Little things on which happiness depends: microRNAs as novel therapeutic targets for the treatment of anxiety and depression

REVIEW

Little things on which happiness depends:microRNAs as novel therapeutic targets for thetreatment of anxiety and depressionRM O’Connor1, TG Dinan2,3 and JF Cryan2,4

1School of Pharmacy, University College Cork, Cork, Ireland; 2Alimentary Pharmabiotic Centre, University College Cork,Cork, Ireland; 3Department of Psychiatry, University College Cork, Cork, Ireland and 4Department of Anatomy,University College Cork, Cork, Ireland

Anxiety and depression are devastating mental illnesses that are a significant public healthconcern. Selective serotonin-reuptake inhibitors are the first-line treatment strategy for thesedisorders, which despite being a significant advantage over older treatments, are hampered bya limited efficacy in a significant subset of patients, delayed onset of action and side effectsthat affect compliance. Thus, there is much impetus to develop novel therapeutic strategies.However, this goal can only be rationally realised with a better understanding of the molecularpathophysiology of these disorders. MicroRNAs (miRNAs) are a newly discovered class ofgene-expression regulators that may represent a novel class of therapeutic targets to treat avariety of disorders including psychiatric diseases. miRNAs are heavily involved in regulatingmany physiological processes including those fundamental to the functioning of the centralnervous system. Evidence collected to date has already demonstrated that miRNA-expressionlevels are altered in patients suffering from depression and anxiety and in pre-clinical modelsof psychological stress. Furthermore, increasing evidence suggests that psychoactive agentsincluding antidepressants and mood stabilisers utilise miRNAs as downstream effectors.Altering miRNA levels has been shown to alter behaviour in a therapeutically desirable mannerin pre-clinical models. This review aims to outline the evidence collected to date demonstrat-ing miRNAs role in anxiety and depression, the potential advantages of targeting these smallRNA molecules as well as some of the hurdles that will have to be overcome to fully exploittheir therapeutic potential.Molecular Psychiatry (2012) 17, 359–376; doi:10.1038/mp.2011.162; published online 20 December 2011

Keywords: anxiety; depression; miRNA; novel drug targets

Introduction

‘Ah, on what little things does happiness depend!’—Oscar Wilde, The Nightingale And The Rose, 1891

Anxiety and depression are devastating mental ill-nesses creating a severe burden on individuals andsociety. It is estimated 9.5% of the US population willreach the criteria for diagnosis of a mood disorder inany given 12 months, whereas this figure is as high as18.1% for anxiety disorders.1 Moreover, the WorldHealth Organisation has predicted by 2020, unipolardepression will become the leading cause of diseaseburden in developed countries (www.who.int/mental_health/management/depression/definition/en/index.html). Currently, the vast majority of drugs

indicated for the treatment of mood disorders targetthe monoamine system, increasing the amount of theneurotransmitters serotonin and/or noradrenalineavailable for signalling in the synaptic cleft.2–5 Thelimitations of such agents include a delayed onset ofaction (weeks–months),6 a significant percentage ofnon-responders7,8 and side effects.9,10 Although therehave been some advances, such as the development ofthe selective serotonin-reuptake inhibitors, whichcarry a lower side-effect profile than their predeces-sors, there has been almost no advancement in thebasic mechanism employed by antidepressants sincethe introduction of the monoamine oxidase inhibitorsin the 1950s.

The current state of pharmacological interventionsfor the treatment of pathological anxiety is equally assobering with little progress made in the five decadesthat have passed since the introduction of thebenzodiazepines in the 1960s.11–13 Currently availabletreatments mainly target the serotonergic andGABAergic neurotransmitter systems;11,14 again theseare of limited efficacy in a significant proportion of

Received 22 July 2011; revised 22 September 2011; accepted 2November 2011; published online 20 December 2011

Correspondence: Dr JF Cryan, Department of Anatomy, UniversityCollege Cork, College Road, Cork, Ireland.E-mail: [email protected]

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the population and also can produce unwanted sideeffects especially dependence.14 As such, there is anunmet medical need for the development of newantidepressant and anxiolytic drugs with novelmechanisms of action. Accordingly, basic researchand drug discovery efforts have begun to move awayfrom this focus on the monoaminergic systems andother potential mechanisms are increasingly beingconsidered. Examples of this includes the growingcorpus of data demonstrating the involvement ofdeficits in cellular plasticity and resilience as con-tributors to the onset of depression (for extensivereviews see ref. 15,16). This is reflected by the factsthat several drugs targeting plasticity and neuro-trophic cascades are currently in various stages ofclinical trials.16 Furthermore, disruptions to circadianrhythm is commonly seen in depressed patients andis considered to be a contributory factor to the onset ofdepression.17,18 Agomelatine, one of the first non-aminergic-based pharmacotherapies approved foruse in major depression,19 targets the melatonergicsystem, a key synchroniser of circadian rhythm.20

Additional neurotransmitters aside from the mono-amines are also being investigated as potential targetsfor the treatment of depression, one such example isthe glutamatergic system, which is becoming thefocus of much research into affective disorders.21–23

Interestingly, case studies have already demonstratedthe capacity of riluzole, which targets the glutamatetransporter to alleviate depressive symptoms.24,25

Research efforts focused on developing anxiolyticsaimed at previously unexploited targets includesresearch centred on the endocannabinoid system,26

substance P27 and neuropeptide Y28–30 among others.Despite this recognition of the need to move away

from the previous overt focus on monoamines, severalbarriers remain that retard drug discovery efforts. Thepaucity of knowledge regarding the molecular patho-physiological mechanisms that underlie such mentalillnesses, and indeed the incomplete understandingof the therapeutically relevant molecular mechanismsemployed by currently available therapeutics arehampering the development of such novel treatmentstrategies.13,31

MicroRNAs (miRNAs)

