC5 and HDAC7. These proteins are co-expressed in cultured adult mouse NSCs, and their expression is reduced upon NSC differentiation. Furthermore, these authors found that the cell-cycle regulator p21 was up-regulated in Tlx knockout mice and ChIP analysis revealed a common Tlx, HDAC3 and HDAC5 binding site in the p21 gene promoter. Moreover, treatment of cultured NSCs with the HDAC inhibitor valproic acid (VPA) induces p21 expression and increased acetylation of H4 at the p21 promoter [89]. Thus, both de-acetylation at the p21 promoter and activation of Tlx are necessary for inhibition of p21 expression. In vitro treatment of adult NSCs with VPA significantly reduced the amount of BrdU labeled cells, indicating a decrease in cell proliferation. Interestingly, both small-interfering RNA (siRNA) targeting Tlx and HDACs had the same effect [89]. Thus, the interaction of Tlx with HDAC3, HDAC5 and HDAC7 seems to be important for the regulation of genes involved in adult NSC proliferation.

A role for histone deacetylation in isolated adult SVZ NSCs is further supported by interesting observations done after treating these cells with the HDAC inhibitors sodium butyrate (NaB) and suberoylanilide hydroxamic acid (SAHA) [90]. Under these conditions, the authors observed impaired proliferation that was accompanied by a profound down-regulation of factors involved in stem cell maintenance and up-regulation of pro-neural factors. For example, the expression of Sox2 and the Notch effector transcription factors Hes1 and Hes5, involved in stem cell maintenance and proliferation, were down-regulated. Under induced differentiation conditions, SVZ NSCs pretreated with the HDAC inhibitor SAHA showed decreased glial and oligodendroglial differentiation compared to non-treated cells while neuronal differentiation was not affected [90]. These results support the role of HDAC activity in SVZ NSC proliferation, as was shown before by Sun and colleagues [89] and provide evidence for an additional role in adult NSC differentiation.

Increased neuronal differentiation at the expense of glial and oligodendroglial differentiation has also been observed in adult hippocampal NSCs treated in vitro with VPA that increased H3 acetylation levels and resulted in increased neuronal differentiation, even when factors favoring non-neuronal cell lineage differentiation were present [91]. Indeed, profound differences were observed when H3 and H4 acetylation levels were compared between NSCs and their progeny. Initially high H3 and H4 acetylation levels were found in undifferentiated NSCs and these levels remained relatively high in cells upon their differentiation into neurons. Lower levels of H3 and H4 acetylation were observed in cells differentiating into astrocytes or oligodendrocytes, suggesting that HDAC activity is crucial for NSC fate decisions. Thus, maintenance of histone acetylation seems important for neuronal lineage progression of adult NSCs, while histone de-acetylation appears important for astrocytic and glial lineage progression.

In vivo, BrdU analysis of the DG of VPA-treated adult rats showed a marked reduction in proliferation, accompanied by a significant increase in BrdU-labeled newborn neurons. Although astrocytic differentiation was unchanged, these results confirmed to a certain extent previous in vitro observations [91]. Similarly, Sun and colleagues [89] showed e.g. that HDAC expression, and thus probably histone acetylation, is decreased after neuronal differentiation of NSCs indicating an important role for histone acetylation in the regulation of NSC differentiation. Additional in vitro evidence supporting this notion was obtained using isolated NSCs from the adult SVZ [92]. In these experiments, treatment of NSC for the SVZ cells with SAHA increased their neuronal differentiation B [92].

