Epigenetics: The neglected key to minimize learning and memory deficits in Down syndrome

Neuroscience and Biobehavioral Reviews 45 (2014) 72–84

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Review

Epigenetics: The neglected key to minimize learning and memory deficits in Down syndrome Alain D. Dekker a,b , Peter P. De Deyn a,b , Marianne G. Rots c,∗ a Department of Neurology and Alzheimer Research Center, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands b Laboratory of Neurochemistry and Behaviour, Institute Born-Bunge, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp, Belgium c Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands

a r t i c l e

i n f o

Article history: Received 18 February 2014 Received in revised form 4 May 2014 Accepted 13 May 2014 Keywords: Down syndrome Mental retardation Alzheimer’s disease Epigenetics DNA methylation Post-translational histone modifications Epigenetic Editing Gene expression

a b s t r a c t Down syndrome (DS) is the most common genetic intellectual disability, caused by the triplication of the human chromosome 21 (HSA21). Although this would theoretically lead to a 1.5 fold increase in gene transcription, transcript levels of many genes significantly deviate. Surprisingly, the underlying cause of this gene expression variation has been largely neglected so far. Epigenetic mechanisms, including DNA methylation and post-translational histone modifications, regulate gene expression and as such might play a crucial role in the development of the cognitive deficits in DS. Various overexpressed HSA21 proteins affect epigenetic mechanisms and DS individuals are thus likely to present epigenetic aberrations. Importantly, epigenetic marks are reversible, offering a huge therapeutic potential to alleviate or cure certain genetic deficits. Current epigenetic therapies are already used for cancer and epilepsy, and might provide novel possibilities for cognition-enhancing treatment in DS as well. To that end, this review discusses the still limited knowledge on epigenetics in DS and describes the potential of epigenetic therapies to reverse dysregulated gene expression. © 2014 Elsevier Ltd. All rights reserved.

Contents 1. 2.

3. 4. 5.

6. 7. 8. 9.

Introduction: epigenetics underlying the cognitive deficits in DS? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epigenetic mechanisms – an overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. DNA methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Post-translational histone modifications and core variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Epigenetic modulation by miRNA and lncRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Altered epigenetic modifications in learning and memory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aberrant DNA methylation is associated with DS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Altered histone tail modifications and core variants are associated with DS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Post-translational histone modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Histone core variants and constitutive chromatin proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MicroRNAs and long non-coding RNAs in DS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epigenetics in DS: a link to Alzheimer’s disease? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epigenetic therapies for cognitive deficits in DS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +31 50 361 01 53. E-mail addresses: [email protected] (A.D. Dekker), [email protected] (P.P. De Deyn), [email protected] (M.G. Rots). http://dx.doi.org/10.1016/j.neubiorev.2014.05.004 0149-7634/© 2014 Elsevier Ltd. All rights reserved.

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1. Introduction: epigenetics underlying the cognitive deficits in DS? With an incidence of approximately 1 in 650–1000 live births, Down syndrome (DS) is the most common genetic intellectual disability in humans (Bittles et al., 2007). In his ‘Classification of Idiots’ (1866), the British physician J.L.H. Down described various recurrent symptoms of the ‘Mongolian type of idiocy’ that he observed among more than 10% of the children that he treated for cognitive impairment (Down, 1995). Down stated that “it is difficult to realize [that the Mongolian type] is the child of Europeans”. Without knowledge about genetics and neurobiology, he was ahead of his time by postulating “that there can be no doubt that these ethnic features are the result of degeneration.” It took almost a century to discover the cause of DS. Lejeune et al. (1959) demonstrated in 1959 that DS was due to a triplication of chromosome 21 (HSA21). In the majority of the cases (over 95%) this is a whole-chromosome trisomy due to meiotic non-disjunction, i.e. a failed separation of one of the paired chromosomes (Antonarakis et al., 2004; Lubec and Engidawork, 2002). Apart from the characteristic facial appearance, this triplication causes various neurological complications of which mental retardation (lower IQ) is the most well-known. DS is characterized by impaired linguistic skills and diminished learning and memory capacities, specifically impairment of the verbal short-term memory and explicit long-term memory (Lott and Dierssen, 2010). In addition to the congenital cognitive deficits, people with DS face accelerated ageing, including early-onset dementia due to Alzheimer’s disease (AD) in 50–70% of the DS population (Zigman and Lott, 2007). This strongly increased risk for AD in DS compared to non-DS individuals has been predominantly attributed to the triplication of the amyloid precursor protein (APP) gene on HSA21, yielding higher levels of amyloid-␤ (A␤), the main constituent of the characteristic plaques in AD (Ness et al., 2012). Despite the fact that 95% of the DS cases is due to a whole-chromosome trisomy, the DS population is characterized by an enormous variability in the type and the severity of clinical features (Roper and Reeves, 2006). This phenotypical variability is strikingly illustrated by the observation that the onset of clinical dementia symptoms in DS differs tremendously. Remarkably, 30–50% of the DS individuals do not develop dementia, despite their full-blown AD-like neuropathology, including A␤ plaques, around midlife (Lott and Dierssen, 2010; Ness et al., 2012; Wilcock, 2012; Zigman and Lott, 2007). As part of the Human Genome Project the complete DNA sequence of HSA21 was elucidated in 2000 (Hattori et al., 2000). Since, many researchers have investigated the overexpressed protein-encoding genes and their effects on learning and memory. Despite increased understanding of the possible underlying genetic mechanisms, it remains very difficult to explain the aforementioned variability among the DS population (Jiang et al., 2013; Prandini et al., 2007). The triplication of HSA21 would theoretically lead to a 1.5 fold increase in gene transcription. However, gene expression studies showed differently. For instance, analysis of HSA21 gene expression in DS lymphoblastoid cells showed that only 22% of the analysed genes had expression levels closely matching this 1.5 fold (class I), compared to control individuals. In particular, 7% had an amplified expression (significantly >1.5; class II), 56% had an expression level that was significantly lower than 1.5 (class III) and 15% of the genes had highly variable expression profiles between subjects (class IV) (Ait Yahya-Graison et al., 2007). Similar results were obtained using the most widely used Ts65Dn mouse model of DS. Ts65Dn mice carry an additional chromosome, consisting of a duplicated part of the mouse chromosome 16 that is translocated to a small segment of the mouse chromosome 17 (Davisson et al., 1990). As a consequence, Ts65Dn mice