IntroductionIn recent years it has become increasingly clear that inaddition to traditional regulatory mechanisms, suchas transcription factor-mediated expression and alter-native splicing, gene expression is also regulatedthrough the activity of non-coding small RNAs. Todate the best characterised of these small RNAs aremiRNAs. Originally discovered when a gene, Lin-4,known to control larval development in C. eleganswas found to be a non-coding RNA transcript.32

Additional work went on to show Lin-4 repressesthe activity of a protein-coding gene, Lin-14, throughsequence-specific binding to the 30 UTR (untranslatedregion) of its mRNA.33 From these original experi-

ments, the field of miRNAs was born and since thenthey have to come to the fore as crucial regulators ofgene expression involved in a staggering number ofphysiological processes.34–42

miRNAs are small (B22 nt) non-coding RNA tran-scripts. They mediate their regulatory effects bybinding to the 30 UTR of mRNAs and targeting themfor degradation or by steric hindrance, which pre-vents access of the cells translation machinery to themRNA.43,44 Additionally, it has been shown thatmiRNAs have the potential to increase translation ofits mRNA targets, however this appears to be muchless common.45 The majority of miRNA genes dis-covered are located in regions of the genome distal topreviously annotated genes, suggesting their expres-sion is under the control of their own regulatoryelements46,47 whereas about 25% have been shown tobe in the intronic regions of pre mRNAs.48 ThesemiRNAs may share transcriptional-control mechan-isms with their mRNA ‘hosts’ leading to a synchro-nised expression. As mentioned, miRNAs are keyregulators of eukaryotic gene expression, with esti-mates of up to 30% of mammalian mRNA transcriptsbeing subject to regulation from miRNAs.43 Further-more, miRNA sequences show a remarkable level ofevolutionary stability, suggesting they are crucial tocorrect physiological functioning.49 miRNAs show ahighly regulated expression both temporally andspatially, which contributes to the unique gene-expression profile in a certain region at a certaintime.50–52 In this way, miRNAs contribute to thedevelopment and maintenance of a specific transcrip-tome particularly suited to a particular tissue at aspecific developmental time point. Early work inC.elegans suggests that cellular levels of individualmiRNA molecules are much larger than other RNAs,such as U6 RNA and mRNAs, again highlighting theirimportance in controlling cellular levels of geneexpression.53

miRNA biogenesismiRNAs have promoters which mainly interact withRNA polymerase II and transcription factors similarto the promoters of protein-coding genes.54–56 Theinitial product of transcription of a miRNA geneis a long RNA transcript known as a pri-miRNA(Figure 1). This pri-miRNA then undergoes nuclearprocessing performed by the type-III endonucleaseDrosha to produce one or several pre-miRNAs, whichare B60–110 nt long transcripts that form hairpinstructures.57,58 Also like protein-coding mRNAs, pri-miRNAs are capped and polyadenylated with differ-ing post-transcriptional-processing steps affectingstability.59 pre-miRNAs are then exported to thecytoplasm via the exportin5-Ran GTP complex60,61

where they undergo further processing by Dicer,another type-III endonuclease.62–65 Dicer-inducedcleavage results in the mature miRNA containing a50 phosphate and a 30 2 nt overhang and a similarstructure made from the opposing strand.48 ThemiRNA is then incorporated into the RNA-induced

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silencing complex (RISC), which allows it to mediateits gene-regulatory activity. The strand with the lessthermodynamically stable 50 end is usually chosen asthe lead strand and incorporated into the ribonucleo-protein complex, whereas the other strand (passengerstrand) is degraded.66

Mechanisms of miRNA-induced regulationThe central catalytic component of RISC is the proteinArgonaute 2, which has endonuclease activity direc-ted against mRNAs.67,68 Currently, the accepted modeldetails miRNA-induced repression of mRNA transla-tion following one of two paths depending on thelevel of sequence complementarity between themiRNA and mRNA target. If there is a sufficientlyhigh level of complementarity between the RISC-incorporated miRNA and the target mRNA thencleavage and degradation of the mRNA will occur,however if the level of complementarity is below acertain threshold then blockade of translation throughsteric hindrance occurs.69,70 This adds another layer

to the scope of gene regulation afforded by miRNAswith levels of sequence complementarity to themRNA dictating if an mRNA is degraded or if trans-lation is simply inhibited which can be relieved ata different time point. This allows a single miRNAto have vastly different effects on mRNA targets in asequence-dependent manner.

An additional feature of miRNAs that increases itsregulatory capacity is the fact that multiple bindingsites for many different miRNAs can exist within the30 UTR of a single transcript and a particular miRNAcan bind to sites on the 30 UTRs on many differentmRNAs.44,71 It has been estimated that each miRNAcan regulate hundreds of individual transcripts.36

This complex structure of gene regulation makesmiRNAs particularly attractive therapeutic drug tar-gets as one miRNA can affect entire gene networksand indeed this may be a particularly attractivestrategy for treating complex disorders, such asdepression and anxiety, where subtle changes tovarious components of a particular system may beeffective. Indeed, in psychiatry there has been arecent departure from having drugs that are veryselective for one therapeutic target towards drugs thatmay have multiple actions.72 However, this posesmajor problems in rational drug discovery efforts.Thus, miRNA-based targets are beginning to offerthemselves as potential strategy to overcome thishurdle.

