epigenetics and microRNAs

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Review

Lost in translation. New unexplored avenues for neuropsychopharmacology: epigenetics and microRNAs

1.

Introduction

2.

Epigenetics

3.

miRNAs

Daniela Tardito, Alessandra Mallei & Maurizio Popoli†

4.

Expert opinion

Laboratory of Neuropsychopharmacology and Functional Neurogenomics, Dipartimento di Scienze Farmacologiche e Biomolecolari and Center of Excellence on Neurodegenerative Diseases, University of Milano, Milano, Italy

Introduction: Mood and anxiety disorders are among the major causes of disability worldwide. Despite clear need for better therapies, efforts to develop novel drugs have been relatively unsuccessful. One major reason is lack of translation into neuropsychopharmacology of the impressive recent array of knowledge accrued by clinical and preclinical researches on the brain. Here focus is on epigenetics mechanisms, including microRNAs, which seem particularly promising for the identification of new targets for alternative pharmacological approaches. Areas covered: First, the current knowledge about epigenetic mechanisms, including DNA methylation, posttranslational modification of histone proteins, focusing on histone methylation and acetylation, and posttranscriptional modulation of gene expression by microRNAs is described. Then evidence showing involvement of epigenetics and microRNAs in the pathophysiology of mood and anxiety disorders as well as evidence showing that some of the currently employed antidepressants and mood stabilizers also affect epigenetic and microRNA mechanisms are reviewed. Finally current evidence and novel approaches in favor of drugs regulating epigenetic and microRNA mechanisms as potential therapeutics for these disorders are discussed. Expert opinion: Although still in its infancy, research investigating the effects of pharmacological modulation of epigenetic and microRNA mechanisms in neuropsychiatric disorders continues to provide encouraging findings, suggesting new avenues for treatment of mood and anxiety disorders. Keywords: antidepressant, epigenetics, microRNA, mood disorders Expert Opin. Investig. Drugs [Early Online]

1.

Introduction

Neuropsychiatric disorders are among the major causes of disability worldwide. A recent estimate, from the European Brain Council of full burden and cost of brain disorders in Europe in 2010, provided startling findings: over 160 million people are affected (over 36% of European population) [1,2]. The burden of disability is over 26% of total burden of all diseases, more than any other group of medical disorders, with total social burden possibly equal or higher than cardiovascular diseases, cancer and diabetes combined. The top four most burdensome disorders are depression, dementias, alcohol use disorders and stroke. Mood disorders (MD), including major depression (MDD) and bipolar disorder (BP), and anxiety disorders (ADs) are estimated to be the most costly diagnostic groups, requiring resources of e113.405 billion each year for MD (with 33.3 million sufferers) and e74.4 billion for AD (with 61.3 million sufferers) [2]. Still 10.1517/13543784.2013.749237 © 2012 Informa UK, Ltd. ISSN 1354-3784, e-ISSN 1744-7658 All rights reserved: reproduction in whole or in part not permitted

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Mood and anxiety disorders are among the major causes of disability worldwide but despite clear need for better therapies, recent efforts to develop novel drugs for their treatment have been relatively unsuccessful. Epigenetics refers to changes in the rate of gene expression that do not involve modifications in DNA sequence. Epigenetic mechanisms such as DNA methylation and posttranslational modification of histone proteins, as well as posttranscriptional modulation of gene expression by miRNAs, have been involved in the pathophysiology of psychiatric disorders. Recent research shows that classical psychotropic drugs, including antidepressants and MSs, may exert their effects partially by inducing epigenetic changes. Epigenetic approaches to treatment of cancer or HC, by means of drugs that are able to modify methylating/ demethylating enzymes or antagomiRs are currently used in clinic or are under scrutiny. The translational application of the recent knowledge in the epigenetics field could open new avenues in neuropsychopharmacology of mood and anxiety disorders.

This box summarizes key points contained in the article.

only about one-third of all individuals affected receive any treatment; up to 70% of patients with MD have residual symptoms, and treatment-resistant MDD is a common clinical occurrence. However, despite clear need for better therapies, recent efforts to develop novel antidepressants have been relatively unsuccessful. Although brain disorders have such a heavy impact on society, an insufficient amount of funding is dedicated to European research on brain disorders, compared to United States (Figure 1) [3]. Just aggravating this situation, several major pharmaceutical companies have recently withdrawn from neuroscience research, due to the high percentage of failure of new drugs for brain disorders, and consequent high cost for development of a new drug, which is around $4 billion and can range as high as $11 billion [4]. Because industry funded nearly half of the budget for research and drug development, this retreat from neuroscience research will only make the situation worse [5]. This seems particularly serious for European research on brain disorders, which is dwarfed compared to US investment in the same field and to other research fields (Figure 1). In addition, this field suffers from high fragmentation and lack of coordination in research [3]. European Union has issued a new general approach for cooperation in research and development which should be implemented in the new Horizon 2020 Program, but it is not known whether the present crisis of the eurozone will allow complete fulfillment of this ambitious program. Aside from this, the present situation of research on brain disorders has been analyzed by several authors and a variety of possible remedies of different nature were proposed, including closer collaboration between neuroscience faculties 2

and medical centers, smaller proof-of-concept trials for compounds with good preclinical evidence, funding from alternative sources (e.g., insurance companies), focusing on endophenotypes rather than complete syndromes, integrative analysis of different datasets from Genome-wide Association Study (GWAS) and converging evidence for gene association in multiple phenotypes (e.g., phenomics), development of pathophysiology-based animal models and others [5-11]. However, a major reason for failure, pinpointed by several authors, is the lack of translation into neuropsychopharmacology of the impressive array of knowledge accrued by both clinical and preclinical research on the brain in the last decade or so. Indeed, this period has witnessed an explosion of new methodologies and findings in the neurosciences, and if one only looks at a short list of the major breakthroughs in this field, it is immediately evident that only a minor part of all this new knowledge has been translationally applied into research on pathophysiology and development of drugs. An incomplete list may include developments in genetics and genomics, particularly array-based methods and highthroughput sequencing; neuroimaging, particularly functional Magnetic Resonance Imaging (MRI); epigenetics, particularly the changes in gene usage induced by environmental factors; posttranscriptional levels of gene expression regulation, particularly microRNAs (miRNAs); live-cell imaging, bioinformatics and systems biology; optogenetics; induced pluripotent stem cells derived from peripheral cells, allowing to obtain differentiated neurons from patients. In this article we focus on two of these breakthroughs, epigenetics and miRNAs, which seem particularly promising for the identification of new targets for alternative pharmacological approaches. For sake of simplicity, our analysis will be restricted to MD and AD, although obviously similar considerations apply to other neuropsychiatric disorders, such as schizophrenia, autism and Alzheimer’s disease. 2.