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are trisomic for about 50% of the genes on HSA21 (Reeves et al., 1995). However, it was demonstrated that many of these genes have transcript levels that significantly deviate from the theoretical 1.5 fold increase (Antonarakis et al., 2004; Kahlem et al., 2004; Lyle et al., 2004). For instance, Lyle et al. (2004) reported that not more than 37% of the genes in Ts65Dn matches the theoretical expression level of 1.5. Accordingly, certain genes are more dosage sensitive than others, thereby contributing in varying extents to the DS phenotypes (Antonarakis et al., 2004). Whereas various studies have tried to identify the crucial phenotype-determining genes, the underlying cause of the gene expression variation has been largely neglected. Conceivably, epigenetic (epi = above (Greek)) mechanisms play a role in gene expression regulation and as such might play a crucial role in the development of the cognitive deficits in DS. An epigenetic trait is defined as “a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence” (Berger et al., 2009). That is, epigenetic mechanisms regulate gene expression without affecting the DNA itself. Importantly, epigenetic marks, including DNA methylation and post-translational histone modifications, are reversible and thus offer a huge therapeutic potential to alleviate or cure certain genetic deficits. Indeed, an increasing body of evidence illustrates the role of epigenetic mechanisms in synaptic plasticity and learning and memory. Memory formation, for example, relies on increased DNA methylation of memory suppressor genes and diminished DNA methylation of memory promoting genes (Day and Sweatt, 2010; Weng et al., 2013). Furthermore, histone acetylation has been shown to play a major role in promoting synaptic plasticity and memory formation and, in turn, inhibition of histone deacetylation has been shown to rescue memory deficits (Graff and Tsai, 2013). Surprisingly, epigenetic mechanisms have been hardly investigated in DS. Most DS studies have focused on genomic aspects, neglecting the increasing body of evidence that demonstrates the contribution of epigenetics to impaired learning and memory. Therefore, this review aims to summarize and evaluate the limited knowledge on epigenetics in the neurobiology of DS, as well as provide additional arguments for its role in learning and memory that are distilled from epigenetic studies of other intellectual disabilities. Importantly, current epigenetic therapies are already used for cancer and epilepsy, and might provide novel possibilities for cognition-enhancing treatment in DS as well. To our knowledge, no studies so far have investigated epigenetic therapy in (mouse models of) DS. However, it offers potentially important new avenues, as classical pharmacological treatment has not been successful yet in diminishing cognitive deficits in DS (Braudeau et al., 2011). To that end, the huge potential of epigenetic therapies (epidrugs and Epigenetic Editing) to reverse deregulated gene expression will be discussed.

2. Epigenetic mechanisms – an overview To enable organized storage of all DNA in the nucleus, DNA in eukaryotic cells is packaged about 10,000 times into a more compact form: chromatin. The basic level of this chromatin is the nucleosome that consists of approximately 147 base pairs of DNA wrapped around a histone core in 1.7 turns. This core is an octamer, containing two copies of each histone type: histone 2A (H2A), H2B, H3 and H4 (Luger et al., 1997), as also shown in Fig. 1. Considering the higher level of compaction, this string of nucleosomes (‘beads on a string’) is folded into a fiber, which in turn, is folded into even more condensed structures (Felsenfeld and Groudine, 2003). Epigenetic mechanisms affect the nucleosomal packaging and thereby alter the accessibility of the DNA for molecular interactions

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Fig. 1. Overview of the major epigenetic hallmarks and associated HSA21-products. DNA is compacted into chromatin, which consists of nucleosomes: 147 base pairs of DNA wrapped around an octamer histone core. Gene expression depends on the chromatin state: open, accessible chromatin (euchromatin) is associated with gene expression and closed, inaccessible chromatin (heterochromatin) is associated with gene silencing. DNMT, DNA methyltransferase; DSCR, Down Syndrome Critical Region; dsDNA, double-stranded DNA; HAT, histone acetyltransferase; HDAC, histone deacetylase; me, methylation; TET, ten-eleven translocation. Adapted from Falahi (in press).

that are required for transcription and replication (Cosgrove et al., 2004; Zentner and Henikoff, 2013). Two chromatin states are typically recognized: (i) accessible, relatively open euchromatin, which is generally associated with active gene expression and (ii) more inaccessible, tightly compacted heterochromatin that is generally associated with silenced gene expression (Fig. 1). Four main epigenetic mechanisms are distinguished that alter this chromatin state: DNA methylation, post-translational histone modifications, nucleosomal core assembly and chromatin remodelling through microRNAs (miRNAs) and long non-coding RNAs (lncRNAs).

novo DNA methylation (Bestor, 2000; Margot et al., 2003). In addition, DNMT3L has no methyltransferase activity, but it has been shown to mediate transcriptional gene repression (Deplus et al., 2002) and stimulate the methylation activity of DNMT3A and 3B by direct binding (Ooi et al., 2007; Suetake et al., 2004). Importantly, female Dnmt3l knock-out mice showed specific hypomethylation of maternally imprinted genes, suggesting that DNMT3L together with DNMT3A/3B mediates de novo DNA methylation of these maternally imprinted genes (Arima et al., 2006; Bourc’his et al., 2001; Hata et al., 2002).

2.1. DNA methylation

2.2. Post-translational histone modifications and core variants

Cytosines preceding a guanine (CpG) are frequently methylated in the human genome. This process of DNA methylation concerns the transfer of the methyl group of S-adenosylmethionine (SAM) to the carbon 5 of a cytosine that precedes a guanine (5 -CpG-3 ), yielding 5-methylcytosine (5mC) (Weng et al., 2013). In general, DNA methylation is associated with the formation of heterochromatin and promoter methylation is associated with repressed gene expression (Baylin and Jones, 2011). DNA methylation is conducted by four different DNA methyltransferases (DNMTs) with different functions. Whereas DNMT1 maintains the methylation marks after DNA replication, DNMT3A and 3B are mainly responsible for de

Specific amino acids of the histone tails that protrude from the nucleosome core are subjected to covalent post-translational modifications, such as acetylation, methylation and phosphorylation. Depending on the type and the position of a modification and the presence of particular effector proteins, a certain (combination of) histone mark(s) is associated with stimulated or repressed gene transcription (Zentner and Henikoff, 2013). For instance, whereas acetylation is generally associated with gene expression, the effect of methylation depends on the specific amino acid and its location. Histone-modifying enzymes maintain these groups on the histone tails: specific enzymes are present for addition (writers)