Measuring miRNA functionSince their discovery almost two decades ago, ourunderstanding of miRNA function has proceededrapidly. This has been partly due to the adaptationof technology originally designed to study the func-tion of larger RNA molecules, which has beensufficiently adapted to allow analysis of miRNAfunction (see Figure 2). Indeed, the rapid elucidationof the pathway used by miRNAs to mediate theirregulatory effects proceeded relatively rapidly, due tothe latter steps being almost identical to those used bysmall interfering RNAs (siRNAs). Much of the workthat will be discussed in detail below uses micro-arrays specifically designed for measuring relativemiRNA quantity within a particular tissue. Theseconsist of short oligonucleotides displaying comple-mentary sequences to known miRNAs spotted onto achip. Fluorescently labelled miRNAs will bind tothese oligonucleotides based on sequence comple-mentarity, and can be visualised using photo-sensi-tive lasers and processing software. This allows thecomparison of multiples (1000 s) of miRNAs betweentwo different samples, for example, control versusdisease state or control versus drug treated. Thisfacilitates ‘fishing’ experiments where researcherscan identify changes to miRNA levels due to a diseasestate or drug treatment. These miRNAs can then beprioritised for follow-up study using modified quan-titative reverse transcriptase-PCR to verify thesechanges. Following from these experiments, research-ers have begun to alter miRNA-expression levels

Figure 1 Biogenesis of microRNAs (miRNAs). miRNAgenes are transcribed by RNA polymerase II producing apri-miRNA. The pri-miRNA is then processed by Drosha toproduce hairpin structures called pre-miRNAs. Pre-miRNAsare then exported from the nucleus to the cytoplasm via theexportin5-Ran complex. In the cytoplasm, they are furtherprocessed by Dicer producing the mature miRNA. Themature miRNA is then incorporated into the RNA-inducedsilencing complex (RISC). The miRNA-RISC complex is thenable to repress translation in a sequence-specific mannereither through degradation of the target mRNA or by blockingaccess of the cells’ translational machinery to the mRNA.


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directly in cell lines and more recently in vivo tofurther characterise the functions of particularmiRNAs. Genetic-association studies have also beenutilised to explore the role miRNAs may have indisease states. Linking polymorphisms present in theregulatory regions governing miRNA expression todisease states would not only initially suggest a rolefor that particular miRNA in a disease state, but mayalso be effective tool used for predicting an indivi-duals likelihood in developing a particular disorder.

Essential to the study of miRNAs and their role invarious disease processes has been the developmentof various bioinoformatic tools designed to facilitatefunctional research into miRNAs. miRBase(www.mirbase.org), the central depositary for miRNAdata, acts as the central database and arbiter ofnomenclature for experimentally verified miRNAs.59

Through the website, primary and mature sequencedata and genomic data can be accessed as well as dataregarding the original discovery and publication ofeach experimentally verified miRNA. This databasehas facilitated the swift growth of the miRNA fieldensuring all researchers are working off the samesequence data and allowing for a uniform nomencla-

ture to facilitate communication. As discussed above,miRNAs bind and repress their mRNA targets basedon sequence complementarity between the twonucleic acids. Different levels of complementarityare required between different regions of the miRNAand mRNA with the seed region (nt 1–8 on themiRNA), requiring full complimentarity and differentparts of the transcript requiring varying levels ofcomplementartity.73–77 Various algorithms (for exam-ple, TargetScan (www.targetscan.org) PicTar (pic-tar.mdc-berlin.de), miRanada (www.microrna.org) toname only a few) have been developed, which predictthe likelihood of a particular miRNA regulating anmRNA based on sequence data as well as additionaldata including evolutionary sequence conserva-tion.76,78 Using these tools to predict the potential ofa miRNA to regulate an mRNA allows for rapidselection of potential mRNAs for follow-up andconfirmatory analysis once a particular miRNA isshown to be altered in a particular experimentaltreatment. This has greatly accelerated research intomiRNAs and the potential biological pathways inwhich they act and will be invaluable in movingmiRNAs from the bench to the bedside.

Figure 2 Common experimental strategies in the search for miRNAs that have the potential to serve as therapeutic targetsfor depression and anxiety disorders. Differential microRNA expression can be identified from different experimental groups(for example, non-disease state versus disease state or non-treated versus treated) obtained from patients, pre-clinical modelsor originate from an in vitro source. A micorarray can be used to assess differences in the levels of a large number of differentmiRNAs between the different experimental groups. The results obtained will then need to be verified using quantitativereverse transcriptase PCR (qRT-PCR). Alternatively, a researcher may chose to use bioinformatic algorithms to prioritise asingle or a particular set of miRNAs for analysis via qRT-PCR (for example, based on predicted miRNA effectors of a gene ofinterest (GOI)). The miRNAs whose differential expression between groups which have been verified through qRT-PCR, canthen be analysed using in vitro assays to confirm target predictions or move directly to pre-clinical models to predictpotential antidepressant or anxiolytic behaviour. From this, individual miRNAs can be prioritised for follow-up work toconfirm their potential therapeutic potential.


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The role of miRNAs in the central nervoussystem (CNS)

It is becoming more obvious that miRNAs areinvolved in the control and maintenance of almostevery aspect of normal physiological functioning withthe CNS being no exception. Early work usingzebrafish demonstrated the miRNA processor Diceris essential to correct development of numeroustissues including the brain, with injection of maturemiR-430 rescuing brain defects in these mutantsdemonstrating that miRNAs have an essential rolein neuronal developmental.79 Specifically, miR-133bhas been shown to be essential for correct maturationof dopaminergic neurons.80 In addition to beingessential for neuronal development, post-mitoticneurons have been shown to express Dicer, suggestingmiRNAs are not only involved in neuronal develop-mental but are also required for continued mainte-nance of neuronal phenotype.81 Furthermore, anabsence of Dicer leads to cell death showing miRNAsare essential for continuous cell survival.82 These datademonstrate the crucial nature of miRNA’s involve-ment in the fundamental process of neuronal differ-entiation and maintenance.

Neurogenesis is an important process in learningand memory83–85 and has been linked to psychiatricdisorders.86 Again miRNAs appear to be at theforefront in regulating this essential process. miR-124-mediated repression of the transcription factorSox9 is essential for the progression of stem cells inthe sub-ventricular zone into fully developed neu-rons87 and another miRNA, miR-137, is also involvedin the development of neural stem cells into matureneurons.88 Clearly, miRNAs control the birth of newneuronal cells and interestingly the concept ofantidepressants mediating part of their benefitthrough neurogenesis is coming more to the fore inresearch.89,90 By targeting miRNAs, a more directroute to inducing neurogenesis may be possible.