Epigenetics

The term ‘epigenetics’ refers to long-lasting changes in gene expression that cannot be accounted for by changes in DNA sequence and that can be influenced by the environment [12-14]. Recent research has involved epigenetic mechanisms in memory formation and synaptic plasticity, adaptation/response to environmental stimuli (e.g., stress), and pathophysiology of psychiatric disorders, including MD [15-18]. MD are complex disorders whose pathogenesis is still essentially unknown, with about 30 -- 40% of heritability in populationbased samples [19], and it is currently accepted that genetic predisposing factors, such as DNA sequence polymorphisms that confer individual vulnerability, and environmental factors concur in the etiology of the disorders [19,20]. In addition to polymorphisms, epigenetic long-lasting modifications of transcriptional activity might explain much of the phenotypic variability in humans (Figure 2) [13]. This could open new therapeutic strategies in the treatment of MD since epigenetic

Expert Opin. Investig. Drugs [Early Online]

Investing as much as the US Investing less than the US


Investing more than the US

New unexplored avenues for neuropsychopharmacology

CONTROL AND CARE OF THE ENVIRONMENT

Concentrated solar thermal Nuclear fusion

Nanosafety Biomass - Bioenergy CO cature and storage 2 NANOTECHNOLOGIES

AGRICULTURE PRODUCTION AND TECHNOLOGY

Photovoltaic Hydrogen and fuel cells Wind energy Polar programs

Geothermal

Cancer

CIVIL SECURITY

SPACE INFORMATION AND COMMUNICATION TECHNOLOGY

BIOTECHNOLOGIES Brain diseases

Low coordination/High fragmentation

Dementia - Alzheimer

Medium

Low fragmentation/High coordination

Figure 1. A scattered public research in the European Research Area. The graph shows the relative size of European public funding compared to the United States for some Science & Technology (S&T) fields. A huge difference exists in terms of the amount of Research & Development (R&D) invested, the degree of existing coordination/fragmentation and performance. Xaxis: degree of coordination among Member State (MS) research programs and of funding and institutional fragmentation. Y-axis: logarithmic ratio of public R&D investment in Europe compared to United States. Size of bubbles is directly proportional to the amount of European public funding (MS + European Commission). Source: Towards joint programming in research: working together to tackle common challenges more effectively. COM (2008) 468. Available at: http://ec.europa. eu/research/press/2008/pdf/com_2008_468_en.pdf

modifications are potentially reversible [21]. As an example, epigenetic drugs are being tested in clinical trials in cancer research [22]. Epigenetic mechanisms DNA methylation, posttranslational modification of histone proteins and regulation of gene expression through small noncoding RNAs are three main epigenetic mechanisms widely studied at present, with the first two being able to change the structure of chromatin itself. Indeed, the condensation state of chromatin modulates gene activity with open euchromatin being associated with gene expression, while closed, condensed heterochromatin is associated with gene silencing [16]. A brief overview of these two epigenetic mechanisms is given below (Figure 3). 2.1

DNA methylation DNA methylation, a rather stable epigenetic mark, is usually associated with gene silencing [23,24] and correct pattern of DNA methylation is essential for normal development, tissue and cell specificity, genomic imprinting and inactivation of X 2.2

chromosome [13,25]. DNA methylation consists in the covalent addition of a methyl group to a cytosine, preferentially found next to a guanosine ([CpG] dinucleotides) [24], with about 70 -- 80% of cytosine within CpG methylated [25]. Unmethylated CpG-enriched region, called CpG islands, can be found in the proximity of a gene, usually close to the gene promoter region [25,26]. About 50 -- 60% of human gene promoters are associated with CpG islands consistent with a role for DNA methylation in gene silencing [13,25]. Conversely, DNA hypermethylation in gene body has been associated with transcriptional activity. DNA methylation is catalyzed by the enzymes DNA methyltransferases (DNMTs) that transfer a methyl group from the donor S-adenosyl-methionine to cytosines. Three distinct DNMTs operate in mammals. DNMT1 is a ‘maintenance’ DNMT that is able to recognize hemimethylated CpG dinucleotides in the newly synthesized DNA strand, thus permitting the inheritance of the methyl marks. Instead, DNMT3a and DNMT3b are ‘de novo’ DNMTs that methylate previously unmethylated CpG [21,24]. The sequence-related DNMT2 protein has been shown to be a de facto RNA


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DNA polymorphisms

GXE interactions

Environmental factors

along with the histone modifying enzymes and the effector proteins constitute the ‘histone code’ [16]. Here we will focus on histone lysine acetylation and methylation -- the two best characterized histone modifications. Histone acetylation Acetylation at lysine residues is generally associated with active gene expression. Indeed, acetylation, neutralizing the positive charge on lysines, disrupts the electrostatic interactions between negatively charged DNA and histone proteins, therefore relaxing chromatin structure [14,17]. Histone acetyltransferases (HATs) mediate histone acetylation. Histone deacetylases (HDACs), the enzymes which remove acetyl groups from histones, are divided into four classes: class I (HDAC1 -- 3, 8), class II (HDAC4 -- 7, 9, 10), class III or sirtuins (SIRT1 -- 7), and class IV (HDAC11) [17,29]. 2.3.1

PHENOTYPE


Genomic effects on epigenome

Environmental effects on epigenome

Epigenetic factors

Histone methylation Histone methyltransferases (HMTs) can mono-, di-, or tri-methylate lysines on histone tails. Histone methylation has been associated with both activation and repression of genes depending on the specific lysine that has been methylated. For example, histone H3 trimethylation at lysine 4 (H3K4me3) has been associated with gene expression, while H3K27me3 or H3K9me3 has been associated with gene silencing [14,18,29]. HMTs are characterized by the Su(var) 3-9, Enhancer of zeste, Trithorax (SET) catalytic domain (with the exception of the HMT Lysine Methyltransferase 4 [KMT4/DOT1L]). Histone lysine demethylases (HDMs) that catalyze lysine demethylation are classified in two classes. The first class is the amine oxidase domain-containing HDMs such as the lysine-specific demethylase 1 (LSD1/KDM1A), the second is the Jumonji-C (JmJC) domain-containing HDM. Each specific lysine residue is the target of distinct HMTs and HDMs, thus representing a complex system of gene expression regulation. The effects on gene expression of methylated histones are probably mediated through effector protein complexes that are able to remodel chromatin [14,18]. 2.3.2

Figure 2. Interaction of genetic background, environmental factors and epigenetic effects in pathophysiology. The traditional Gene x Environment scheme, envisaging that phenotype (including many diseases) is determined by the interaction of genetic background (e.g., DNA polymorphisms) and environmental factors (e.g., adverse life events, stress, etc.), must be integrated with the results of epigenetic studies. The phenotype is not only determined by genotype (DNA sequence) and environment (external impact) but also determined by environmental effects on the epigenome (activating or repressing genes) and genomic effects (e.g., polymorphisms) on the epigenome.

methyltransferase. Several recent reports suggested that DNA may be subjected to active demetylation [27,28], although the molecular mechanisms by which this occurs are still not completely understood. Two mechanisms have been proposed to explain the role of DNA methylation in gene silencing. First, methylated CpG in gene promoters will interfere with binding of transcription factors (TFs), thus reducing transcription. Second, methylated DNA-binding proteins (MBPs), such as MeCP2, will bind to regions with high density methylated CpG. MBPs are able to recruit other proteins such as histone modifying enzymes that in turn cause the condensation of chromatin and gene silencing [13,21]. Histone modifications Histone N-terminal tails are subjected to various covalent modifications, such as acetylation, methylation, phosphorylation, ubiquitylation, SUMOylation, ADP-ribosylation, deimination and proline isomerization [17,21]. These covalent posttranslational modifications are able to modify the condensation state of chromatin, thus altering gene expression. The global combinatorial pattern of histone modification 2.3