A.D. Dekker et al. / Neuroscience and Biobehavioral Reviews 45 (2014) 72–84

or removal (erasers) of particular histone marks, e.g. acetylation is increased by histone acetyltransferases (HATs) and reduced by histone deacetylases (HDACs). Histone modifications form docking platforms for chromatin-associated effector proteins that alter the chromatin structure. It is important to note that these histone modifications are not static, but rather the equilibrium of continuous addition and removal of these chemical groups by epigenetic writers and erasers (Ueda et al., 2006). Next to modifications of the histone tail, histone core variants can also influence gene expression. Incorporation of different histone variants into the nucleosomal core affects the chromatin structure (Luger et al., 2012). Most variants have been discovered for H2A and H3 and were reported to have a diverse role in gene expression regulation, e.g. incorporation of macroH2A is associated with gene repression (Creppe et al., 2012; Luger et al., 2012). 2.3. Epigenetic modulation by miRNA and lncRNAs Finally, endogenously expressed miRNAs and lncRNAs also function as epigenetic modulators by recruiting enzymes that mediate the condensation of heterochromatin (Farazi et al., 2008; Mercer and Mattick, 2013; Morris, 2009). These non-protein-encoding RNAs seem to act as scaffolds or guides to direct epigenetic writers, erasers and readers to specific sites in the genome (Della Ragione et al., 2014). In particular, miRNAs consist of approximately 22 nucleotides and might be involved in mediating the condensation of heterochromatin through methylation of H3 lysine 9 (H3K9) and H3K27, next to their function in mRNA cleavage and degradation and translational repression (Morris, 2009; van den Berg et al., 2008). Furthermore, various endogenous miRNAs have been reported to epigenetically silence the expression of human target genes by directing components of repressive chromatin-modifying complexes to the promoters of these genes (Li, 2013). In addition, lncRNAs consist of at least 200 nucleotides and are best characterized for their role in epigenetics: they modulate the chromatin state by binding to chromatin-modifying proteins (Mercer and Mattick, 2013). For instance, Khalil et al. (2009) demonstrated that many lncRNAs bind to the histone methylase polycomb repressive complex (PRC) 2. Indeed, targeted depletion of certain PRC2-associated lncRNAs by means of small interfering RNA resulted in altered gene expression (Khalil et al., 2009). 3. Altered epigenetic modifications in learning and memory The importance of the epigenetic mechanisms in synaptic plasticity, learning and memory is increasingly being unraveled, a field of research sometimes referred to as ‘cognitive epigenetics’ (Day and Sweatt, 2011). Findings from human intellectual disabilities, as well as from various animal models have demonstrated that epigenetics is essential for learning and memory (Day and Sweatt, 2011). For instance, the presence of high levels of DNMTs, as well as methyl-CpG-binding proteins in neurons suggests a role for DNA methylation in neuronal functioning (Sanchez-Mut et al., 2012). Indeed, the expression patterns of DNMTs change depending on the stage of neurodevelopment, suggesting that DNA methylation is involved in neurogenesis. For example, whereas DNMT3b expression is predominantly confined to early neurogenesis, DNMT3a is mainly expressed in later embryonic development and post-natal tissues (Feng et al., 2005, 2007). Furthermore, Rett syndrome, a progressive neurodevelopmental disorder in females that results in mental retardation, is caused by a single mutation in the methyl-CpG-binding protein 2 (MeCP2) that recognizes

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methylated DNA (Amir et al., 1999). More recent evidence suggests that neuronal plasticity also depends on neuronal activity-induced hydroxymethylation of certain critical genes (Ma et al., 2009). Hydroxymethylation, an early intermediate of DNA demethylation, is mediated by the ten-eleven translocation (TET) protein family (Tahiliani et al., 2009). Interestingly, a recent study showed that Tet1 knock-out mice presented deficits in hippocampal neurogenesis and impaired learning and memory (Zhang et al., 2013). Next to DNA methylation, post-translational histone modifications also relate to learning and memory processes. For instance, contextual fear conditioning, a hippocampus-dependent form of associative learning, is associated with increased phosphorylation of H3 serine 10 (H3S10), dimethylation of H3K9 and trimethylation of H3K4 in the CA1 region of the rat hippocampus (Chwang et al., 2006; Gupta et al., 2010). Furthermore, histone acetylation is strongly associated with promoting synaptic plasticity and memory formation, whilst histone deacetylation has been related to memory deficits (Graff and Tsai, 2013). For example, deregulated acetylation of H4K12 has been associated with memory impairment in aged mice (16 months), which was overcome by administration of HDAC inhibitors (Peleg et al., 2010). In agreement, histone acetyl transferases (HATs) are crucial for memory formation, as is demonstrated by the mental retardation in Rubinstein-Taybi syndrome that is caused by a loss of function mutation in the CBP/P300 histone acetyl transferase (Barrett and Wood, 2008). Moreover, recent evidence indicates that histone lysine methylation is associated with learning-dependent synaptic plasticity and hippocampus-dependent long-term memory formation as well (Jarome and Lubin, 2013). Finally, mounting evidence suggests a role for miRNAs and lncRNAs in neurodevelopment, synaptic plasticity and learning and memory (comprehensively reviewed in Della Ragione et al., 2014; Saab and Mansuy, 2014). Importantly, these non-coding RNAs have been implicated in the formation of long-term memories, which is based on activity-dependent gene transcription and translation in the brain. MiRNAs and lncRNAs can regulate this by affecting epigenetic marks through recruitment of epigenetic enzymes, besides their direct effect on transcriptional and translational processes (Della Ragione et al., 2014; Saab and Mansuy, 2014). Due to the genetic base pair mutations in epigenetic factors, Rett syndrome and Rubinstein-Taybi syndrome are classified as chromatin diseases (Berdasco and Esteller, 2013). Although such mutations have not been documented in DS, it might be regarded, in part, as a chromatin disease as well. Indeed, a growing body of evidence demonstrates that the triplication HSA21, via the subsequent overexpression of various genes, directly deregulates cellular epigenetic mechanisms in DS (Table 1). In turn, these disrupted epigenetic processes are associated with altered gene expression profiles and thus provide obvious candidates that might contribute to the cognitive deficits in DS. A substantial number of those genes is located on a specific part of HSA21, frequently referred to as the Down Syndrome Critical Region (DSCR). It was discovered after the observation that, apart from whole-chromosome trisomies of HSA21, partial trisomies also induced typical DS phenotypes. Therefore, triplication of this part of chromosome 21, which roughly corresponds to the q22 band, is already sufficient to yield the majority of characteristic DS traits, including a component of the mental retardation (Korenberg et al., 1990; Sinet et al., 1994). However, more recent cases of partial trisomies with the clinical features of DS have demonstrated the contribution of other HSA21 regions and excluded certain DSCR genes, thus arguing against a single critical region (Cetin et al., 2012; Korbel et al., 2009; Korenberg et al., 1994; Ronan et al., 2007). Despite the demonstrated involvement of epigenetics in learning and memory processes, only a few epigenetic studies have been

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Table 1 Aberrant epigenetic mechanisms due to overexpressed HSA21-linked proteins in DS. HSA21 product

Class of gene expressiona

Primary function

Downstream epigenetic effector

Epigenetic consequence

References

DNMT3L

Unknown

DNA methyltransferase

DNMT3A, DNMT3B

DNA methylation and histone deacetylation

Deplus et al. (2002) and Ooi and Wood (2007)

CBS

Class I

Homocysteine conversion

SAM depletion

DNA and histone methylation

Infantino et al. (2011), and Wallace and Fan (2010)

SIRT1 (HDAC)

Histone deacetylation

Guo et al. (2010)

DYRK1A

Class I

Kinase

CREB and CBP/P300 (HAT)

Histone acetylation

Barrett and Wood (2008) and Weeber and Sweatt (2002)

SWI/SNF complex

Histone modifications

Lepagnol-Bestel et al. (2009) BRWD1

Class IV

RUNX1

Class III

ETS2

Class III

H2AFZP

Unknown

H2BFS

Class III

CHAF1B

Class III

HMGN-1

Class I

miRNA-155

Unknown

miRNA-802

Unknown

miRNA-99a

Unknown

miRNA-125b-2

Unknown

let-7c

Unknown

lncRNAs

Unknown

Transcriptional regulator

Huang et al. (2003)

Bakshi et al. (2010)

Transcription factor

Histone variant

CBP/P300 (HAT)

Histone acetylation

Unknown

Unknown

Sun et al. (2006) Sanchez-Mut et al. (2012) Gardiner and Davisson (2000) and UniProtKB/Swiss-Prot (2013)