Synaptic plasticity is an essential process facilitat-ing learning and memory and disruption may lead topsychiatric disorders. Early evidence for the rolemiRNAs have in plasticity came from work whichshowed Fragile-X mental retardation protein, whichis a synaptic translational repressor, interacts withmiRNAs and components of the RISC pathwayincluding Dicer and Argonaute 1.91 Further workshowed components of the RISC pathway are essen-tial for synaptic protein synthesis highlightingmiRNAs involvement in synaptic plasticity.92 miR-134is localised to the synaptodendritic compartment ofrat hippocampal neurons and regulates Limk1, whichitself regulates the size of dendritic spines that aresites of postsynaptic excitatory transmission.93 Stu-dies since have demonstrated specific miRNAs areenriched in synapses and control local proteinsynthesis and likely have a role in plasticity.94,95

Although the field is still relatively in its infancy,early data is placing miRNAs right at the centre ofmany of the fundamental processes that underscore

correct functioning of the CNS. Disruptions to theseprocesses have been linked to the development ofanxiety and depression,15,86,96 and it is likely thatmiRNAs fine tune these biological functions throughsubtle yet additive effects on mRNA functions.Dysregulation of miRNA function would lead toincorrect regulation of their mRNA targets leading toincorrect physiological functioning in a whole host ofsystems. By targeting this aberrant miRNA function-ing, therapeutic benefits may be possible.

Obstacles encountered in attempts to utilise nucleicacids for therapeutic purposes

Despite the promise and excitement generated by thepotential of utilising miRNAs for therapeutic pur-poses, there are several pitfalls that may prevent theiruse. Some of these difficulties have already beenencountered in incipient attempts to utilise nucleicacids as therapeutics. Initial gene-therapy attempts,where the aim was to restore the function of adefective gene through the addition of a functionalcopy, generated great excitement in the last decade ofthe previous century. At this time, huge leaps weremade with molecular techniques designed to mani-pulate and deliver nucleic acids to cells.97 The firstgene therapy trial, designed to correct a genetic defectpresent in severe combined immunodeficiency dis-order resulted in success in two different patients.98,99

However, the success of this initial attempt proveddifficult to replicate with the majority of successiveattempts failing, eventually culminating in disaster in1999 when two out of ten patients developedleukaemia-like conditions due to the therapy.100 Thiswas a huge setback to the field of gene therapy andexposed many of the flaws that dogged early attempts,these included the generation of a severe immuneresponse,100 poor integration of vectors as well as theflipside of this double-edged sword, where lentiviralvectors inserted into and disrupted native genes.101

Although the initial benefits promised by genetherapy have not yet been reached, some of theseproblems have at least been partially overcome with ahost of therapies currently at various stages of clinicaltrials.102,103 Another approach utilising nucleic acidsto provide potential therapeutic benefit, which alsoarrived to huge fanfare, is the use of antisense viasiRNA molecules. As mentioned above, these aresimilar to miRNAs except that they display perfectcomplementarity to their respective targets and tendto be from exogenous sources.104,105 This method ofgene silencing also seems not to have lived up toinitial expectations suffering from problems withdelivery105 as well as the activation of an immuneresponse.106 However, despite these problems, thissiRNA-based approach is far from dead in thewater with proof of concept studies in non-humanprimates proving effective in combating the replica-tion of viruses involved in serious pathologicalconditions.107–109


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In retrospect, it was probably impossible for initialattempts at the therapeutic utilisation of nucleic acidsto live up to the near astronomical expectations thatsurrounded the field. However, researchers are begin-ning to overcome problems associated with this typeof approach and with more realistic expectations onwhat these treatments can deliver there is still roomin pharmaceutical research for this strategy. miRNAsas relative latecomers to the field of gene therapy willhopefully benefit from the lessons learned both intechnical terms and in keeping expectations realisticfor such a fledgling technology. Moreover, as nativeregulators of gene expression they may not suffer fromproblems such as an exaggerated immune responseand their subtle effects on gene expression may makethem more amenable to a therapeutic purpose.

Therapeutic potential of miRNAs

Preclinical research has already demonstrated thepotential of altering miRNA function to produce atherapeutically relevant outcome in rodents forvarious different types of disorders, including tumourformation,110–112 cardiac disorders113 and allergy-induced asthma.114 A number of pharmaceuticalcompanies have emerged where their pipeline istotally based on miRNA-based targets (for example,Regulus (www.regulusrx.com), Santaris pharma a/s(www.santaris.com), MiRNA therapeutics (www.mir-narx.com)). Recently this approach has proven suc-cessful in primates with initial work showingintraperitoneal injection of a miR-122 LNA (lockednucleic acid; see section Antagomir-based knock-down) antisense molecule significantly reduced le-vels of miR-122.115 Additional work revealed miR-122to be essential for infection by the hepatitis C virusand high doses of the anti-miR-22 compound over acourse of 14 weeks significantly attenuated viralreplication as measured by viral RNA levels.116

Furthermore, this was achieved with no serious sideeffects and with no adaptive response from the virus.This was the first demonstration that the modulationof miRNA function may be an effective and safetherapeutic strategy in primates. Currently the firstmiRNA-based therapeutic, a miR-122 inhibitor, basedon the above research is in phase-2 clinical trials(www.santaris.com/product-pipeline).