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Epigenetics in mood and anxiety disorders Numerous lines of evidence have implicated epigenetic mechanisms in MD and AD [14,16]. As mentioned above, stress, especially early life stress, is a crucial factor in MD pathophysiology. In seminal studies, Weaver et al. demonstrated the long-lasting epigenetic effect of maternal care on offspring. In adulthood, the progeny of poorer maternal caregiver dams display an anxious phenotype, higher corticosterone in response to stress, higher DNA methylation at the splice variant 17 of the glucocorticoid receptor (GR) promoter in hippocampus, lower histone H3 acetylation at lysine 9 (H3K9ac), reduced binding of the TF nerve growth factor-induced protein A (NGFI-A) to GR promoter and reduced GR17 mRNA expression, compared to progeny reared by high maternal care provider dams [30,31]. 2.4



H3K4me3

Active chromatin

H3K9ac

Co-regulators

TF

Promoter

Pol ll complexes Histone acetylation DNA demethylation

DNMTs HMTs

HATs


HDACs

HDMs MBPs

Histone deacetylation DNA methylation

DMEs

H3K9me3 H3K27me3

Inactive chromatin

Figure 3. Epigenetic mechanisms. DNA methylation and posttranslational modification of histone proteins are two major epigenetic mechanisms involved in chromatin remodeling. Open chromatin allows binding of TF and RNA Pol II complexes and is associated with active gene expression, while condensed chromatin is associated with gene silencing. Methylated CpG dinucleotides at gene promoters interfere with TF binding and are recognized by MBPs. MBPs recruit other proteins such as HDACs, DNMTs, HMTs and other corepressors that induce chromatin condensation and gene silencing. Histone trimethylation at lysine 9 (H3K9me3) and at lysine 27 (H3K27me3) are histone marks associated with inactive chromatin. Conversely, unmethylated promoters have a greater affinity for HATs, histone demethylases (HDMs), DNA demethylases (DMEs), and histone marks associated with active chromatin, including acetylated H3 at lysine 9 (H3K9ac) and trimethylated H3 at lysine 4 (H3K4me3). Black filled circles represent methylated CpG dinucleotides; white filled circles represent unmethylated CpG dinucleotides. Modified from Zhang X, Ho SJ. Mol Endocrinol 2011;46:R11-R32.

Society for Endocrinology (2011). Reproduced by permission.

Interestingly, a postmortem study of suicide completers showed that individuals with a history of childhood abuse or neglect had higher GR DNA methylation levels and lower GR mRNA expression in hippocampus compared to controls or suicides that did not experience childhood maltreatments, suggesting that the same epigenetic mechanism may work in rodents and humans [32]. Other preclinical studies highlighted how early life stress could impact the stress response through epigenetic programming. Maternal separation in mice was found to induce long-lasting increase in hypothalamic arginine vasopressin (AVP) expression, due to hypomethylation in the AVP gene enhancer region containing the MeCP2 binding sites. This hypomethylation was accompanied by increased secretion of corticosterone and increased immobility in the forced swim test (FST) [33]. Roth et al. found reduced levels of total

brain-derived neurotrophic factor (BDNF) (i.e., coding exon 9) mRNA levels and increased methylation at BDNF-4 and BDNF-9 exons in prefrontal cortex (PFC) of adult rats exposed to early life maltreatment [34]. Chronic social defeat (CSD) stress, an animal model of MDD, is able to modify acetylation and methylation state of histones [14]. Indeed, mice subjected to CSD showed decreased expression of BDNF-3 and BDNF-4 transcripts in hippocampus, associated with long-lasting increase in repressive H3K27me2 at respective promoters [35]. In a different animal model, the knock-in mouse carrying the Val66Met human polymorphism of BDNF gene, a recent study showed increased H3K27me3 at BDNF-6 promoter, accompanied by reduced transcription of corresponding transcript [36]. In addition to preclinical studies a number of clinical studies showed that epigenetic changes are involved in MD.


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For example, postmortem analysis of brains from BD (and schizophrenia) patients showed decreased expression of reelin and glutamic acid decarboxylase of 67 kDa (GAD67) mRNAs in cortical GABAergic neurons correlated with increased expression of DNMT1. Upregulation of DNMT1 has been proposed to be responsible for the transcriptional repression of reelin and GAD67 promoters [37]. Another study showed that in the frontal cortex of a subset of MDD suicides the expression level of tyrosine kinase receptor B truncated isoform 1 (TrkB.T1) (a BDNF receptor isoform mostly expressed in astrocytes) was significantly lower with respect to controls, and that this downregulation was associated with methylation at specific CpG dinucleotides near coding region of the gene and with increased H3K27 methylation [38,39]. In addition, analysis of PFC from postmortem brains of BP and MDD patients showed an increase in H3K4me3 at promoters of the synapsin genes, which are involved in presynaptic function and neurotransmitter release [40]. Epigenetics and psychotropic drugs for mood and anxiety disorders

2.5

In this section we highlight the available evidence showing that traditional antidepressants are able to alter the epigenetic landscape. Chronic treatment with the tricyclic antidepressant imipramine was shown to restore BDNF levels, reduced in a social defeat animal model of depression (see Section 2.4), by decreasing HDAC5 levels, thus increasing H3 acetylation at BDNF-3 and BDNF-4 promoters. Interestingly, chronic imipramine did not reverse social defeat stress-induced H3K27me2 increase, suggesting that stress may lead to stable repression mark at BDNF promoters. In addition, hippocampal overexpression of HDAC5 in defeated mice prevented the epigenetic effects of imipramine at BDNF promoters [35]. Amytriptyline was shown to partly reverse DNA hypomethylation in cultured astrocytes, by reducing DNMT1 activity [41]. In a recent study with the Flinders Sensitive Line rat, a genetic animal model of depression, chronic treatment with the selective serotonin reuptake inhibitor (SSRI) escitalopram reversed DNA hypermethylation at the p11 gene promoter. This was associated with increased p11 expression and reduced DNMT1 and DNMT3a expression [42]. A study showed that the SSRI fluoxetine reversed dentate gyrus (DG) H3K9me3 decrease following 21-day chronic restraint stress in rats [43]. In addition, monoamine oxidase (MAO) inhibitors are able to inhibit H3K4 demethylation (see Section 2.6.3). The anticonvulsant and mood stabilizer (MS) valproate has been shown to inhibit classes I and II HDACs [29]. Mice treatment with valproate was able to reduce methylation of reelin and GAD67 promoters induced by previous methionine (a methyl donor) administration. This accelerated demethylation was accompanied by increased H3 acetylation at both reelin and GAD67 promoters [44]. In addition, valproate was shown to enhance extinction of conditioned fear (a type of learning related to 6

human AD and phobias) in mice. This was accompanied by increased H4 acetylation around BDNF-1 and BDNF-4 promoters, and increased expression of BDNF-4 mRNA in mouse PFC subjected to partial extinction training [45]. Drugs regulating epigenetic mechanisms as potential therapeutics for mood and anxiety disorders