Constitutive chromatin protein

Multiprotein complex with MDB-1 and HP-1

Methylation-mediated transcriptional repression

Reese et al. (2003)

Constitutive chromatin protein

CBP/P300 (HAT)

Histone acetylation

Ueda et al. (2006)

MeCP2

Activated or repressed gene transcription

Abuhatzira et al. (2011) Abuhatzira et al. (2011), and Samaco and Neul (2011)

MicroRNA

Unknown

Epigenetic modulator

Unknown

The National Center for Biotechnology Information (2013)

Mercer and Mattick (2013)

a

Gene expression classification based on Ait Yahya-Graison et al., 2007. Due to the triplication of HSA21 in DS, an increased expression level of 1.5 fold is expected in DS compared to non-DS controls. Four classes of genes were reported with expression levels that were around 1.5 (class I), significantly higher than 1.5 (class II), significantly lower than 1.5 (class III) or highly variable (class IV).

conducted in DS. To the extent that it is known, the subsequent sections discuss the contribution of each of the four epigenetic mechanisms to the cognitive deficits in DS, particularly focusing on epigenetic alterations due to any overexpressed HSA21 gene product, i.e. also those outside the DSCR (Table 1). 4. Aberrant DNA methylation is associated with DS Although relatively little is known about altered DNA methylation patterns in DS, some studies indicate that people with DS have different DNA methylation compared to the general population, as specified below. First of all, DNMT3L is encoded on HSA21 (Gardiner and Davisson, 2000). As DNMT3L stimulates methylation by DNMT3a and DNMT3b, its overexpression might thus yield aberrant DNA methylation patterns, thereby potentially contributing to the cognitive deficits in DS. To our knowledge, the first report on the presence of differential DNA methylation in DS was published in 2001, demonstrating increased genome-wide hypermethylation of lymphocyte DNA in DS children with a full trisomy 21, compared to their normal siblings (Pogribna et al., 2001). In agreement, Chango et al. (2006) identified six DNA fragments that were hypermethylated in eight DS subjects compared to eight healthy controls. However, the applied technology did not allow for determination of the specific DNA sequence (Chango et al., 2006). Then, in 2010, a high

throughput screen for differentially methylated genes in DS was conducted using DNA that was extracted from total peripheral white blood cells and isolated T-lymphocytes (Kerkel et al., 2010). Compared to non-DS controls, a range of stable, gene-specific alterations in CpG methylation patterns was observed, which was independent of the differential cell counts. Strikingly, these genes were found on autosomes, other than HSA21, indicating the influence of an additional copy of HSA21 on the epigenetic marks of other chromosomes. Many of the differentially methylated genes are involved in the development and functioning of white blood cells, which is supportive of the fact that DS is characterized by immune system deficiencies, amongst others resulting in the high frequency of infections (Ram and Chinen, 2011). Recently, Jones et al. (2013) examined differential DNA methylation in cheek swabs of ten adult DS subjects and ten age-matched, healthy controls. 3300 CpGs were reported with DNA methylation levels that differed more than 10% between both groups. Interestingly, cognitive function was assessed using the Dalton Brief Praxis test and correlated to the DNA methylation results. Five differentially methylated probes correlated with cognitive functioning as well, indicating the relation between cognitive impairment due to trisomy 21 and altered DNA methylation. Two of those probes were observed in the TSC2 gene, which has been associated with the tau pathology of AD – the second major cognitive deficit in DS (Jones et al., 2013).

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Fig. 2. Schematic illustration of the CBS-induced depletion of SAM and its effects on nuclear and mitochondrial DNA methylation. Simplified: CBS is encoded on HSA21 and therefore overexpressed in DS, leading to the increased conversion of homocysteine into cystathionine. Accordingly, less homocysteine is available for conversion into the SAM-precursor methionine, yielding decreased SAM levels and a subsequently reduced DNA methylation capacity. Although hypomethylated mtDNA was indeed observed (Infantino et al., 2011), various studies reported hypermethylated nuclear DNA in DS (Chango et al., 2006; Infantino et al., 2011; Pogribna et al., 2001). Interrupted lines indicate that intermediary components are omitted. CBS, cystathionine ␤-synthase; mtDNA, mitochondrial DNA; SAM, S-adenosylmethionine.

Interestingly, reduced levels of the methyl donor SAM have been described in DS. Despite the aforementioned reports on hypermethylation in DS, this suggests a reduced cellular methylation capacity (Infantino et al., 2011; Pogribna et al., 2001). The decreased SAM levels are attributed to overexpression of the HSA21-encoded cystathionine ␤-synthase (CBS) in DS. CBS is a central enzyme in the one-carbon metabolism, catalysing the conversion of homocysteine into cystathionine. This is the first step in the transsulfuration pathway, which results in the synthesis of the antioxidant glutathione (Fig. 2). As a consequence, less homocysteine is available for the methionine cycle in which homocysteine in converted into methionine, the precursor of SAM (Pogribna et al., 2001). Therefore, reduced SAM levels and the subsequently reduced methyl transfer to DNA is a likely mechanism underlying aberrant DNA methylation patterns in DS. In addition to nuclear DNA methylation, SAM is also required for methylation of cytosines in mitochondrial DNA (mtDNA) by the mitochondrial DNMT1, the only catalytically active DNMT found in mitochondria so far (Infantino et al., 2011; Shock et al., 2011). Importantly, despite a more than 50% increased expression of the SAM carrier, which transports SAM into the mitochondrion, significantly decreased mitochondrial SAM levels were found in lymphoblastoid cells of DS compared to controls (Infantino et al., 2011). Whereas previous studies reported hypermethylation of nuclear DNA in DS (Chango et al., 2006; Infantino et al., 2011; Pogribna et al., 2001), the opposite was demonstrated for mtDNA (Fig. 2). Compared to age-matched controls, mtDNA is hypomethylated in DS, suggesting an impaired mitochondrial methylation capacity in DS that, in turn, could lead to mitochondrial dysfunction (Infantino et al., 2011). Indeed, mitochondrial dysfunction has been convincingly demonstrated in DS, including reduced expression of genes encoding mitochondrial enzymes (Conti et al., 2007; Lee et al., 2003) and impaired ATP synthesis (Valenti et al., 2010). The latter is likely to affect many epigenetic processes that require ATP as a substrate, including the ATP-dependent production of SAM for DNA methylation (Wallace and Fan, 2010). The case of altered DNA methylation in DS is reinforced by a recent study that demonstrated global hypermethylation in placental villi samples derived from DS fetuses compared to normal villi, thereby illustrating that epigenetic changes are already present in early development (Jin et al., 2013). Indeed, DNA methylation provides a new diagnostic method for the detection of DS. In contrast to the commonly used invasive (and risky) sampling procedures to obtain fetal genetic material, a novel non-invasive prenatal detection method was recently developed using free fetal DNA in the