HDAC2 is specifically important for neuronal maturation in both the adult SGZ and SVZ. HDAC2 is highly expressed in dividing cells within these areas. Low HDAC2 expression is associated with NSC quiescence, while higher expression levels are found in transit amplifying cells and HDAC2 remains present upon differentiation [93]. Deletion of HDAC2 in mice reduces total HDAC activity in the OB and hippocampal areas accompanied by a significant reduction in newborn neuron numbers and increases in cell death. In contrast, there was a significant increase in the proliferation rate of transit amplifying cells, as determined by the amount of cells in the S-phase of the cell cycle. This increased proliferation but defective neuronal generation in HDAC2 deficient mice is thought to result from the lack of gene repression by HDACs. The transcription factor Sox2 is expressed in wild type NSCs and its expression decreases upon progression to neuroblasts. However, in HDAC2 deficient mice, Sox2 expression was observed in neuroblasts present in the DG. This observation indicates that insufficient histone deacetylation of genes that are usually repressed by HDAC2 in cells differentiating towards the neuronal fate, like Sox2, may impair their maturation but increase their proliferation capacity. Importantly, although deletion of HDAC2 impaired neuronal maturation in the adult brain, deletion of HDAC2 did not change neurogenesis during embryonic development. Therefore, the requirement for HDAC2-dependent regulation of proliferation-related genes, allowing proper neuronal differentiation, seems specific for adult neurogenesis [93]. Thus, although several epigenetic mechanisms regulating embryonic neurogenesis are conserved into adulthood, also new mechanisms seem to emerge that regulate adult NSCs specifically.

The activity of several HATs has been studied in vivo as well [94]. The Querkopf (Qkf) protein is a member of the MYST family of HATs and it is a transcriptional activator with histone acetylase activity. During embryonic development, Qkf is expressed throughout the brain but its expression is restricted to neurogenic areas in the adult brain. In the SVZ of the adult brain Qkf is expressed in type A, B and C NSCs. A 90% reduction in Qkf transcription is observed in mice carrying hypomorphic Qkf alleles. This reduction is associated with decreased NSC proliferation and alterations in the proportions of the cell types derived from them, suggesting that defective neurogenesis in the OB of adult Qkf-deficient mice may result from a decrease in the proliferative NSC population and alterations in the cell progeny derived from it [94]. In addition, isolation of SVZ NSCs from Qkf-deficient mice showed impaired neuronal differentiation in vitro, while Qkf overexpression increased neuronal differentiation [94]. This indicates that the level of Qkf, and presumably of Qkf-mediated histone acetylation, regulates neuronal differentiation of adult NSCs in the SVZ. A similar impairment in neuronal differentiation was observed in isolated cells in vitro[94]. In conclusion, these results suggest a role for histone acetylation in neuronal differentiation, in line with previous studies where increased acetylation induced by HDAC inhibition increases neuronal differentiation.

Histone methylation and adult NSCs

Adult neurogenesis is under tight epigenetic control of histone methylation that is regulated by two antagonistic complexes. The Polycomb-group (PcG) protein complex, that promotes histone 3 lysine 27 tri-methylation (H3K27me3), and the Trithorax-group (TrxG) protein complex, that promotes histone 3 lysine 4 tri-methylation (H3K4me3). Both are part of an evolutionarily conserved chromatin remodeling system that silences or activates gene expression, respectively. Together, these histone methylation events regulate the establishment and maintenance of different cell states in NSCs [51, 54, 95].

The PcG member B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) is required for postnatal NSC self-renewal. In vitro, Bmi-1 overexpression in NSCs isolated from the adult mouse SVZ increases neurosphere formation and self-renewing capacity of these cells [96]. Moreover, when differentiation was induced after five culture passages, the differentiation capacity of wild type NSCs was very low, while Bmi-1 overexpressing NSCs produced both glia and neurons under the same experimental conditions. Both immature and mature neuronal markers were expressed in these cultures. In vivo overexpression of Bmi-1 showed a similar increase in NSC proliferation in the SVZ and RMS [96]. This indicates that increased H3K27me3 induced by Bmi-1 overexpression could affect the expression of genes important for NSC proliferation and differentiation both in vitro and in vivo. In support, proliferation within the SVZ is decreased in adult Bmi-1 deficient mice [97]. In addition, NSCs isolated from Bmi-1 deficient mice showed decreased proliferation and self-renewal capacity in vitro, compared to wild type cells [97]. Although direct histone methylation measurements were lacking in this study, Bmi-1 is part of the PcG complex that catalyzes H3K27 tri-methylation, indicating that impairment of repressive histone methylation due to loss of Bmi-1 may be responsible for the results observed. Interestingly, Bmi-1 deficiency has been associated with increased expression of cell-cycle inhibitors such as p16 (Ink4a) and p19 (Arf), and accurate repression of these genes by Bmi-1 represents a critical mechanism by which Bmi-1 drives NSC self-renewal [98].