Challenges in delivering miRNAs to the CNS

General aspectDespite the above data demonstrating the modulationof miRNA function is a feasible therapeutic strategy,the leap from knocking down miRNA function in theperiphery to the brain is considerable. The idealtherapy would be an active compound suitable fordispersal in a form that can be administered orally,survives passage through the gastrointestinal system,crosses the blood–brain barrier and produces thedesired effect in the desired region, all while remain-ing non-toxic. The modulation of miRNA functioning

within the CNS will likely be achieved throughoverexpression or reducing function (knockdown)using an antisense oligonucleotide. However, deliv-ery to the brain of viable nucleic acids is particularlydifficult compared with peripheral organs. Theblood–brain barrier, a network of tight junctionsformed by endothelial cells, eliminates the passingof the majority of molecules from circulation into thebrain, making the systemic approach (detailed above)for the delivery of nucleic acids almost impossible.117–119

The brain is also an extremely heterogenous organwith a large amount of cellular diversity, achievingthe desired expression of a specific nucleic acid in aspecific part of the brain has historically provedchallenging.118 However, some recent work hasdemonstrated the possibility of using vectors underthe control of cell-specific promoters to produceknockdown limited to particular a region.120 miRNAsas therapeutic tools suffer from similar practicaldrawbacks, which other nucleic acids have alsosuffered. Broadly speaking there are three mainhurdles, stability of the nucleic acid in the blood,inability of miRNAs to cross many biological mem-branes and mediating cell-specific delivery.121,122 Thedesign of efficient delivery mechanisms (vectors)has been essential to the development of nucleicacids, for example, siRNA, as potential therapeuticsand miRNAs are no exception. Broadly speakingvectors intended for the delivery of nucleic acids canbe classified as either viral or non-viral. Viral vectors(retroviruses, adenoviruses and so on) are efficientfacilitators of transfection allowing for integration ofthe vector into the host chromosome producing stableexpression.123 However, these are difficult to produceon a large scale and even more importantly they arehampered by a serious safety burden detailedabove.100,123,124 Synthetic and non-viral methods arebeing actively pursued as potential strategies to over-come these problems,124 but these delivery methodsalso need to overcome many barriers to becometherapeutically viable.124 In addition to the choice ofvector, another critical choice is the route of adminis-tration. As mentioned above, an orally active thera-peutic is the most desired but also most challenging.Other routes that can be considered are more invasiveand obviously the potential benefits and risks willhave to be carefully considered. These include intra-venous injections, osmotic minipumps and directinjections to the brain. Currently it is possible toenvisage that while such invasive strategies may bebeneficial for the treatment of some severe neurologi-cal disorders such as Huntington’s Disease, their wide-spread utility for psychiatric disorders is limited. Thatsaid, there is an increasing move towards employinginvasive technologies such as deep-brain stimulationto treat severe treatment refractory patients with majordepression or anxiety disorders.125–127

The utilisation of miRNAs as therapeutics will haveto overcome additional difficulties, which may beunique to each particular miRNA and any therapiesutilising miRNAs will each have to be considered on


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an individual basis. The establishment of an appro-priate dose of a particular miRNA may be a particu-larly complex issue as repression by miRNAs ismodest up to a point at which increased levels of aparticular miRNA generate a type of molecular switchwhere protein production is highly repressed.128

Moreover, the levels of repression induced by specificmiRNAs varies greatly among cell type,128 furtheradding to the need to deliver the miRNA to a specificcell or group of cells. Furthermore, due to thesequence-specific manner by which miRNA mediatetheir regulatory effects,78 caution will need to be takento ensure non-target mRNAs are not repressed todeleterious consequences by altering the levels of aparticular miRNA.

There are two basic choices when deciding onmodulating the function of a particular miRNA. Thiswill either be knocking down or increasing the levelsof a particular miRNA available to regulate aparticular mRNA.

Antagomir-based knockdownIn vivo knockdown of mRNA targets using antisensenucleotides in rodent models has already beenachieved in the periphery129 and moreover, thebrain,130–133 which has led to therapeutically relevantbehaviour outputs. In these studies, siRNA constructsdesigned to target the mRNA of genes previouslyimplicated in the neurobiology of depression,DAT131,132 GRM7130 and serotonin transporter(SERT)131,133 were administered to the rodent brain.This produced an hyperlocomotor response followingreduction of DAT expression,131,132 an antidepressanteffect in the forced-swim test after targeting SERTmRNA,131,133 whereas knocking down of GRM7mRNA led to an impairment to Pavlovian fearlearning.130 To achieve the in vivo knockdown ofmiRNAs, Krutzfeldt et al.134 developed antagomirs,chemically modified, cholesterol-conjugated single-stranded RNA analogues (LNAs) complementary tomiRNAs, which bind to and prevent miRNAs frommediating their gene-silencing effects. These havealready been used to knockdown miRNAs in both theperiphery134 and brain.135,136 However, success inrodent models does not always translate to the humansituation, thus the recently demonstrated knockdownof miRNAs in primates is a major step forward towardmiRNA-based therapeutics in humans.115 The factthat altering miRNA expression in the brain produceda therapeutically relevant behavioural change,135,136 isextremely promising, however, it needs to be notedthat knockdown of miRNAs were achieved throughinvasive surgical techniques, which would only betherapeutically relevant in severe disease cases.Another drawback with this type of approach wouldbe the requirement to continuously administer theseantagomirs to achieve a sustained knockdown.

OverexpressionDelivery of RNAi molecules in vivo has been possiblefor years, with the use of viral vectors driven by strong

promoters being a popular choice.137,138 This techni-que has already proved successful with miRNAs inrodent models producing therapeutically relevantoutcomes in the areas of drug addiction and depres-sion-like behaviour.135,139

The altering of miRNA expression has greatpotential not only in treating disease states where aclear aberrant miRNA expression is present but alsoin treating disorders where protein-coding genes areimplicated. It may be miRNAs subtle effects on thetranslation of mRNAs may make them uniquelysuited to treating disorders where only a mild changeto expression is desired.

Evidence demonstrating the potential of targetingmiRNAs to treat depression

Acute and chronic stress alter miRNA-expressionprofiles in a brain region-dependent fashionStress is one of the major predisposing factors foranxiety and mood disorders. Chronic stress and stressin early life can create pathological changes in thebrain that impair behavioural and physiologicalresponses.140–142 These changes can predispose cer-tain individuals to psychiatric diseases, such asdepression and anxiety disorders.96,143–145 If miRNAshave a role in the adaptive (and maladaptive)response to a psychological stressor then it may bethe case that targeting miRNAs may be aviable strategy for the treatment of stress, whichmay predispose individuals to mood and anxietydisorders.