2.6

As discussed above, epigenetic modifications have been associated with MD and AD in both preclinical and clinical studies. Moreover, psychotropic drugs have been shown to induce epigenetic changes in several brain areas. Taken together these findings suggest that development of molecules targeting epigenetic mechanisms (i.e., DNA methylation or histone posttranslational modifications) could open new treatment options for psychiatric disorders. We focus our discussion on three classes of potential drugs in the following sections: the DNMT inhibitors (DNMTi), the HDAC inhibitors (HDACi), and compounds that target HMT and HDM activity (Table 1). DNA methyltransferase inhibitors DNMTi, especially cytidine analogs, have been mainly characterized for their anticancer activity in cell culture models and in several human malignancies (Table 1). DNMTi can be classified in two classes: the first includes nucleoside analogs that are incorporated into DNA and catalytically inhibit DNMT, the second is formed by the non-nucleosides DNMTi. Two nucleoside analogs, 5-azacytidine (5-azaC) and 5-aza-2-deoxycytidine (5-azaD or decitabine), have been approved for the treatment of myelodysplastic syndrome [46]. Recent research highlighted that modulation of DNMT function is involved in rodent behavior. For instance, DNMT3a2 expression was shown to regulate cognitive abilities and decrease with aging [47]. A study showed that both systemic and intra-hippocampal administration of 5-azaC and 5-azaD have antidepressant-like effect in the FST and tail suspension test (TST), widely used to assay compounds with antidepressant activity. In addition to behavioral changes, this study also showed increase of hippocampal BDNF expression [48]. Interestingly, since the nucleoside analogs need to be incorporated into DNA to inhibit DNMTs, to explain their effects on postmitotic neurons, DNA-independent mechanisms of inhibition have been postulated [49]. A study mentioned above showed that 7-day treatment with the nucleoside analog zebularine was able to decrease DNA methylation at promoter 4 of BDNF gene and rescue expression of total BDNF mRNA in an animal model of early life maltreatment [34]. This result suggests that early life stress-induced changes in behavior and related epigenetic marks are potentially reversible by DNMTi in adulthood. Non-nucleoside DNMTi are also currently being developed and used in preclinical research in oncology [46]. Among them, the antiarrhythmic procainamide and RG108 have been employed as DNMTi in neuroscience basic research [48,50,51]. 2.6.1



Table 1. Compounds targeting epigenetic mechanisms. Compounds


DNMT inhibitors AzaC AzaD Zebularine RG108 HDAC inhibitors SB

Type

Targets methylation methylation methylation methylation

Ref.

Cytidine analog Cytidine analog Cytidine analog Non-nucleoside inhibitor

DNA DNA DNA DNA

Class I and class II HDAC inhibitor

[35,52-54,120]

[22,58] [122] [59] [22]

Trichostatin A


MS-275

Selective class I HDAC inhibitor

SAHA HMT/HDM inhibitors DZNep BIX-01294 E67-2 SL11144


Histone acetylation; Histone methylation Histone acetylation; DNA methylation Histone acetylation; DNA methylation Histone acetylation

EZH2 HMT inhibitor G9a HMT inhibitor JHDM1D HDM inhibitor LSD1 HDM inhibitor

H3K27 methylation H3K9 methylation H3K9 demethylation H3K4 demethylation

[48] [48] [34] [48]

[30] [44,55,121] [55]

AzaC: 5-azacytidine; AzaD: 5-aza-2-deoxycytidine; DNMT: DNA methyltransferase; DZNep: 3-Deazaneplanocin A; HDAC: Histone deacetylase; HDM: Histone demethylase; HMT: Histone methyltransferase; SAHA: Suberoylanilide hydroxamic.

Histone deacetylase inhibitors HDACi can be divided into different chemical classes, the main ones being the short-chain fatty acids (such as valproic acid and butyrates), hydroximates (such as suberoylanilide hydroxamic acid [SAHA] and trichostatin A) and benzamides (such as MS-275) [29]. Sodium butyrate (SB), a weak, nonselective, inhibitor of classes I and II HDAC, has been shown to possess antidepressant-like properties. In a study cited above, SB given systemically for 21 days to mice subjected to social defeat paradigm showed modest antidepressant-like effects [35]. In another study, SB administered chronically, alone or in combination with the SSRI fluoxetine, was able to decrease immobility score in the TST [52]. Similar results were obtained in a recent study by Yamawaki et al., who found that chronic SB was able to reduce immobility both in TST and FST [53]. However, another study did not find any changes in mouse models of depression following SB treatment [54]. Central infusion of trichostatin A, a class I and class II HDAC inhibitor, was able to reduce DNA hypermethylation, increase H3K9ac and NGFI-A at GR17 promoter and reverse behavioral and biochemical consequences of early life stress observed in adult rats reared by poorer maternal caregiver dams [30,31]. A recent study employed two HDACi, SAHA (a selective inhibitor of classes I and II HDACs) and MS-275 (a selective inhibitor of class I HDACs) to evaluate their potential role as antidepressants. Continuous infusion with the two HDACi into the nucleus accumbens of mice subjected to social defeat was shown to produce antidepressant-like effects in several behavioral tests, to increase levels of H3K14ac and to induce patterns of gene expression, measured by microarrays, similar 2.6.2

to those induced by the antidepressant fluoxetine [55]. In another study, SAHA was able to prevent repressive histone modifications induced at the metabotropic glutamate receptor 2 by atypical antipsychotics and improve their therapeuticlike effects [56]. Moreover, SAHA was shown to facilitate fear extinction, by increasing levels of acetylated histones at the promoter of the N-methyl-D-aspartate (NMDA) receptor subunit 2B [57]. Compounds targeting histone methyltransferase/histone demethylase activity

2.6.3

The fine tuning between histone acetylation and histone methylation has been suggested to be of paramount importance in neurobiology of psychiatric disorders [14,18]. Although, targeting histone acetylation may be a good strategy for the treatment of MD/AD, there are some limitations. For example, HDACi may also inhibit acetylation of nonhistone proteins and this could interfere with several cellular functions; moreover, some HDACi induce side effects such as cardiac toxicity, deficits in hematopoiesis and memory formation [18,22]. Given the complexity and specificity of HMTs and HDMs, recent research is currently focusing on how to exploit the mechanisms of histone methylation/demethylation to develop drugs that can selectively target this specific histone posttranslational modification [14,18]. Interestingly, LSD1/ KDM1A (a HDM that specifically targets H3K4) is structurally similar to MAO; the MAO inhibitor antidepressants phenelzine and tranylcypromine are able to block LSD1/KDM1A activity and, therefore, increase H3K4 methylation. This suggests that compounds with LSD1 inhibitor activity may have antidepressant properties [29]. Another target of HMTs and HDMs is H3K9. Both G9a protein (an H3K9-specific