Recent observations have shown that the TrxG member mixed-lineage leukemia 1 (Mll1) is required for adequate neurogenesis progression [99]. Mll1-deficient NSCs purified from the SVZ survived, proliferated and efficiently differentiated into glial lineages but their neuronal differentiation was impaired. In Mll1-deficient cells, the expression of the early proneural Mash1 and gliogenic Olig2 expression was preserved, but Dlx2, a key downstream regulator of SVZ neurogenesis, was not detected. In line with these observations, neurogenesis could be rescued by Dlx2 overexpression, demonstrating the crucial role of Mll1 in controlling Dlx2 expression and thus progression towards a neuronal phenotype. Indeed, ChIP analysis showed direct interactions of Mll1 with the Dlx2 gene promoter and Dlx2-regulatory sequences were bivalently marked by both H3K4me3 and H3K27me3 in Mll1-deficient cells. This bivalent histone methylation pattern resulted in the Dlx2 gene failing to properly activate, demonstrating the relevance of epigenetic regulation of Dlx2 in controlling adult neurogenesis in the SVZ [99]. In vivo, Mll1 deficiency decreases the size of neurogenic regions in the postnatal brain including neuronal number, with a sharp decrease in the amount of newly formed neurons in the OB. However, in the SVZ, DCX positive cells are increased in number, indicative of an impaired migratory capacity. Moreover, continuous expression of transit-amplifying cell characteristics in these DCX expressing neuroblasts suggests that gene repression upon differentiation was impaired, which may provide a plausible explanation for the impaired differentiation and migration observed in Mll1 deficient neuroblasts [99]. Thus, Mll1 expression and histone methylation catalyzed by the TrxG complex seems to be an important regulator of postnatal neurogenesis in the mouse SVZ.

Wu and colleagues [62] have demonstrated the ability of DNMT3a to interact with histone methylation. Whereas DNA methylation at promoter regions generally prevents the binding of transcription factors and inhibits gene expression, Wu and colleagues [62] showed that DNMT3a activity correlates with increased expression of neurogenic genes. The increased expression of these neurogenic genes seems to be mediated through an interaction between DNA methylation and histone methylation. ChIP analysis showed that loss of DNMT3a increased binding of the PcG complex Polycomb repression complex 2 (PRC2) to DNMT3a targets, which was accompanied by increased H3K27me3 levels and decreased target gene expression. This effect was specific for DNMT3a targets since binding of PCR2 and H3K27me3 levels did not change at non-DNMT3a targets. In support of this conclusion, restoration of DNMT3a activity functional rescued by introduction of wild type DNMT3a reversed the abnormally increased H3K27me3 levels and PRC2 occupancy at down-regulated DNMT3a target genes in the DNMT3a knock-out NSCs. These results indicate that methylation by DNMT3a may antagonize the repression of gene activity mediated by PcG complex binding and H3K27me3 establishment in NSCs [62] and support a function for DNMT3a in the repression of genes regulating NSC self-renewal and activation of neurogenic genes, thereby regulating neuronal differentiation.

MicroRNAs and adult NSCs

Functional studies of different miRNAs demonstrate their importance for different stages of adult neurogenesis. Let-7b, miR-9, miR-106b, miR-137, miR-184 e.g., are involved in the proliferation of adult mouse NSCs. An additional role for miR-9, miR-34a, miR-137 and miR-184 as well as for miR-124 have been found in neuronal differentiation. Moreover, miR-137 is involved in synaptogenesis and miR-132 regulates both synaptogenesis and neuronal network integration of adult mouse NSCs [100], while miR-34a and miR-125b modulate dendritogenesis and spine morphology [102]. We here focus on well-studied miRNAs with a key role in adult neurogenesis, e.g. miR-34a, that was recently implicated in aging and neurodegeneration in Drosophila, is an essential miRNA, particularly in the developing brain [103].