maternal peripheral blood. This method analyses differences in DNA methylation of HSA21 regions between the mother and her child and is based on the occurrence of fetal-specific differentially methylated regions (DMRs) on HSA21. Ratios of such DMRs for fetus over mother will be different for a trisomic child, which has an additional copy of the differentially methylated HSA21 locus, compared to a non-DS fetus. Comparing these methylation ratios for a combination of DMRs between normal and DS cases enabled correct non-invasive prenatal diagnosis of DS (Papageorgiou et al., 2011). Recently, an additional study validated two of these DMRs as potential fetal-specific epigenetic markers (Lim et al., 2014). To which extent DNA methylation patterns are altered in the DS brain, particularly brain areas involved in learning and memory, remains to be elucidated. In that context, the hydroxylation of 5mC into 5-hydroxymethylcytosine (5hmC), an early intermediate of DNA demethylation, might be very relevant: TET proteins mediate this hydroxylation and were shown to be downregulated in the DS placenta, which possibly contributes to the aforementioned hypermethylated regions (Guo et al., 2011a,b; Jin et al., 2013). Interestingly, 5hmC has the highest prevalence in mature neurons compared to other mammalian cells and global 5hmC expression increases with ageing, particularly at genes involved in neurological disorders, such as AD (Weng et al., 2013). The relationship between 5hmC and learning and memory is still in its infancy. However, its high expression in neurons and increased levels during ageing and AD, seem to indicate a relevant role for 5hmC and TET. In conclusion, increasing evidence indicates aberrantly methylated DNA in DS. Although DNA methylation is generally regarded as a stable epigenetic modification, recent findings have elucidated that intermediate DNA modifications are present, especially in neurons (Weng et al., 2013). The reversibility of DNA methylation in neurons might offer a great potential for the treatment of neurological disorders, such as the cognitive deficits and AD in DS.

5. Altered histone tail modifications and core variants are associated with DS Next to DNA methylation, post-translational histone tail modifications also alter the chromatin structure and thus likely the gene expression profiles. Furthermore, the chromatin structure is affected by constitutive chromatin proteins and by incorporation of different histone core variants.

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5.1. Post-translational histone modifications As described before, post-translational histone tail modifications affect synaptic plasticity, learning and memory. To our knowledge, no studies so far have profiled post-translational histone modifications in DS. Despite the lack of direct proof for altered histone marks in DS, there is an increasing body of evidence suggesting a strong contribution of histone modifications to the neurological deficits observed in DS and other intellectual disabilities. In this respect, five DSCR genes (DYRK1A, ETS2, HMGN1, BRWD1 and RUNX1) have been currently identified that influence particular histone modifications, suggesting abnormal modifications in DS. Firstly, the DSCR gene DYRK1A contributes to learning deficits in DS (Smith et al., 1997). DYRK1A is a member of the Dual specificity tyrosine-phosphorylated and regulated kinase (DYRK) family. This highly conserved subfamily of protein kinases catalyzes autophosphorylation on tyrosine residues and phosphorylation of serine/threonine residues on exogenous substrates (Becker and Joost, 1999). Studies in Drosophila and mice have revealed that the DYRK1A protein is necessary for normal brain development in a dose-sensitive way (Fotaki et al., 2002; Tejedor et al., 1995). Lepagnol-Bestel et al. (2009) demonstrated that increased DYRK1A expression in primary murine cortical neurons reduced dendritic growth and complexity (Lepagnol-Bestel et al., 2009). In addition, transgenic mice that overexpressed DYRK1A showed significant impairment in cognitive flexibility and spatial learning. This indicates a causative role of DYRK1A overexpression in mental retardation in DS (Altafaj et al., 2001). Interestingly, DYRK1A has a bipartite effect on histone modifications. First of all, DYRK1A directly phosphorylates the Thr522 of the SIRT1 histone deacetylase, thereby promoting deacetylation and possibly deteriorating cognitive capacities (Guo et al., 2010). In addition, DYRK1A phosphorylates the cyclic AMP response element-binding protein (CREB) at serine 133, thereby inducing the recruitment of the CREB binding protein (CBP/P300). CBP/P300 is a histone acetyl transferase that promotes CREBmediated expression of genes (Weeber and Sweatt, 2002; Yang et al., 2001). A loss of function mutation in CBP/P300 results in the aforementioned Rubinstein-Taybi syndrome that includes mental retardation (Barrett and Wood, 2008; Bartholdi et al., 2007). In addition to DYRK1A, two other HSA21 proteins influence the activity of CBP/P300: the erythroblastosis virus E26 oncogene homolog 2 (ETS2) and the nucleosome-binding high-mobility group N1 (HMGN1) (Sun et al., 2006; Ueda et al., 2006). Therefore, it is conceivable that the HAT/HDAC balance is deregulated in DS, causing aberrant histone acetylation patterns that affect learning and memory processes. Apart from its direct phosphorylation effects, DYRK1A also alters gene expression via the neuron-restrictive silencer factor (NRSF, also known as REST). NRSF regulates the expression of a range of neuronal genes involved in, amongst others, ion channels, neurotransmitter receptors and synapses (Canzonetta et al., 2008; Schoenherr and Anderson, 1995; Sun et al., 2005). NRSF represses transcription of these neuronal genes in non-neuronal cells by binding to the neuron-restrictive silencer element (NRSE). Besides non-neuronal cells, NRSF is also present in undifferentiated neuronal progenitors. However, it ceases to be expressed in differentiated neurons, thereby enabling gene expression. Therefore, NRSF has been termed as ‘a master negative regulator of neurogenesis’ (Schoenherr and Anderson, 1995). In DS, NRSF levels seem to be perturbed. For instance, decreased NRSF expression was observed in the aforementioned placental villi samples from DS fetuses compared to non-DS villi (Jin et al., 2013). Moreover, various NRSF-regulated genes were repressed in neurospheres derived from fetal DS brain cells, whilst