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HMT) and H3K9me2 were decreased in nucleus accumbens of mice subjected to social defeat, while G9a overexpression in nucleus accumbens exerted antidepressant-like effects [14]. In addition, transgenic mice overexpressing SET Domain Bifurcated 1 (SETDB1) (another H3K9-specific HMT) in forebrain areas showed antidepressant-like behavior [14,18]. H3K27 could also be a possible target. Indeed, as discussed above, several reports involve H3K27me3 in the neurobiology of psychiatric disorders [35,36,38]. Specific targets in HMTs and HDMs that could be used to develop new drugs are their catalytic domains such as the SET domain for the HMTs and the amine oxidase and JmJC domains for HDMs [18]. Several new compounds of this kind are currently being studied in preclinical trials in cancer research. Different examples of this approach are the drug 3-Deazaneplanocin A (DZNep), an enhancer of zeste homolog 2 (EZH2, an HMT that specifically targets H3K27) inhibitor that targets leukemia cell [58] and BIX-01294, initially developed as potent inhibitor of G9a HMT [17]. In addition, analogs of BIX-01294 have been shown to selectively inhibit JmJC domain containing histone demethylase 1 homolog D (JHDM1D) (a JmJC H3K9 demethylase) [59]. 3.

miRNAs

The miRNAs are a large family of small (20 -- 22 nucleotides) noncoding RNAs, abundantly present in a variety of organisms from algae to humans, with a key role in the posttranscriptional regulation of gene expression. Their discovery dates back to 1993 with the identification of lin-4, a small ribonucleotide molecule involved in the regulation of ‘larva to adult switch’ in C. elegans [60]. Nowadays, mirBase, the primary online repository for all miRNA sequences and annotation, annotate 2,042 human, 1,281 mouse and 723 rat mature miRNAs [61,62]. In mammals, miRNAs are predicted to control the activity of ~ 50% of all protein-coding genes. Many miRNAs are expressed in a tissue-specific or developmental stage-specific manner, thereby contributing to cell-type-specific profiles of protein expression. Functional studies indicate that miRNAs participate in the regulation of almost every cellular process and therefore it is not surprising that changes in their expression or function are associated with many human pathologies [63-65]. miRNAs regulate protein synthesis posttranscriptionally by base-pairing to target mRNAs. Generally, miRNAs inhibit protein synthesis either by repressing translation or by inducing deadenylation and degradation of target mRNAs but were also reported to activate translation [65-69]. Individual miRNAs have the potential to target hundreds of different mRNAs, and a single mRNA could be modulated by several different miRNAs, thus implying a coordinate and fine-tuned expression of proteins in a cell and even in particular cell compartments [68,70]. In this regard it has been discovered that in neurons the whole machinery for miRNA biogenesis and function is present in different 8

compartments: neuronal somata, dendrites and axons, thus suggesting their involvement in local control of protein translation in response to synaptic activation or inhibition [70-74]. In the next sections we will summarize the processes involved in miRNA biogenesis and function, and then we will review the available data on the involvement of miRNAs in the pathophysiology and pharmacotherapy of MD and AD. miRNA biogenesis and its regulation The miRNAs can be encoded within both protein coding and noncoding transcriptional units and can be located within introns or exons. Intragenic miRNAs are synthesized together with the ‘host’ gene, using the host promoter, whereas intergenic miRNAs use their own promoters with CpG islands, TATA box sequences, initiation elements and histone modifications, suggesting control by TFs, enhancers, silencing elements and chromatin modifications [68,75-77]. Interestingly, miRNAs are often organized in interrelated clusters (within 0.1 -- 50 kb from each other) that coordinately control target mRNAs, thus regulating pathways of common cellular responses [78]. The majority of miRNAs are transcribed by RNA polymerase II to primary miRNA (pri-miRNA) transcripts, highly complex double-stranded stem loop structures of 100 -- 1,000 nucleotides in length, then processed to pre-miRNA, a »60 -- 70 nucleotide precursor, by a complex containing the RNAse-III type endonuclease Drosha and its cofactor, DiGeorge syndrome critical region 8 (DGCR8), as well as other cofactors. Pre-miRNAs, once exported in the cytoplasm by exportin-5, are cleaved in a »20 bp miRNA/miRNA* duplex by the RNase-III type enzyme Dicer and its cofactor, the mammalian TAR RNA-binding protein (TRBP). In mammals, Dicer is supported by Argonaute 2 (Ago2), a RNaseH-like endonuclease that cleave the 3’ arms of some pre-miRNAs. The ‘guide’ strand of the miRNA duplex is then loaded into the RNA-inducing silencing complex (RISC), whereas the other strand (miRNA*) is released and degraded, although in some cases both strands can associate with RISC to target distinct sets of mRNAs (Figure 4) [67,68,70,75,79]. The miRNA biogenesis can be modulated by several activating or repressing proteins that interact with Drosha, Dicer or miRNA precursors. Editing of miRNA precursors, by altering base-pairing, the structural properties of the transcripts, affecting both Drosha- and Dicer-mediated processing, or inhibiting the cytoplasmic export of pre-miRNAs, can perturb miRNA functioning with important consequences on target recognition [68,75,80]. Similarly, single nucleotide polymorphisms (SNPs), as well as point mutations within the precursors, in the mature miRNAs or in the mRNA target could alter the processing or target recognition. 3.1

RISC complex and miRNA function -- target regulation

3.2

Within the RISC complex, composed of a set of wellknown proteins and components whose functions have not



miRNA gene nucleus

pri-miRNA 5′

3′ Drosha DGCR8


pre-miRNA

pre-miRNA

cytoplasm Dicer TRBP

miRNA:miRNA* Secretion RISC

RISC AAA

mRNA cleavage

exosome AAA

Translational repression

Translational stimulation

mRNA degradation P-body sequestration

Figure 4. miRNA biogenesis and function. miRNAs are processed from precursor molecules (pri-miRNAs), which are either transcribed from independent miRNA genes or portions of introns of protein-coding genes. Pri-miRNAs (folded into hairpin structures) are then processed by a microprocessor complex containing the RNase-III type endonuclease Drosha and its partner DiGeorge syndrome critical region gene 8 (DGCR8) to pre-miRNAs (~ 70-nucleotide hairpins). pre-miRNAs are transported to the cytoplasm by exportin5, where they are cleaved by Dicer, a RNAse-III type enzyme complexed with TAR RNA binding protein (TRBP) to yield ~ 20-bp miRNA duplexes. miRNAs are then assembled into a complex called miRNA-induced silencing complex (RISC) whose key components are proteins of the Argonaute (AGO) family and other proteins that function as regulatory factors or effectors (see text for a complete description). Generally, miRNAs lead to posttranscriptional gene silencing by inducing mRNA cleavage or degradation, or altering protein production by competing with translation initiation factors and/or abrogating ribosome assembly (translation initiation), or by recruiting cellular factors that will target the degradation of the growing polypeptides (translation elongation), and/or sequestering targets into cytoplasmic P bodies. It has also been reported that miRNAs can stimulate mRNA translation. Moreover, recently miRNAs have been found in exosomes, extracellular vesicles released by most cells, containing both coding and noncoding RNAs, which can transfer genetic information to recipient cells. Reprinted from: Developmental Cell. Vol 18(4), Liu N, Olson EN. MicroRNA Regulatory Networks in Cardiovascular Development. Pag: 510-525. Copyright (2010), with permission from Elsevier.

been fully clarified, miRNAs interact with their targets through base-pairing. The initial bases of miRNA sequences (bases 2 -- 8), namely the seed sequences, are the only bases that generally pair with perfect complementarity to the target mRNA, mainly at the 3¢ untranslated region, and are responsible for definition of target specificity [65,70,81]. The RISC complex (Figure 4) is involved in at least three different functions: inhibition of translation initiation and/ or elongation, cotranslational protein degradation, premature termination of translation and mRNA deadenylation (resulting in mRNA degradation) [63,68,69].