MiR-34a regulates neuronal differentiation via Notch signalling by repressing the γ-secretase inhibitor numb homolog (Drosophila)-like (NUMBL) [104]. Overexpression of miR-34a increases neurite elongation of mouse NSCs [105]. MiR-34a modulates the expression of synaptic targets including synaptotagmin-1 and syntaxin-1A while its target SIRT1 may mediate the effects on neurite elongation. Overexpression of miR-34a further alters hippocampal spinal morphology, and subsequent electrophysiological function of dendritic spines [106].

MiR-125b is another brain-enriched miRNA, abundantly expressed in the foetal hippocampus under physiological circumstances [107–109]. MiR-125b levels increase during in vitro differentiation of embryonic stem cells [110]. Moreover, miR-125b is downregulated in cerebellar neuronal progenitors, increasing with differentiation, thereby allowing cell maturation and growth inhibition [111]. MiR-125b functions by suppressing Nestin expression, thus modulating proliferation and differentiation of neural stem and progenitor cells, as well as migration of the cell types derived from them [112]. Furthermore, the regulatory function of miR-125b on dendritogenesis could be partly attributed to the fact that a subset of its repressed targets, such as itchy E3 ubiquitin protein ligase (ITCH) and diacylglycerol O-acyltransferase 1 (DGAT1), in turn antagonize neuronal genes in several neurogenic pathways. Therefore, their translational repression by miR-125b suggests a positive role for miR-125b in neurite outgrowth and differentiation [113].

MiR-132 is a brain-enriched miRNA centrally involved in the regulation of neuronal plasticity upon neuronal activation [114]. Overexpression of miR-132 in cultured hippocampal neurons demonstrates that miR-132 modulates short-term synaptic plasticity [115], while overexpression in vivo triggers an increase in dendritic spine density [116]. MiR-132 has been proposed to differentiate neuronal stem cells specifically into dopaminergic neurons via a direct posttranscriptional repression of the nuclear receptor subfamily 4, group A, member 2 (NR4A2, also known as Nurr1) [117]. MiR-132 is also required for normal dendritic maturation in newborn neurons in the adult hippocampus and indirectly participates in CREB-mediated signaling [118]. More specifically, CREB-induced transcription of miR-132 results in a decrease of MeCP2 expression and a subsequent decrease in brain-derived neurotrophic factor (BDNF) due to de-repression of REST [119]. On the other hand, miR-132 expression is greatly enhanced via the ERK1/2 pathway by neurotrophins, such as BDNF, thus forming a negative regulatory feedback loop [120].

Although MiR-124 is abundantly expressed in the adult brain, its expression in different isolated cell types of the adult mouse SVZ indicates an important role in neuronal differentiation. While expression was absent in both type B and C cells, miR-124 expression was observed at the transition from type C transit amplifying cells to type A neuroblast cells. Upon further differentiation, expression increases [121]. Separation of the neuroblast population based on their cell cycle stage indicated by a DNA dye shows increasing miR-124 levels from S/G2-M phase to G0/G1 phase. Thus, miR-124 expression increases at the transition from type C to type A cells and furthermore increases upon cell cycle exit of the neuroblasts. An in vitro knock-down of miR-124 decreases the amount of neuroblasts exiting the cell cycle, while the amount of proliferating type C and A cells increases. This indicates that miR-124 expression is specifically important for the transition from proliferating neuroblasts to differentiated neuroblasts that have left the cell cycle. Computational analysis of miR-124 targets identified the Sox9 transcription factor, that is involved in NSC self-renewal, the Notch-ligand Jagged-1 and the transcription factor Dlx2. MiR-124 targeting of Sox9 was studied in more detail [121]. While differentiating NSCs expressing miR-124 still express Sox9 mRNA, Sox9 protein expression is repressed. This observation supports post-transcriptional repression of Sox9 by miR-124 at the transition from proliferating to differentiating neuroblast cells.