non-NRSF-regulated genes with similar functions were unaffected (Bahn et al., 2002). Furthermore, Canzonetta et al. (2008) reported a 30–60% reduced NRSF expression in the transchromosomic TgDyrk1A mouse model of DS, resulting in increased transcript levels of downstream targets (Canzonetta et al., 2008). This inverse correlation, however, was lost in another transgenic mouse model of DS that overexpressed DYRK1A (Lepagnol-Bestel et al., 2009). In agreement, silencing the third copy of DYRK1A by RNA interference rescued NRSF levels, thereby confirming the important role of DYRK1A in NRSF-mediated gene regulation (Canzonetta et al., 2008). Importantly, DYRK1A regulates NRSF by binding to the SWI/SNF chromatin remodeling complex (Lepagnol-Bestel et al., 2009). This complex uses ATP to mobilize nucleosomes and rearrange the chromatin structure and induces the expression of multiple other genes involved in histone modifications, e.g. the histone methyl transferase L3MBTL2, the histone demethylase JARID1D and the HDAC interactor NCOR (Lepagnol-Bestel et al., 2009; Lu and Roberts, 2013; Sanchez-Mut et al., 2012). Therefore, the overexpression of DYRK1A in DS is likely to affect a range of epigenetic mechanisms, thereby strongly indicating that epigenetic marks are presumably altered in DS compared to the non-DS population. Recently, Altafaj et al. (2013) administered short hairpin RNA against DYRK1A to Ts65Dn mice, resulting in normalized DYRK1A protein levels, improved synaptic plasticity and partial amelioration of the hippocampal-dependent search strategy in the Morris water maze (Altafaj et al., 2013). In the same year, De la Torre et al. (2014) reported that the DYRK1A inhibitor epigallocatechingallate (EGCG), a green tea flavonol, rescued visuospatial memory (Morris water maze) and object recognition memory (novel object recognition test) in both Ts65Dn and TgDyrk1A mice. Moreover, a pilot study showed that EGCG-treatment (3 months) had positive effects on visual memory recognition and working memory, as well as quality of life and social functioning, in young adult DS subjects, compared to placebo controls (De la Torre et al., 2014). Again, this demonstrates the important contribution of DYRK1A to learning and memory deficits in DS. Interestingly, cancer studies currently investigate EGCG as an epidrug, since it can inhibit DNMT activity. Indeed, re-expression of genes that were silenced through promoter methylation has been observed (Fang et al., 2003; Nandakumar et al., 2011). Besides the known effects of EGCG on DYRK1A, it might thus improve memory formation via altering the methylation profile of DS individuals as well, which would emphasize the potential of epidrugs (further discussed in Section 8) in decreasing cognitive symptoms in DS. In addition to DYRK1A, two other DSCR-encoded proteins interact with the SWI/SNF complex, thereby altering histone modifications and likely gene expression: BRWD1 and RUNX1. The bromodomain and WD repeat domain containing 1 (BRWD1) modulates the chromatin by binding through its two bromodomains and by associating with the SWI/SNF complex (Huang et al., 2003). Moreover, the Runt-related transcription factor 1 (RUNX1) forms multiprotein complexes at target gene promoters to which the SWI/SNF subunits BRG1 and INI1 bind. RUNX1 is associated with histone modifications that are typical of euchromatin, such as dimethylated H3K4 and acetylated H4, again illustrating the role of epigenetics in DS (Bakshi et al., 2010). Unfortunately, the role of the SWI/SNF complex has not been investigated in DS yet. However, mounting evidence indicates the involvement of this chromatin-remodeling complex in neurodevelopment and hence might be critical in the cognitive deficits in DS. For instance, the expression of the SWI/SNF subunit BRG1 is enriched in the brain and the spinal cord of mice embryos (Randazzo et al., 1994) and the dorsal neural tube of chick embryos

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(Schofield et al., 1999). In zebrafish, Eroglu et al. (2006) revealed that BRG1-deficiency leads to impaired neurogenesis and neural crest cell differentiation (Eroglu et al., 2006). Furthermore, the contribution of the SWI/SNF complex to cognitive deficits is reinforced by the finding that the X-linked alpha-thalassemia mental retardation (ATRX) syndrome is caused by mutations in the gene that encodes the SWI/SNF protein ATRX (Gibbons et al., 1997; Villard et al., 1996). Therefore, aberrant functioning of the SWI/SNF complex due to overexpressed HSA21 products might lead to mental retardation in a similar way. Finally, post-translational histone modifications might be altered due to mitochondrial dysfunction in DS. Mitochondria are the major cellular source of high energy intermediates like acetylcoenzyme A, nicotinamide adenine dinucleotide (NAD+), SAM and ATP, which are respectively involved in acetylation, deacetylation, methylation and phosphorylation of histones (Wallace and Fan, 2010). Consequently, aberrant mitochondrial production of these high energy intermediates in DS, for example due to the overexpression of CBS, is likely to cause alterations in post-translational histone marks in DS. Interestingly, a recent study revealed that incubation of DS lymphoblasts and fibroblasts with EGCG counteracted mitochondrial dysfunction. In particular EGCG stimulated mitochondrial biogenesis and rescued ATP synthase catalytic activity and oxidative phosphorylation (Valenti et al., 2013), probably restoring the levels of one or more high energy intermediates. In addition to its inhibitory effect on DYRK1A, EGCG might thus improve learning and memory by rescuing mitochondrial functioning in DS. 5.2. Histone core variants and constitutive chromatin proteins HSA21 encodes the H2A histone family member Z pseudogene 1 (H2AFZP) and the H2B histone family member S pseudogene (H2BFS) (Gardiner and Davisson, 2000; Sanchez-Mut et al., 2012; The National Center for Biotechnology Information, 2013). Whereas H2BFS has been described as component of the nucleosomal core, it is currently unknown whether the H2AFZP encodes a protein (Sanchez-Mut et al., 2012; UniProtKB/Swiss-Prot, 2013). Both their effect on gene regulation needs to be established as well. In addition to these histone variants, two DSCR encoded constitutive chromatin proteins contribute to nucleosome assembly: CHAF1B and HMGN1. The Chromatin assembly factor 1B (CHAF1B) protein is involved in nucleosome assembly onto newly replicated DNA by recruiting H3 and H4 (Kaufman et al., 1995; Verreault et al., 1996). Reese et al. (2003) showed that CHAF1B forms a multiprotein complex with the methyl-CpG binding protein (MBD)-1 and heterochromatin protein (HP)-1, thereby again demonstrating the involvement of epigenetics in DS (Reese et al., 2003). A second constitutive chromatin protein that is overexpressed in DS is HMGN1. As described before, HMGN1 affects post-translational histone modifications, in particular it inhibits phosphorylation of H3S10 and H3S28 and enhances H3K14 acetylation via CBP/P300 (Abuhatzira et al., 2011; Ueda et al., 2006). In addition, HMGN1 has been described to regulate the expression of MeCP2. MeCP2 is highly expressed in the brain and can activate or repress gene transcription (Brink et al., 2013). Altered MeCP2 activity may result in mental retardation and learning disabilities (Abuhatzira et al., 2011; Samaco and Neul, 2011). Abuhatzira et al. (2011) reported transcript levels of HMGN1 to be increased with 50% and transcript levels of MeCP2 to be decreased with 30% in brain tissue of DS patients compared to non-DS age-matched controls. In mice, it was found that altered HMGN1 protein levels resulted in histone modifications in the MeCP2 promoter and a modified chromatin structure (Abuhatzira et al., 2011). Therefore, overexpressed HMGN1 might disturb normal learning and

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memory processes through reduced MeCP2 protein expression and subsequently altered epigenetic marks.