The principal components of RISC are Dicer (involved in the processing of pre-miRNAs and in the upload of miRNAs in RISC), the Ago proteins and the glycine-tryptophan protein of 182 kD (GW182) proteins. A major role for Ago is the recruitment of other proteins within RISC, such as GW182 proteins, the key players of miRNA-mediated translational repression and mRNA degradation. The recruitment of Ago to targeted mRNAs can induce deadenylation of the polyadenylated 3¢-end thus eliciting mRNA degradation, and can also affect the formation of functional ribosomes by competing with translation initiation factors and/or


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abrogating ribosome assembly (translation initiation). The Ago--miRNA complex can also lead to degradation of the growing polypeptides (translation elongation) by recruiting peptidases or posttranslational modifying enzymes [82]. miRNAs in brain: a role in mood and anxiety disorders


3.3

In the past few years growing evidence has supported a key role for miRNAs in central nervous system (CNS) development and homeostasis. It has been reported that almost 50% of all identified miRNAs are expressed in mammalian brain, where they appear to be differentially distributed not only in specific areas but also within neurons and distinct neuronal compartments. This highly specific compartmentalization has been shown to be a key factor in the control of local protein expression, synapse development and function. A role for miRNAs in neurogenesis, neuronal differentiation and survival, as well as in neuroplasticity, is now well established, although further work is needed to better clarify these aspects (reviewed in Refs [72-74,83,84]). The unique mode of functioning of miRNAs, that is, the ability of a single miRNA to target several different mRNAs often belonging to specific functional networks, has prompted research toward the study of potential involvement of miRNAs in the pathogenesis and pharmacotherapy of neuropsychiatric disorders. Numerous preclinical investigations showed that different stress paradigms may influence the expression of miRNAs and some of their targets in different brain areas, thus further supporting an involvement of miRNAs in MD and AD (reviewed in Refs [85,86]). Briefly, an increase in miR-18a levels, which targets the GR, was found in the hypothalamus of a stress-sensitive rat strain [87]. miR-34c was found to be markedly upregulated after acute and chronic stress in mice and downregulated in Dicer ablated cells [88]. Differential modifications in miRNA expression were demonstrated in relation to normal (non-learned helplessness) versus aberrant (learned helplessness) response to a repeated shock paradigm in rats [89]. It was also suggested that stress-inducible cognitive impairments could be attributable to cholinergic-mediated induction of miR-132 in hippocampus [90]. Since the first suggestion by Perkins et al. [91] of a possible involvement of noncoding RNAs in the genetic liability to schizophrenia, several lines of evidence of dysregulated miRNAs in other neuropsychiatric disorders have been provided. Perkins et al. were the first to describe an altered expression profile of miRNAs in PFC of schizophrenic and schizoaffective individuals [92]. Subsequently, it was reported that an altered profile of miRNA expression is also present in postmortem PFC from bipolar patients. Kim et al. found that 15 miRNAs were differentially expressed with respect to healthy controls: seven were upregulated and eight were downregulated. Target analysis for four validated miRNAs yielded prediction of 2,666 genes; several of them were from networks related to nervous system function and disease [93]. Moreau et al. reported altered levels of 24 miRNAs in 10

postmortem brain from schizophrenic or bipolar subjects [94]. Notably, most of the differentially expressed miRNAs were underexpressed in both the schizophrenia and bipolar groups relative to controls, in line with previous results [92,93], but only a few differentially expressed miRNAs were in common among the various studies. More recently, an overall decrease in miRNA expression was observed in PFC of depressed suicides, with significant modifications in 21 miRNAs [95]. Some of the predicted targets for altered miRNAs were Vascular Endotelial Growth Factor (VEGF) receptor, Neuropilin-1, Growth-associated Protein 43 (GAP43), Synaptosomalassociated protein 25 (SNAP25), synaptojanin-1, synaptotagmin-1, Lim kinase-1, B-cell lymphoma 2 (Bcl2) and DNMT3b. In addition, other signaling proteins, ion channels, and ubiquitin ligases were identified. Several of these proteins have been previously linked to MD, thus suggesting a role for miRNAs in the pathogenesis of MDD, or at least in depressed suicides [85]. Further support for involvement of miRNAs in MD was given by studies investigating the presence of genetic variations in miRNA genes or in miRNA processing genes in affected patients. First, the ss178077483 polymorphism in the pre-miRNA-30e was positively associated with MDD risk [96]. Another study reported a significant association between the T allele of the rs76481776 polymorphism in pre-miR-182 and late insomnia in depressed patients. Transfection of the mutated form of pre-miR-182 in cells leads to overexpression of the mature miRNA, together with a reduced expression of genes known to be involved in regulation of clock period and entrainment [97]. Although the functional data remain to be confirmed in patients, these findings suggest that the described SNP in pre-miR-182 could have functional implications in the posttranscriptional modulation of circadian rhythms. Finally, a recent work identified association between specific variants in DGCR8 and Ago1 and depression in a case-control study [98]. Although more preliminary, some data suggest involvement of miRNAs also in pathological AD. It was reported that the truncated isoform of neurotrophin-3 receptor gene (NTRK3) is regulated by at least five miRNAs, a common SNP in NTRK3 is associated with the obsessive compulsive disorder (OCD) hoarding type and two rare variants affecting the miRNA-mediated regulation of NTRK3 were identified in patients with panic disorder (PD) [99]. A genetic association study revealed that two SNPs (in miR-22 and miR-339) are significantly associated with PD, whereas other SNPs (tagging miR-138-2, miR-488, miR-491 and miR-148a) are associated with different PD phenotypes but did not reach significance. Bioinformatic analyses and functional experiments showed that these miRNAs modulate the expression of several genes previously implicated in anxiety, thus suggesting that alteration in miRNAs may affect physiological pathways related to the development of AD [100]. Finally, an intriguing study recently showed that miR-144/144* and miR-16 levels are significantly increased



in peripheral blood of healthy students after a nationally administered examination for academic promotion in Japan. Changes in miR-144* and miR-16 levels significantly correlated with circulating interferon-g and/or Tumor Necrosis Factor a (TNF-a) levels from immediately after to 1 week after the examination, thus suggesting that these miRNAs could be part of an integrated response to stress in healthy subjects [101]. To the best of our knowledge, thus far no studies have investigated the presence of altered miRNA expression in postmortem brain or peripheral tissues from patients with AD. miRNAs and psychotropic drugs for mood and anxiety disorders