One of the first studies to highlight the potentialimportance of miRNAs in the stress response demon-strated chronic immobilisation stress in rats producedregion-specific changes to miRNA levels.146 miRNAswere differentially affected between the CA1 area ofthe hippocampus and central nucleus of the amygdalafollowing restraint stress. Furthermore, acute (1 day)and chronic restraint stress (14 days) produceddifferent miRNA-expression profiles in the tworegions with very little overlap.146 The authors notethat chronic stress produced a greater number ofsignificant changes to miRNA levels than acute stressand that most of these changes represented decreases.In contrast to these results, a similar experimentfound differing results when they analysed miRNAexpression in the pre-frontal cortex of CD-1 micefollowing restraint stress.147 In these studies, a largeincrease (as opposed to a decrease in the previousexperiment discussed) in expression levels ofmiRNAs following a single restraint stress (1 sessionof 2 h), however, only a modest change followingrepeated restraint stress (5 sessions over 5 days).147 Asource of the contrasting results between these twoexperiments may be methodological differences asboth studies used a different experimental speciesand very different restraint procedures, with Meersonet al.146 subjecting rats to 14 days of restraint for 4 h aday, and Rinaldi et al.147 using CD1 mice and a muchmilder restraint procedure consisting of 2 h for 5 days.


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Additionally, the original microarray genechips usedfor the profiling study were very different in bothstudies (Meerson et al.146 utilised the mirVana oligoset to construct an array in house, whereas Rinaldiet al.147 used a miRCURY LNA microRNA array;Exiqon A/S, Vedbaek, Denmark), which could affectthe final results.

Although currently there is conflicting data sur-rounding which miRNAs are altered as well as themagnitude of changes (both in number of miRNAsand the levels as to which these are changed), it isbecoming increasingly clear that psychological stressimpacts on miRNAs levels with further experimentsbeing required to achieve more definitive conclusionson which particular miRNAs are altered and howthis leads to downstream changes affecting cellularfunction.

It is tempting to speculate that these changes arepart of an adaptive homoeostatic response by whichan increase to selective miRNAs serves the dualfunctions of receding changes to mRNA levels, thatmay lead to detrimental effects if expressionremained high and also to activate a program of geneexpression that will work in concert to produce anadaptive response to the stressor.

Both of these studies demonstrate stress-inducedchanges to miRNA levels are brain-region specificwith very little overlap occurring between thedifferent brain regions analysed (CA1 region of thehippocampus, central nucleus of the amygdala andpre-frontal cortex) both within and between thestudies. This would not be unexpected as these verydifferent stress-induced miRNA profiles would leadto very different gene-expression patterns, whichwould likely be uniquely suited to that particularbrain regions function in the stress response. Thisregion specific and differential response to acute andchronic stress is mirrored in mRNA-expressionpatterns148–152 so it may be that changes to miRNAexpression serve to influence mRNA expressionpatterns, which can in turn influence miRNA-expres-sion patterns creating an intricate regulatory loop.

Chronic immobilisation stress is not the only stressmodel where the role of miRNAs has been investi-gated. Maternally separating rat pups from theirmothers is used as a model of early life stress andleads to a host of physiological alterations,153–155

increased 5-HT turnover and an exaggerated hypotha-lamic–pituitary–adrenal axis response to restraintstress.156 Uchida et al.156 went on to demonstratematernally separated animals have increased expres-sion of the transcription factor REST4 in the pre-frontalcortex and it has been previously demonstrated thatREST4 regulates several brain-enriched miRNAs.157,158

Moreover, several of these miRNAs including mir-132,-124-1, -9-1, -9-3, -212 and -29a are increased in thepre-frontal cortex of maternally separated rats.156 Onecan speculate that the stress-induced increase toREST4 leads to a host of gene-expression changesincluding the upregulation of the above-mentionedmiRNAs. These will then go on to repress their target

mRNAs leading to a host of downstream changesgreatly amplifying the original signal.

The above data suggest changes in miRNA expres-sion represent an integral part of the stress response.It is likely that miRNA levels change in response tostress to act in an adaptive nature altering gene-expression patterns to help combat the psychologicalstressor. However, exaggerated or prolonged levels ofstress could possibly lead to prolonged changes tomiRNA expression, which may ultimately lead tomaladaptation. In stress disorders where a clearchange to miRNA levels has been identified, it maybe possible to normalise the changed miRNA levelsreducing the prolonged effects of stress. Furthermore,miRNAs may also be used to fine tune expression ofmRNAs involved in stress broadening their scope as atherapeutic tool.

Genetic differences in miRNA expression influence thecoping responseThe genetic loci of miRNAs are subject to similarregulatory controls as mRNAs, being located inpolycistroinic units under the control of promotersregulated by transcription factors and mainly beingtranscribed by RNA polymerase III.54–56 Therefore, itis very likely that individual differences in miRNA-expression patterns exist and these will influence anindividual’s reaction to a stressor. Some early pre-clinical work has demonstrated support for thishypothesis.

The stress-sensitive F344 rat strain displays anexaggerated HPA axis response over 14 days ofrestraint stress, whereas the control Sprague Dawleystrain demonstrates a gradual decrease in HPAactivity.159 This is in line with other data demonstrat-ing F344 rats display both an exaggerated-HPAresponse to chronic mild stress160 and increasedanxiety in the elevated plus maze.161 This strain wasfound to have increased levels of the miRNA miR-18ain the paraventricular nucleus (PVN) of the hypotha-lamus, which was found to target the glucocorticoidreceptor (GR). Increased levels of miR-18a in the PVNis a likely contributing factor to the genetic suscept-ibility to stress displayed by these rats.