6. MicroRNAs and long non-coding RNAs in DS In the recent years, growing evidence implicates miRNAs and to a lesser extent lncRNAs in various neurodevelopmental disorders with intellectual disability, such as Fragile X, Prader–Willi and Rett syndromes (Della Ragione et al., 2014; Saab and Mansuy, 2014). Despite the limited knowledge about the role of miRNAs and lncRNAs in DS, it has become clear that various non-coding RNAs are encoded on HSA21. Five miRNAs are located on HSA21 and therefore likely overexpressed in DS: miRNA-99a, miRNA-125b-2, miRNA-155, miRNA-802 and let-7c (Sanchez-Mut et al., 2012; Sethupathy et al., 2007; The National Center for Biotechnology Information, 2013). Although the contribution of these five miRNAs to DS is far from understood, new evidence implicates some of them in pathways that are altered in DS. For example, miRNA-155 is implicated in the endosomal pathway, which is dysregulated in DS, e.g. shown by the significantly enlarged early endosomes in DS pyramidal neurons (Cataldo et al., 2000). In that respect, it is interesting that miRNA-155 downregulates the CCAAT/enhancer binding protein ␤ (C/EBP␤), a transcription factor that regulates the expression of the Sorting nexin 27 (SNX27). SNX27 regulates endosomal receptor recycling, particularly promoting glutamate receptor recycling from early endosomes to the (synaptic) plasma membrane (Joubert et al., 2004; Wang et al., 2013). Indeed, DS brain samples presented increased expression of miRNA-155 and decreased expression of C/EBP␤ and SNX27. Importantly, SNX27 knock-out mice showed synaptic dysfunction and deficits in learning and memory, while overexpression of SNX27 in Ts65Dn mice rescued these impairments (Wang et al., 2013). Therefore, the overexpression of miRNA-155 in DS is likely to result in altered endosomal protein sorting and decreased expression of glutamate receptors at the synaptic membrane, thus affecting normal synaptic functioning (Wang et al., 2013). Furthermore, studies into Rett syndrome revealed that miRNA155 and miRNA-802 target MeCP2 (Samaco and Neul, 2011). Therefore, the downregulation of MeCP2 in DS might be due to these miRNAs, thereby potentially contributing to mental retardation and learning and memory impairment (Abuhatzira et al., 2011; Nagarajan et al., 2006; Samaco and Neul, 2011). Interestingly, Qiu et al. (2012) recently reported that MeCP2 is an important factor in homeostatic negative feedback after neuronal activation: MeCP2 binds to the promoter of the glutamate receptor 2 (GluR2) and subsequently inhibits its expression by recruiting a repressor complex that includes HDAC1. This homeostatic response is required to maintain the neural network and is disrupted in the mecp2 knockout mouse model for Rett syndrome (Qiu et al., 2012). Interestingly, Ts2 mice, another segmental trisomy model of DS, present glutamatergic deficits in the hippocampus. The reduced hippocampal glutamate levels related to impaired hippocampal synaptic plasticity and deficits in spatial memory, suggesting a role for abnormal glutamatergic neurotransmission in the cognitive symptoms in DS (Kaur et al., 2014). Therefore, the miRNA-155 mediated downregulation of SNX27 and possible downregulation of MeCP2 through miRNA-155 and miRNA-802 might cause glutamateric aberrations and related learning and memory deficits in DS. Resembling miRNAs, various regulatory roles in gene expression have been demonstrated for lncRNAs, including modulation of the chromatin state by binding to chromatin-modifying proteins (Della Ragione et al., 2014; Mercer and Mattick, 2013). A lncRNA database search revealed that a substantial number of lncRNAs with yet unknown function is encoded on HSA21 (Bhartiya et al., 2013).

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Table 2 Currently approved epidrugs. Epidrug (active ingredient)

Trade name

5-Azacytidine

Vidaza

Decitabine Vorinostat Romidepsin

Istodax

Valproic acid

Various

Class

FDA approval

EMA approval

19-05-2004

17-12-2008

Dacogen

02-05-2006

20-09-2012

Zolinza

06-10-2006

Withdrawn

05-11-2009

Refused

Various approval dates

Various approval dates

DNMT inhibitor

Indication Myelodysplastic syndromes

Cutaneous T-cell lymphoma HDAC inhibitor Epilepsy Bipolar disorder

EMA, European Medicines Agency; FDA, US Food and Drug Administration. References: Mack (2010) and Nebbioso et al. (2012). Approval dates were obtained from: European Medicines Agency (2014) and US Food Drug Administration (2014).

The importance of lncRNAs in synaptic plasticity and learning and memory demonstrates the necessity to investigate the role of these and other lncRNAs in DS.

histone acetylation and histone methylation (for a comprehensive review see Veerappan et al., 2013). Therefore, various epigenetic marks are likely altered in demented DS individuals as well.

7. Epigenetics in DS: a link to Alzheimer’s disease?

8. Epigenetic therapies for cognitive deficits in DS

In addition to their congenital learning and memory impairments, the cognitive capacities of people with DS are likely to deteriorate later in life due to a higher risk for early-onset AD. The triplication of the APP gene has been regarded as the main cause of this strongly increased risk for AD. Indeed, people with DS present higher levels of APP and its splicing product A␤, which characterizes the main pathological hallmark of AD: the extraneuronal A␤ plaques (Ness et al., 2012). Interestingly, post-mortem analysis revealed that almost all individuals with DS have a full-blown AD-like neuropathology around mid-life. As previously described, 30–50% of the DS individuals do not show signs of dementia, despite the presence of A␤ plaques and intracellular neurofibrillary tangles of hyperphosphorylated tau protein. In addition, in those who get AD, the age at which the first clinical symptoms appear varies tremendously (Lott and Dierssen, 2010; Ness et al., 2012; Wilcock, 2012; Zigman and Lott, 2007) Therefore, solely focusing on the consequences of the overexpressed APP on HSA21 is too limited to explain this considerable inter-individual variability of AD in DS. Clearly, other factors determine why one DS individual becomes demented and the other not, while both present this extensive pathology in the brain. Which factors are essential contributors to AD in DS is currently far from understood. However, it is conceivable that different expression levels of these factors determine the presence or absence of clinical dementia symptoms. Again, epigenetic mechanisms affect gene expression, and are thus potential therapeutic targets to interfere with AD in DS. Importantly, various overexpressed HSA21 genes have been attributed a role in the progression of AD, possibly via their downstream epigenetic effects. For instance, besides phosphorylating APP at Thr668 (Ryoo et al., 2008), DYRK1A is also able to phosphorylate multiple sites of tau proteins, thus contributing to AD pathology. Indeed, these tau sites were found to be hyperphosphorylated in adult DS brains (Liu et al., 2008). Whether DYRK1A-mediated histone (de)acetylation and the SWI/SNF complex contributes to this is currently unknown. Furthermore, various studies show altered expression of miRNAs in AD (Tan et al., 2013), e.g. miRNA-125b is increased in the AD brain, possibly relating to the overexpressed miRNA-125b2 in DS (Lukiw, 2007). Disturbed miRNA-125b2 levels in DS might thus relate to the strongly increased risk for AD in DS. Although epigenetic studies in DS are in its infancy, an increasing body of evidence demonstrates aberrant epigenetic modifications in AD in the general population, including DNA methylation,