3.4

The first evidence of a possible involvement of miRNAs in the action of psychotropic drugs was provided by Zhou et al., who showed that chronic treatment with the MS lithium or valproate induced significant modifications in the expression levels of a number of miRNAs in rat hippocampus (Table 2) [102]. Interestingly, nine of the miRNAs (let-7b, let-7c, miR-105, miR-128a, miR-24a, miR-30c, miR-34a, miR-221 and miR-144) were modulated by both drugs. The authors utilized a ‘convergent genomic approach’ to prioritize the investigation on the predicted target genes that, based on the available data at the time of the investigation, resulted to be 1,654. Six candidates were common to both the predicted miRNA effectors and the BP risk gene lists, and many of the putative target genes were involved in neurite outgrowth, neurogenesis, and signaling of Phosphatase and Tensin Homolog (PTEN), Extracellular-regulated Kinase (ERK) and wingless-type MMTV integration site member (Wnt)/b-catenin pathways. The ability of lithium to modify miRNA expression was confirmed by a study in which 13 selected miRNAs were analyzed in lymphoblastoid cell lines (LCLs) from bipolar patients and control subjects treated with or without lithium in culture [103]. Significant changes were found in 7 of the 13 miRNAs tested after 4 days of treatment; 4 of the 7 significant miRNAs, miR-34a, miR-152, miR-155 and miR-221, consistently changed expression after 16 days of treatment. Interestingly, miR-221 and miR-34a (upregulated in this study) were previously shown to be downregulated in rat hippocampus in response to lithium [102], suggesting a tissue-specific effect on common targets. A restricted analysis on the miRNAs upregulated by lithium showed a significant inverse co-regulation in 29 and 10 predicted targets for miR-221 and miR-34a, respectively [103]. More recently, a study focused on the effects of drugs used for the treatment of bipolar mania (mainly a combination of MS and antipsychotics) on miR-134, known for its role in remodeling the neuronal structures as a consequence of synaptic activity, in peripheral blood of patients before and after treatment. Plasma miR-134 levels were reduced in drugfree patients when compared with controls; 2 or 4 weeks of treatments increased miR-134, but at both times miR-134

levels remained lower than in controls [104]. Although with some limitations (i.e., number of patients, different drugs or drug combinations), this study suggests that miR-134 may be a potential peripheral marker of acute mania and associated with treatment response. A seminal study from Baudry et al. was the first to demonstrate a possible role for miRNAs in the action of antidepressants [105]. It was reported that miR-16, which among other targets also regulates the serotonin transporter (SERT), is expressed at higher levels in noradrenergic as against serotonergic cells; its reduction in noradrenergic neurons causes de novo SERT expression. In mice, prolonged exposure to fluoxetine, whose primary mechanism of action is related to the inhibition of SERT, increased miR-16 levels in serotonergic raphe nuclei, which in turn reduced SERT expression. Upon infusion into raphe, fluoxetine lead to release of the neurotrophic factor S100b, which acts on noradrenergic cells of the locus coeruleus by decreasing miR-16, and thus turning on the expression of SERT in noradrenergic neurons. Although several aspects remain to be clarified, this data suggest a role for miR-16 in the therapeutic action of SSRI antidepressants, creating new serotonin sources through the switch of noradrenergic neurons toward a serotonergic phenotype. In this context, antidepressant effects on the miRNome expression in both depressed patients and in rat hippocampus were recently investigated by means of whole-miRNome quantitative analysis. A clinical study reported changes in miRNA blood expression induced by 12 weeks of treatment with the SSRI escitalopram in depressed patients. Out of 755 analyzed, 30 miRNAs were differentially expressed after treatment (28 miRNAs upregulated and 2 downregulated). Many of these miRNAs have a key role in neuroplasticity and stress response and some of them were previously associated with the pathogenesis of psychiatric disorders and the mechanism of action of psychotropic drugs (i.e., let-7d, let7e, miR-26a, miR-26b, miR-34c-5p, miR-128, miR-132, miR-494 and miR-22*). miRNA targets prediction, and functional annotation analysis showed a significant enrichment in several pathways associated with neuronal function (such as neuroactive ligand-receptor interaction, axon guidance, long-term potentiation (LTP) and depression), supporting the hypothesis that these miRNAs may be involved in antidepressant action [106]. In a second report the effects of treatments for different time lengths (3, 7 and 14 days) with fluoxetine or desipramine, a tricyclic antidepressant with predominant action on the noradrenaline reuptake, were investigated in rat hippocampal miRNome. Interestingly, a significant time-associated miRNA modulation by antidepressants was found: an effect was already observed after 3 days, and was more marked after 7 days of treatment. It is noteworthy that some miRNAs were similarly modulated by both drugs, thus suggesting common targets in their mechanism of action. Bioinformatic analysis for target prediction and functional annotation produced lists


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D. Tardito et al.

Table 2. Evidence of miRNA modulation by treatments with drugs for mood and anxiety disorders.


Drugs/treatment duration

Tissue

miRNA analyzed/technique

Lithium and valproic acid (3 weeks)

Rat hippocampus

662 miRNAs (human, mouse, rat; microarray)

Lithium (4, 8 and 16 days)

LCLs from bipolar patients (in vitro treatment)

13 selected miRNAs (qPCR)

MSs and antipsychotics (2 and 4 weeks)

Blood from bipolar patients before and after treatments Rat brain, cells in culture

miR-134 (qPCR)

Blood from major depressed patients before and after treatment Rat hippocampus

Whole human miRNome (TaqMan low density array)

Fluoxetine (acute and prolonged) Escitalopram (12 weeks)

Fluoxetine and desipramine (3, 7 and 14 days)

Ketamine (acute injection), ECS (10 days), fluoxetine (21 days)

Rat hippocampus

miR-16 (different experimental approaches)

Whole rodent miRNome (TaqMan low density array)

miRNA profiling

Results

Ref.

37 miRNAs modified by lithium; 31 by valproic acid; 9 in common 7 miRNAs differentially expressed after 4 days of lithium; none after 8 days; 5 (out of 7) after 16 days; Treatments significantly increase miR-134 levels in patients without reaching control levels miR-16 modulates SERT expression in noradrenergic and serotonergic cells 28 miRNAs upregulated; 2 miRNAs downregulated

[102]

Different miRNAs modulated in a time-dependent manner by fluoxetine and desipramine; some miRNAs in common between the two drugs Fluoxetine, ECS and ketamine modified 2, 10 and 15 hippocampal miRNAs, respectively

[107]

[103]

[104]

[105]

[106]

[108]

SERT: Serotonin transporter; ECS: Electroconvulsive shock therapy.

of several target genes and associated pathways mainly involved in neuronal plasticity: axon guidance, Wnt and Mitogen-activated Protein Kinase (MAPK) signaling, LTP, among others [107]. Finally, changes in rat hippocampal miRNA expression were also found after treatment with acute ketamine (a NMDA receptor antagonist shown to induce a rapid and persistent antidepressant effect), electroconvulsive shock therapy and fluoxetine, thus further supporting a role for miRNAs in the therapeutic molecular mechanisms of antidepressants [108]. miRNAs and associated mechanisms as potential therapeutics for mood and anxiety disorders

3.5

The ability of miRNAs to regulate gene expression and, in particular, the fact that a given miRNA may regulate the expression of hundreds of target mRNAs, often associated in gene networks, makes them extremely attractive therapeutic drug targets for treating complex disorders, such as MDD and AD, where subtle changes to various components of a particular system may be effective [86]. The findings showing altered miRNA expression/regulation in patients with MD or AD together with the evidence that drugs currently used in the treatment of these disorders modulate miRNA expression further encourage following this direction. 12