Similarly, repression of mRNA translation by miR-9 is important for neuronal differentiation. Expression of this miRNA has been observed in the neurogenic regions of the brain [122]. Although different miR-9 targets have been identified to regulate this process, miR-9 expression, like miR-124, increases upon neuronal differentiation. Functional analysis of miR-9 in isolated adult mice forebrain NSCs supports its role in neuronal differentiation. While miR-9 overexpression reduced NSC proliferation and increased neuronal differentiation, miR-9 knock-down showed opposite effects. MiR-9 overexpression was accompanied by a reduction in expression of the Tlx receptor that is involved in NSC maintenance, as discussed before. ChIP analysis showed that miR-9 targets Tlx at its 3′UTR, inducing translational inhibition. MiR-9 thus negatively regulates Tlx expression and reduces NSC proliferation but increases neuronal differentiation [122]. Another miRNA targeting Tlx is Let-7b. Increased expression has been observed upon neuronal differentiation similar to miR-124 and miR-9. A knock-down of Let-7b enhances NSC proliferation and decreases neuronal differentiation, while again overexpression shows the exact opposite [123].

Additional functions resulting from the combined actions of miR-9 and miR-124 in neuronal fate progression were demonstrated in a reprogramming study of isolated human fibroblasts [124]. Here the authors showed that miR-9 and miR-124 are capable of inducing a neuronal fate conversion. Combined expression of these miRNAs with transcription factors important for neurogenesis enhanced the rate of conversion of these cells into the neuronal lineage, which was accompanied by an increased maturation of the differentiated neurons. Strikingly, neurogenic transcription factor expression alone did not induce the conversion of these fibroblast cells into the neuronal fate [124]. Thus, the combination of miR activity regulating gene translation and the regulation of gene expression by different transcription factors act together to induce a neuronal fate conversion. This study emphasizes the importance of these miRNAs in the induction of neuronal fate.

Other miRNAs regulate different stages of adult neurogenesis acting on different targets during the process of neuronal maturation [125]. Adult mice hippocampal NSCs were isolated and used to identify lineage specific miRNAs. To this aim, miRNA expression patterns of differentiated astrocytes and neurons were compared by qPCR and miRNAs specifically enriched in the neuronal lineage were further investigated. Following this approach, miR-137, specifically enriched in neurons, was implicated in neuronal maturation. In vivo overexpression of this miRNA in newborn neurons of the adult mouse DG decreased their dendritic complexity, dendritic spines and length of the maturated neurons. This indicates that the maturation process in the miR-137 overexpressing cells was impaired. The increase in miR-137 seems to disrupt the sequential events of neuronal maturation leading to structural alterations. In vitro analysis of miR-137 expression confirmed enrichment in dendrites of differentiated neurons, indicating a role in development of these dendrites as was observed in vivo [125]. Underscoring its importance in neurogenesis, miR-137 targets the mind bomb 1 (MIB1) protein, an ubiquitin ligase essential in neurodevelopment [125] and miR-137 post-transcriptionally represses the expression of Ezh2, a histone methyltransferase and Polycomb group protein, resulting in a global decrease in histone H3K27me3. Furthermore, miR-137 is epigenetically regulated by MeCP2, a DNA methyl-CpG-binding protein, a mechanism we discussed before and in the next section [126]. Although in-depth mechanistic studies of miRNA functioning will have to be done in order to understand the complete regulation network, overall, the studies discussed in this section suggest that miRNAs are capable of regulating NSCs at different stages. Subsequent identification of miRNA targets might contribute to unravel the control of neurogenesis at the molecular level.