When a group of rats are subjected to repeatedinescapable shock, a subgroup will fail to showescape-orientated behaviour even when the possibi-lity of escape exists.162,163 This phenomenon is termedlearned helplessness (LH) and has been used as amodel of the vegetative symptoms of depression.162–166

It has been previously shown that Holtzman rats whodo not develop this LH behaviour (NLH (non-learnedhelplessness) rats) show numerous hippocampalmRNA changes when compared with LH and naı̈verats.167 This increased mRNA response in NLH ratsmay represent a coping mechanism that serves toproduce a physiological response allowing it toovercome stress. A similar pattern has been demon-strated in the hippocampal miRNA response withNLH rats showing a large change to miRNA levels(mainly a decrease), whereas LH rats showed a similar


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yet blunted response pattern.168 This demonstratesthat underlying genetic differences in miRNA-expres-sion pattern are associated with an individual’sability to develop a behavioural-coping response toa particular stressor, further work, involving addi-tional behavioural paradigms and the modulation ofmiRNA levels in vivo need to be conducted tostrengthen (or reject) the case for a causative relation-ship between miRNA-expression levels and behav-ioural coping responses (or lack thereof). If one exists,it may be possible to use miRNAs to induce a patternof gene expression, which would allow for bettercoping in a stressful situation.

Predicted mRNA targets of miRNAs involved in thestress response modulate physiological response tostressAlthough expression-profiling studies following astressor can indicate the potential involvement ofa miRNA in the adaptive response to stress, it isultimately their regulatory effects on theirmRNA targets that dictate their influence on cellularfunction.

Following miRNA-expression profiling, bioinfor-matic analysis has been designed to predict themRNA targets of the miRNAs showing alteredexpression response to stress. Interestingly, one ofthe most commonly found class of targets aretranscription factors.146 A similar picture emergedfrom other studies focused on the role of miRNAsinvolved in LH behaviour where a large number oftranscription factors are also regulated.168 This over-representation of transcription factors as targets maymake miRNAs very attractive therapeutic as alteringtheir expression may serve to alter the expression of awhole network of genes. A striking example of thisregulation of transcription factors by miRNAs isCreb1, which regulates a host of cellular func-tions.169–171 Of the miRNAs altered in LH comparedwith NLH rats, four (all of which were upregulated)are predicted to regulate Creb1.168 Furthermore, it isknown that LH have reduced levels of Creb1, whichcan be reversed by chronic antidepressant treat-ment.172 Although the authors do point out that theincreased levels of hippocampal miRNAs targetingCreb1 in LH rats are unlikely to be the soledetermining factors of Creb1 levels,168 it is likely theyplay a role in its downregulation. In this instance, theupregulation of miRNAs that target Creb1 likelycontributes to the decreased Creb1 levels seen in LHrats. Using antagomirs to reduce the inhibitory effectsof these miRNAs may be a strategy that would mimicsome of the downstream effects of antidepressants,which serve to increase Creb1 levels.

As discussed above, both miR-134 and miR-183display increased expression in the amygdala due toacute stress, whereas chronic stress decreases miR-134 expression while leaving miR-183 unchanged.146

miR-134 and miR-183 share the splicing factor, SC35as an mRNA target confirmed through in vitro studies.Previously it has been shown that SC35 is increased

in the pre-frontal cortex of mice subjected to 4 days ofswim stress173 and was shown in vitro to promote thesplicing of AchE (acetylcholinesterase) from thedominant AChE-S to the stress-induced AChE-Rvariant. A reduction in amygdalar miR-134 wouldrelieve the repression of SC35 expression, which inturn would induce a selection towards the increasedlevels of stress-induced AChE-R variant. Initial targetdata is suggesting miRNAs likely regulate a largenumber of gene-expression regulators, includingtranscription and splicing factors. Although alteringa miRNA may produce a small initial regulatory effecton its initial target mRNA, this may lead to a muchgreater downstream effect due to altering the expres-sion of other regulators of gene expression.

As mentioned above, F344 rats possess lowerexpression of the GR protein but not mRNA in thePVN of the hypothalamus.159 A reduction in GRwould lead to a decreased level of negative feedbackon the HPA axis contributing to the exaggerated HPAresponse seen in the strain. Sequence data predictsthe 30 UTR of GR mRNA possess multiple miRNA-binding sites,174 and in addition Uchida et al.159

demonstrate miR-18a negatively regulates GR incultured neuronal cells. Furthermore, they showF344 rats have increased miR-18a expression in thePVN compared with the SD strain. Taken together,this data would suggest that increased miR-18a in thePVN of F344 rats inhibits translation of GR mRNA toprotein, thus contributing to the exaggerated HPAresponse. Knocking down of miRNAs may be a viablestrategy to increase GR levels, which would lead to anoverall suppression of the HPA response.

The above data demonstrate miRNAs, which showexpression changes due to stress, are predicted toregulate mRNA targets, which have themselves beenimplicated in stress-induced depressive behaviour.Altering the levels of these miRNAs will lead tochanged levels of these mRNAs, which may producetherapeutically relevant benefits.

Human genetic studies highlighting the potential roleof miRNAs in depressionGenetic evidence is beginning to show geneticdifferences in miRNA expression contribute to thesusceptibility to the onset of depression. The firststudy to examine the potential link between poly-morphisms in miRNA genes showed a statisticallysignificant link between a polymorphism in the miR-30e gene and the onset of major depressive disor-der.175 Although this was a very ethnically limitedgroup, these results were an important indication thatdifferences with miRNA genes can contribute to theonset of psychiatric disorders. Functional dataregarding miR-30e show it can suppress cell growth,176

and it has previously been demonstrated the samepolymorphism was also linked with the onset ofschizophrenia.177 Furthermore, post-mortem studiesreveal increased levels of miR-30e in patientswith schizophrenia.178 So although it is by nomeans confirmed that miR-30e has a role in brain


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development, early evidence is pointing to it havinga significant role in the genesis of mental disordersand may be an interesting target to consider fortherapeutic intervention.