Although there is clear evidence for miRNA involvement in brain function and dysfunction, the specific role of each miRNA and its targets relevant to a particular phenotype are only beginning to be clarified. Two main experimental strategies, overexpression and loss of function, currently used in this field could also represent the starting points to develop miRNA-based therapies for neuropsychiatric disorders. Both sense and antisense miRNAs and artificial miRNAs have been employed in in vivo studies in mammals. Caution should be taken when using overexpression to avoid that artificial increases in miRNA concentration lead to inhibition of mRNAs that are not physiological targets. Antisense oligonucleotides complementary to mature miRNA sequences (antagomiRs) are the backbone of miRNA silencing. AntagomiRs can have different chemical modifications that allow RNAse protection and regulate their pharmacological properties, such as pharmacokinetics. A more recent development in this field are the locked nucleic acid (LNA) antisense nucleotides that confer higher stability of RNA duplex and appear to be more effective in binding miRNAs than the previous antagomiRs, with increased membrane permeability and less sensitivity to degradation by endonucleases, and can be administered in vivo to animal models through different routes (i.e., intracerebroventricular delivery) [64,109,110]. Another recent approach use fully LNA-modified phosphorothioate




oligonucleotides complementary to the miRNA seed region (tiny LNAs) that may inhibit individual miRNAs as well as miRNA families in both cultured cells and different tissues in mice, but are not able to reach the brain following systemic delivery [111]. Another strategy that offer several advantages, such as the delivery of transgenes by viral vector in cells difficult to transfect and able to inhibit families of miRNAs, are miRNA sponges (miR-SP), introduced first by Ebert et al. [112]. miR-SP consist of DNA constructs producing RNAs with repeated sequences complementary to a miRNA or a miRNA family. The sponges hybridize with the endogenous miRNAs and impede the association with the target mRNAs. miR-SP can be placed downstream of promoters, thus conferring spatiotemporal control of miR-SP deployment, and recently miR-SP transgenics have been tested in mice CNS, where their ability to simultaneously inhibit miRNA family members has been shown [113]. 4.

Expert opinion

Epigenetic drugs are already used in the clinic. Food and Drug Administration (FDA) approved two DNMTi, 5-azaC and 5-azaD, for treatment of myelodysplastic syndromes, respectively, in 2004 and 2006 and the HDACi SAHA and romidepsin for treatment of cutaneous T cell lymphoma in 2006 and 2009 [22]. Additional epigenetic drugs are currently under scrutiny. A quick search of the NIH website for clinical trials with the keyword ‘epigenetic’ yielded 107 trials, most of which for different kinds of malignancies [114]. Some authors have foreseen a promise for personalized epigenetic treatments, and indeed current technology developments now make it possible to draw epigenetic maps of human genomes, which may greatly help in understanding pathophysiology of complex diseases [115]. The first genome-wide DNA methylation scan in MDD was recently reported, suggesting (and confirming) a role for increased cholinergic transmission in MDD [116]. Perhaps the greatest potential for epigenetic drug target discovery lies in HMTs and HDMs; it was estimated that these enzymes are in the order of 100 in human genome [18]. This makes regulation of histone methylation/demethylation a very attractive therapeutic target, which may allow reaching the required level of selectivity for targeting different areas and cell populations in brain. It is also worth noting that only a subset of histone posttranslational modifications have been explored under this respect (see Section 2.6), while the complete ‘histone code’ probably contains a much wider range of possible pharmacological manipulations. Finally, it should also be reminded that many traditional drugs (antidepressants and antipsychotics) also have epigenetic modifications in their mechanism (see Section 2.5). This suggests that, by thoroughly analyzing their mechanisms, we may understand better as to what are the relevant targets that are linked to therapeutic effect. Although still less developed than epigenetic-based pharmacology, miRNA-based pharmacology holds just as

much promise for future therapies. An LNA inhibitor of liver-specific miR-122, required by Hepatitis C (HC) virus for replication [117], has reached phase IIa of development for treatment of HC. Tissue specificity increases the chances that targeting one particular miRNA will affect at lower extent other organs, where that miRNA is expressed at very low levels. However, targeting miRNAs to the brain poses additional problems. The blood--brain barrier represents a major obstacle for molecules negatively charged like nucleic acids, with a size roughly corresponding to 7 or 15 kDa for miRNA antagonists or mimics, respectively [110]. Therefore, miRNA delivery route is still a major problem for CNS. Alternatives to systemic or intracerebral delivery, that is, routes that could perhaps be feasible only for grossly invalidating neurodegenerative diseases (e.g., Huntington’s disease), are currently investigated. Viral vectors have been used in animal models, but their application to humans is limited by safety problems. Interesting alternative routes are carbon nanotubes and exosomes; siRNA delivery with carbon nanotubes has been successfully used in a rodent model of ischemia [118]. Exosomes and microvesicles are extracellular vesicles released by most cells, specifically equipped to mediate intercellular communication via the transfer of genetic information, including both coding and noncoding RNAs. Stem cellderived microvesicles appear to be naturally equipped to mediate tissue regeneration under certain conditions. Interestingly, exosomes loaded with siRNA have been recently used to target the APP-cleaving enzyme Beta-secretase 1 (BACE1) in mouse brain [119]. Development of safe and efficient routes for delivery will be an essential step for exploitation of the therapeutic potential of miRNAs in CNS. Aside from problems related to delivery, the wide spectrum of regulation inherent in miRNA mechanisms, with a single miRNA regulating up to hundreds of genes and with several miRNAs co-regulating a single gene, represents at the same time an advantage and a challenge for miRNA-based therapeutics. This means that by targeting a single miRNA it is possible to modulate entire gene networks, but also that the mechanism is liable to produce unwanted effects in related gene networks that may hinder the therapeutic application. However, the recent findings that only relatively small subsets of miRNAs are essential for viability and basic behavior in C. elegans, and for gross neural patterning in zebrafish, may suggest that miRNA function is more related to adaptive mechanisms in circuit formation, refinement and function than to basic mechanisms involved in embryogenesis (discussed in Ref. [113]). This, and the findings showing that different antidepressants and MS have marked effects on the miRNome (see Section 3.4), may suggest that it is possible to target miRNA-regulated mechanisms linked to behavior (e.g., mood and anxiety) without affecting at the same time basic and survival mechanisms.

Acknowledgment D Tardito and A Mallei contributed equally to this work. This work was partly supported by a grant from Fondazione


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Cariplo (N 2701-2009) to Daniela Tardito, by the fellowship Dote Ricerca: FSE, Regione Lombardia to Alessandra Mallei and by fellowship from Regione Lombardia (Progetto Nepente, decreto 4779 of 05/14/2009) to Daniela Tardito and Alessandra Mallei. Bibliography


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Affiliation Daniela Tardito PhD, Alessandra Mallei PhD & Maurizio Popoli† PhD † Author for correspondence Laboratory of Neuropsychopharmacology and Functional Neurogenomics, Dipartimento di Scienze Farmacologiche e Biomolecolari and Center of Excellence on Neurodegenerative Diseases, University of Milano, Via Balzaretti 9 -- 20133 Milano, Italy Tel: +39 02 5031 8361; Fax: +39 02 5031 8278; E-mail: [email protected]

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