Epigenetic interplay in the regulation of adult NSCs

In addition to gene expression regulation, miRNAs also interact with, and regulate epigenetic mechanisms such as DNA methylation and histone modifications, with possible consequences for AD [127]. These interactions are considered central to understanding the regulation of gene-expression networks during neurogenesis. For instance, two epigenetic regulators that have been found to interact are MBD1 and miR-184. MBD1 knock-out in vivo and acute knock-down of MBD1 in vitro induce significant increases in miR-184 expression [128]. In contrast, in vitro overexpression of MBD1 decreases miR-184 expression. Indeed, the genomic region surrounding the miR-184 gene contains high CpG-rich areas and ChIP analysis of wild type NSCs showed MBD1 binding surrounding the miR-184 genomic area. The increase in miR-184 expression observed in MBD1 deficient NSCs was accompanied by increased H3K4me3 and H3K9Ac and decreased H3K27me3 surrounding the miR-184 genomic region [128]. These results indicate that MBD1 may regulate miR-184 expression by interacting with histone modification mechanisms. MBD1 seems to antagonize H3K4me3 and thereby inhibit miR-184 expression using a mechanism different from the DNMT3a-mediated antagonism of H3K27me3 discussed in previous sections [62]. Overexpression of miR-184 showed repression of astroglial and neuronal lineage genes and decreased differentiation of adult NSCs in vitro. Moreover, NSC proliferation and neurosphere formation were increased. In vivo, BrdU analysis after miR-184 overexpression in the DG indicated an increase in NSC proliferation while the percentage of differentiating cells was decreased [128]. As Zhao and colleagues [64] and Singh and colleagues [61] showed before, MBD1 regulates neuronal differentiation. These results suggest that the regulation of neuronal differentiation mediated by miR-184 may involve its regulation by MBD1 and modifications of histone marks.

Thus, the interplay between MBD1, miR-184 and histone modification mechanisms seem to maintain, at least in part, the balance between NSC proliferation and differentiation. Moreover, as discussed before, MBD1 targets FGF2, important for NSC proliferation [65]. Repression of this growth factor by MBD1 is necessary for proper neuronal differentiation, adding an additional player within this molecular network regulating neuronal differentiation of adult NSCs. In support of this hypothesis, activity dependent DNA demethylation by Gadd45b increases expression of a growth factor similar to FGF2 [81]. Based on the previous discussion, these complex interactions between epigenetic mechanisms could explain, at least in part, the release of repression on proliferation/differentiation genes through histone modifications and decreased MBD1 binding due to DNA demethylation.

AD and DNA methylation

Initial epigenetic investigations pertaining to AD focused on DNA methylation of the APP gene and illustrate the complexity and difficulty of investigating the epigenetics of the multifactorial and heterogeneous affliction that is AD. West and colleagues [147] observed hypomethylation of the APP gene promoter in an AD patient, whereas Barrachina and colleagues [148] did not find any significant AD-related abnormalities in methylation of the APP promoter region. They also did not find any abnormal methylation patterns in the MAPT and PS1 genes, even when looking at different stages of the disease. While this group did report the presence of high and low methylated CpG sites in and around the APP promoter region, Brohede and colleagues [149] found no methylation at all at the investigated CpG site in the APP gene. Interestingly, Tohgi and colleagues [150] have found an age-related decrease in cytosine methylation in the promoter region of the amyloid precursor protein (APP) gene in the human cerebral cortex. Additionally, they observed abnormal cytosine methylation in the promoter region of the tau gene in the aged human cerebral cortex [151].

Although it remains to be elucidated whether the APP gene is specifically regulated by DNA methylation or not, strong evidence suggests that DNA methylation is disrupted in AD. Pioneering studies have shown that S-adenosylmethionine (SAM), a methyl donor crucial for DNMTs activity, is severely reduced in AD [152]. Later, the relation of this finding with actual DNA methylation was corroborated by the detection of decreased global DNA methylation in the AD brain [153, 154]. Additional studies have specifically investigated the hippocampus, one of the brain regions strongly affected by AD and found increased levels of 5-mC [155] and DNMT3a [63] in the hippocampus of aging mice, but reduced 5-mC levels in APP/PS1 transgenic mice (Chouliaras et al., submitted, 2014) and in the hippocampus, entorhinal cortex and cerebellum of AD patients [156, 157]. Furthermore, DNA methylation in AD seems to particularly involve DNMT3a, as the presence of a tagSNP in the DNMT3a gene correlated with cognitive decline in MCI patients (Chouliaras et al., submitted 2014).

Remarkably, Aβ itself has been shown to affect DNA methylation [158]. Aβ seems to induce global DNA hypomethylation, while its effect on specific genes is more complex. Indeed, the NEP gene seems to be hypermethylated under influence of Aβ, repressing its transcription [158]. This interaction between Aβ and NEP might be of crucial importance for AD pathology, as the NEP gene encodes for neprilysin, one of the primary enzymes involved in Aβ degradation.