Despite various ongoing clinical trials in DS (U.S. National Institutes of Health, 2013), no treatment is currently available to prevent or alleviate the impaired learning and memory nor the early-onset AD in DS. Education and a stimulating environment might (slightly) improve cognitive capacities. However, mental retardation and the progression of AD in DS cannot be ameliorated or prevented yet. To that end, various studies have tried to develop pharmacological treatments to improve cognition in DS. In DS mouse models, such as the widely used Ts65Dn mice, promising cognitive-enhancing results have been described (Wiseman et al., 2009). However, none of the investigated pharmacological treatments for DS patients have reached the market. Moreover, almost all clinical trials with drugs for AD in the general population have failed so far. The US Food and Drug Administration (FDA) has approved symptomatic AD treatment with cholinesterase inhibitors, such as donepezil. However, these drugs only provide short-term relief by improving cognition and to a certain, rather limited extent behaviour, but do not interfere with the underlying neurodegeneration (Mangialasche et al., 2010). Interestingly, a 10-week administration of donepezil (2.5–10 mg/day) to DS children of 10–17 years of age failed to show cognitive improvement (Kishnani et al., 2010). To our knowledge, other AD drugs, such as those targeting the accumulation of A␤, did not receive a market approval or are still in the process of clinical trials (Mangialasche et al., 2010; U.S. National Institutes of Health, 2013). Hence, investigating new targets and therapies is important. Here, we have pointed out the major role that epigenetics play in synaptic plasticity and learning and memory. Various aberrant epigenetic modifications contribute to intellectual disabilities, and hence, might be involved in the cognitive deficits in DS. In contrast to the triplication of HSA21, epigenetic marks are reversible, and thus ideal targets to alleviate certain features in DS. Therefore, drugs that inhibit epigenetic enzymes, so-called epidrugs, offer a promising new way of treatment as alternative for, or to act synergistically with, classical pharmacology. The field of epigenetics is booming, especially in cancer research, and provides new approaches to address a wide variety of diseases. Currently, the FDA has approved four epidrugs against cancer (Table 2): the DNA methyltransferase inhibitors Vidaza (5-azacytidine) and Dacogen (decitabine) with an indication for myelodysplastic syndromes and the HDAC inhibitors Zolinza (vorinostat) and Istodax (romidepsin) with an indication for cuteneous T-cell lymphoma (Mack, 2010; Nebbioso et al., 2012). In addition, it was demonstrated that valproic acid, which is already used against epilepsy and bipolar disorders for years, acts as a HDAC

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inhibitor and has anti-cancer properties as well (Papi et al., 2010). Previously, studies showed that HDAC inhibitors may rescue memory impairments, thereby arousing many researchers’ interest in histone acetylation and finding specific HDAC inhibitors to combat learning and memory deficits (for a comprehensive review see Graff and Tsai, 2013). Nevertheless, to our knowledge, no other epidrugs than valproic acid are currently approved for neurological complications. However, a wide array of other clinical trials is ongoing for neurological disorders, including a trial in DS with the aforementioned EGCG. Furthermore, additional studies investigate new indications for the approved epidrugs. An up-to-date review is provided in Nebbioso et al. (2012). Despite the promising results of the approved treatments (Table 2), epidrugs have genome-wide and non-chromatin effects, thereby altering a range of biological processes. In that respect, specific targeting of genes or proteins in specific tissues is the biggest challenge. In combatting the cognitive deficits in DS, specific targeting of (a part of) the third copy of HSA21 in the brain is required without affecting peripheral epigenetic modifications. Very recently, the complete third copy was silenced in vitro using the large non-coding RNA molecule X inactive specific transcript (XIST), which endogenously silences the second X-chromosome in females. Jiang et al. (2013) introduced an inducible XIST transgene into the DYRK1A locus in induced DS pluripotent stem cells. As a consequence, stable heterochromatin marks were observed (repressive histone marks and DNA methylation), leading to chromosome-wide transcriptional silencing. As confirmation it was shown that transcription of e.g. DYRK1A and APP was repressed (Jiang et al., 2013). Future studies should demonstrate if this method would be successful in in vivo models as well. Such innovative approaches open new avenues to pinpoint pathways or genes underlying the DS phenotype. In this respect, the upcoming technology of Epigenetic Editing offers a targeted approach to silence (combinations of) individual genes, such as DYRK1A and APP, thereby validating their role in DS. Epigenetic Editing comprises the targeting of particular epigenetic enzymes (writers or erasers) to specific genes with the use of lab-engineered DNA binding domains that target the endogenous gene of interest (de Groote et al., 2012). Such engineered domains, for instance designer zinc finger proteins, are subsequently fused to an epigenetic enzyme with desired properties, e.g. a certain DNMT (Rivenbark et al., 2012; Siddique et al., 2013), DNA demethylase (Chen et al., 2014) or histone modifier (Falahi et al., 2013). As a consequence, epigenetic modifications at the target gene are actively overwritten, thereby causing long-term modulation of gene expression, either stimulating or repressing its expression (de Groote et al., 2012). Therefore, Epigenetic Editing represents a promising method to specifically modulate the expression of one or more genes, thereby opening new avenues to ameliorate the cognitive deficits in DS. The question then is, which targets? In this review we have considered epigenetic alterations due to overexpression of certain HSA21 genes (summarized in Table 1). Obviously, these genes would serve as first group of targets. However, it is conceivable that non-HSA21 products influence the epigenetic mechanisms in DS as well. Unfortunately, hardly any epigenetic profiling studies have been conducted in DS. Therefore, future studies should investigate epigenetic marks in DS, preferentially including subgroups with and without AD and groups of different ages. 9. Conclusion Down syndrome is the most common genetic intellectual disability. Despite the promising results in mouse models of DS and various ongoing clinical trials, no (preventive) treatment for the two major cognitive hallmarks – congenital mental retardation

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and early-onset AD – is currently available. Findings in other intellectual disabilities, including Rett syndrome and Rubinstein–Taybi syndrome, have elucidated the role of epigenetics in synaptic plasticity and learning and memory. The field of epigenetics is booming in cancer research. However, cognitive epigenetics is still in its infancy, despite mounting evidence to suggest a crucial role for epigenetics in learning and memory and their related deficits. DS is characterized by extensive inter-individual variability. The triplication of HSA21 would theoretically result in a 1.5 fold increased expression level. However, the transcript levels of various HSA21 genes deviate from this, thereby contributing to the DS phenotypes in varying extents. Whereas most researches have tried to identify these genes, the underlying cause of this gene expression variation has been largely neglected. In that respect, epigenetic mechanisms are of essence as they regulate genome function and thus might play a crucial role in cognitive deficits in DS. Although the role of epigenetics in DS is currently far from understood, an increasing body of evidence indicates the involvement of DNA methylation, post-translational histone modifications, nucleosomal core assembly and chromatin remodelling through miRNAs and lncRNAs in DS. As is summarized in Table 1, various overexpressed HSA21 gene products are epigenetic modulators, thereby deregulating epigenetic mechanisms in DS. In turn, these disturbed mechanisms might contribute to the observed learning and memory deficits. Importantly, epigenetic marks are reversible, and thus may offer great therapeutic potential to prevent or improve the cognitive symptoms in DS. Most promising is the technique of Epigenetic Editing in which specific epigenetic enzymes, such as a DNMT or HAT, are recruited to specific genes by means of a lab-engineered DNA binding domain. Thereupon, epigenetic modifications are actively overwritten, causing a potentially persisting modulation of gene expression. Conceivably, partial repression of overexpressed HSA21 genes can yield physiological expression levels that might alleviate the cognitive deficits in DS. In short, it has become clear that one cannot ignore the involvement of epigenetics in intellectual disabilities, including DS. Although the amount of studies on epigenetics in DS is limited, these studies indicate disturbed epigenetic mechanisms due to overexpression of multiple HSA21 genes. Regarding the aforementioned cognition-enhancing therapies, future studies should identify the aberrant epigenetic marks in DS compared to the general population and between DS with and without AD, in order to subsequently overwrite these marks by using Epigenetic Editing.

Conflict of interest The authors have declared no conflict of interest.

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