Although the consequences of aberrant DNA methylation associated with AD remain to be fully elucidated, some affected genes have been identified. Siegmund and colleagues [159] found SORBS3 to be hypermethylated, while S100A2 was hypomethylated, possibly reflecting an acceleration of the age-related changes in the normal brain. SORBS3 encodes a cell adhesion molecule and decrements in its expression seem to contribute to the synaptic abnormalities associated with AD [160]. Increased expression of S100A2, which encodes a calcium binding protein, is associated with corpora amylacea formation [161]. In addition, Scarpa and colleagues [162] showed that PS1 was hypomethylated. As the protein encoded by PS1 is part of the enzymatic complex responsible for Aβ production, increased PS1 expression may enhance Aβ formation. Of note, one study comparing human postmortem frontal cortex genome-wide DNA methylation profiles between late-onset AD and 12 cognitively normal controls found widespread, albeit modest, discordant DNA methylation independent from DNA methylation changes with age [163].

AD and DNA hydroxymethylation

DNA hydroxymethylation is not as well studied as DNA methylation, and neither in relation to AD. Nevertheless, studies of DNA hydroxymethylation in the hippocampus suggest a pattern similar to DNA methylation: increasing levels with normal aging [155, 164, 165], but strongly decreased levels in APP/PS1 mice (Chouliaras et al., submitted 2014) and AD patients [156, 157]. Interestingly, Münzel and colleagues showed that levels of 5-hmC increase with age [164]. The importance of DNA hydroxymethylation in AD is further stressed by the discovery of a single nucleotide polymorphism (SNP) in the TET1 gene, which protein catalyzes the conversion of 5-mC into 5-hmC, associated with late onset AD [86, 166]. While the functional impact of changes in DNA hydroxymethylation associated with AD largely remain to be explored, the findings discussed in this section further support the notion of a widespread failure of the epigenetic regulatory system in AD.

AD and histone modifications

Besides DNA methylation, a growing body of evidence suggests that alterations in histone acetylation are among the basic molecular mechanisms underlying AD pathogenesis. Histone acetylation is significantly lower in the temporal lobe of AD patients compared to aged controls [167]. Furthermore, Marques and colleagues [168] showed that increased levels of beta-secretase 1 (BACE1), a protease that cleaves APP in the amyloidogenic pathway, are seen in peripheral blood mononuclear cells of AD patients and increased BACE1 promoter accessibility are associated with increased histone H3 acetylation. These findings are supported by other observations showing aberrant histone acetylation levels in animal models of AD [169]. Interestingly, there is some evidence that dysregulation of histone H4 lysine 12 (H4K12) acetylation is implicated in learning impairment in aged mice. Peleg and colleagues [170] observed that differential gene expression and abnormal H4 acetylation were associated with impaired memory function in contextual fear conditioning in aged mice. Interestingly, these deficits were counteracted by the application of HDAC inhibitors into the hippocampus [170]. Importantly, chronic systemic inhibition of HDAC reverts the cognitive deficit observed in APPswe/PS1dE9 transgenic mice in the contextual fear conditioning model [171]. Unfortunately, the identity of the specific HDAC(s) that is responsible for the memory impairments remains unknown because these studies have mostly used non-selective HDAC inhibitors.

More recent studies have indicated that HDAC2, crucially involved in the regulation of memory and synaptic plasticity, might be directly implicated [172]. Gräff and collaborators investigated the role of HDAC2 in AD [173]. Using CK-p25 mice as model for AD-like neurodegeneration, they found a significant increase of HDAC2 in the hippocampus and prefrontal cortex of these mice. In contrast, no significant changes in HDAC2 expression were detected in the amygdala, an area not affected by neurodegeneration in this animal model. When these authors investigated the functional impact HDAC2 dysregulation, they found that H2bK5, H3K14, H4K5 and H4K12 were all hypoacetylated in CK-p25 mice. Importantly, increased HDAC2 binding and hypoacetylation negatively correlated with activated RNA Polymerase II binding and mRNA expression in genes related to learning, memory and synaptic plasticity [173]. These observations were confirmed by HDAC2 knockdown, which successfully restored synaptic plasticity and cognitive performance in CK-p25 mice. In addition, Gräff and colleagues [173] investigated the effects of two neurotoxic stimuli associated with AD, hydrogen peroxide and Aβ, on HDAC2 expression in primary hippocampal neurons. They found that these noxious stimuli increased HDAC2 levels in cells, an event likely resulting from glucocorticoid receptor (NR3C1) activation in response to the neurotoxic stimuli, thus linking AD hallmarks to aberrant epigenetic regulation possibly mediated by NR3C1. Finally, Gräff and colleagues [173] validated their findings in postmortem human brain samples from sporadic AD cases at different Braak stages. These experiments revealed that HDAC2 levels are significantly increased in the hippocampus and entorhinal cortex, areas known to be affected in AD. Moreover, HDAC2 levels were elevated in all Braak stages, including I and II, indicating that deleterious HDAC2 activity might be one of the earlier events in the development of AD.

AD and microRNAs

Apart from their involvement in regulating neurogenesis in normal conditions mentioned in previous sections, miRNAs have also been shown to be involved in AD pathogenesis. We and others have recently reviewed the experimental evidence supporting this conclusion [127], so we only discuss some relevant examples here. For instance, miR-15, miR-16, miR-132 and miR-497 have been associated with tau regulation, whereas miR-106a, miR-106b, miR-107, miR-124, miR-137, miR-153, miR-195 and miR-520c have been linked to APP metabolism and Aβ production [174]. More specifically, a role for miR-132 in the regulation of alternative splicing of tau exon 10 has been demonstrated by studying its repression of the polypyrimidine tract binding protein 2 (PTBP2) transcript. This repression interfered with the physiological phosphorylation of tau, thus linking aberrant miR-132 functioning to possible disease state [175]. In the same study, members of the miR-16 family (miR-16, miR-15, miR-195 and miR-497) were identified as regulators of ERK1 and therefore tau phosphorylation in neuronal cells in vitro, including primary rat neurons. An additional link between miR-16 expression and AD pathology was introduced by Liu and colleagues [176]. In this study, miR-16 overexpression reduced APP levels in the brains of senescence-accelerated mouse prone 8 (SAMP8) mice, another animal model of age-related behavioral deterioration and AD-associated neurodegeneration that displays deficits in learning and memory [177].

Regulation of Aβ production further implicates miRNA function in AD via different mechanisms. For instance, endogenous miR-106a, miR-153, and miR-520c downregulate APP levels in human neurons by directly targeting the 3′ UTR of the APP mRNA [178, 179] and thus reducing Aβ levels. Suppression of BACE1 translation by miR-195 and miR-124 also reduces Aβ production [180, 181], while miR-137 and miR-181c indirectly regulate Aβ production via modulation of serine palmitoyltransferase (SPT) levels [182]. Lastly, the expression of certain miRNAs is affected by the presence of Aβ. miR-106b expression appears to be induced in APPswe/PS1dE9 brains due to increased Aβ42 oligomers [183], whereas miR-9 and miR-181c are downregulated in cultured hippocampal neurons exposed to Aβ, providing another link to the pathogenesis of AD [184].

Interestingly, while some of the miRNAs implicated in AD are also involved in other neurodegenerative diseases, such as Mild Cognitive Impairment (MCI) or Parkinson’s Disease (PD), some seem to be more specific to AD itself. Recently, Leidinger and colleagues pinpointed a ‘12-miRNA signature’ in AD using next generation sequencing (NGS) to trace miRNAs from blood samples of 44 AD patients and 22 age-matched healthy controls [185]. The signature consisted of miRNAs that were differentially expressed strictly in AD, including miR-26a, -26b, -103a, -107, -112, -151a, -161, -532, -1285, -5010, let-7d and let-7f, thereby providing a tool to distinguish AD from other neurodegenerative diseases with a reasonable accuracy [185]. Of note, many of these 12 miRNAs may have distinct roles in neurodevelopmental pathways, such as neurite outgrowth, synaptic formation and neuronal migration, portraying the complex nature of AD and its implications in neuronal development.