Disruption to the circadian rhythm is being viewedmore and more as a contributing factor the onset ofdepression.179,180 A genetic-association study hasshown a polymorphism in the pre-miR-182 gene tobe significantly linked with major depression and lateinsomnia.181 Interestingly, this miRNA was predictedto regulate the CLOCK gene, which itself has beenlinked to insomnia and depression,182–184 which theauthors confirmed in vitro through the use of aluciferase reporter.181 These data indicate aberrantmiRNA expression may contribute to the dysregula-tion of genes associated with maintaining the correctcircadian rhythm and through this may contribute tothe onset of depression. Targeting these miRNAs mayhelp to reset the circadian rhythm and reducedepressive symptoms.

It may be possible to alter the expression ofmiRNAs for which an altered expression profile hasbeen shown to influence the onset of depressivedisorders. By normalising the levels of these miRNAs,it may be possible to produce therapeutically relevantoutcomes.

Specific miRNAs are the targets of psychoactivedrugs

As discussed above, our current knowledge regardingthe molecular underpinnings of the therapeuticallyrelevant mechanisms underlying current treatmentsfor psychiatric disorders remain incomplete.Although in many cases the immediate biochemicaleffect is known (for example, inhibiting the reuptakeof serotonin and/or noradrenaline), the downstreammolecular effects, which likely mediate much of thetherapeutic benefit, remain unknown. It is nowbecoming clear that alteration to miRNA levels maybe one of the therapeutically relevant downstreameffects of antidepressants. The fact that currentlyavailable therapeutics for the treatment of mooddisorders function to alter miRNA levels supportsthe idea of targeting them directly to producetherapeutically beneficial effects.

One of the first studies to investigate if miRNAs arepotentially targets of psychoactive drugs investigatedif chronic treatment with the mood stabilisers,lithium and sodium valproate, could influencehippocampal miRNA levels in Wistar Kyoto rats.71 Itwas found that lithium altered 37 miRNAs whereassodium valproate altered 31. Interestingly of these, asimilar change was seen in 9 miRNAs suggestingthese miRNAs may be especially relevant to themood-stabilising effects of these drugs.71 To prioritisepredicted targets for follow-up analysis, the authorscross referenced this list with the Welcome Trust CaseControl Consortium list for likely targets involved inbipolar disorder and found six that were alsoregulated by the miRNA targets (CAPN6, DPP10,

ESRRG, FAM126A, GRM7 and THRB). Of these, itwas found that hippocampal protein levels of GRM7,DPP10 and THRB were upregulated, which was inline with microarray data showing several miRNAspredicted to regulate these mRNAs were downregu-lated. Indeed, early work conducted in our laboratoryhas begun to shed light on the effect of lithium andchronic immobilisation stress on miRNA function.We found that chronic immobilisation stress alteredhippocampal miR-34c levels and amygdalar miR-15alevels with lithium reversing these changes but onlyin a stress-specific fashion.185 It may be that certainpsychoactive drugs mediate their benefit by reversingdisease-induced changes to miRNA expression inspecific brain regions. To further add credence to thehypothesis that these mood stabilisers functionthrough altering the levels of specific miRNA levels,it will be necessary to alter these miRNAs in vivoand examine their effect in pre-clinical models ofdepression.

miR-16 has been shown to regulate the SERT in vivousing a luciferase reporter assay,139 and interestingly,has been shown to be involved in determining if aneuroectoderaml cell line follows a serotonergic ornoradrenergic fate.139 In cells that differentiate intoserotonergic cells, miR-16 levels remain unchanged,however, cells that develop along the noradrenergicpathway show increased miR-16 levels and reducedSERT levels.139 It is likely that miR-16 contributes tothe development and maintenance of a noradrenergicphenotype by repressing translation of the SERT (andpossibly other) mRNAs. Furthermore, infusion offluoxetine into the raphe nucleus increases miR-16expression, which would contribute to the selectiveserotonin-reuptake inhibitors inhibitory effects onSERT function through repression of mRNA transla-tion.139 When miR-16 was infused directly to theraphe it was found to have an antidepressant-likeeffect in the forced-swim test (FST).139 Interestingly,when miR-16 levels were analysed in the locuscoeruleus following chronic fluoxetine treatment theywere found to be decreased.139 Moreover, reducingmiR-16 in the locus coeruleus using a miR-16antagomir led to an antidepressant effect in theforced-swim test.139 This was a very significantfinding, demonstrating that one of the most com-monly used antidepressants likely employed alteringof miRNAs as a therapeutically beneficial mechan-ism. Furthermore, fluoxetine differentially affectedmiR-16 levels in the raphe nucleus and locuscoeruleus. These early data demonstrate targetingspecific miRNAs in specific brain regions may be amore direct route to achieving an antidepressanteffect. It also highlights the regional specificity thatmay be required as increasing a miRNA in one regionmay have the opposing effect in a different region.This poses a major challenge for drug development.

miRNAs have been shown to be involved not onlyin the therapeutically relevant mechanisms of psy-choactive drugs but also in the negative effectsincluding mediating drug dependence. It was found


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that striatal miR-212 was increased in rats trained toself administer cocaine, and that this increasedecreased the motivational properties of the drug byincreasing the stimulatory effects of the drug on CREBsignalling. Moreover, overexpression of miR-212 inthe striatum decreased cocaine-seeking behaviour,whereas knockdown of striatal miR-212 increasedthe addictive effects of cocaine. miRNAs may repre-sent a novel avenue of therapeutics for treating drugaddiction.135,136,186

The above data showing currently used medica-tions for the treatment of psychiatric disordersemploy alterations to miRNA levels as possibledownstream therapeutic mechanisms are extremelypromising. By directly targeting these miRNAs, it maybe possible to produce a more robust antidepressiveeffect possibly more rapidly and with reduced sideeffects.

miRNAs and anxiety

To date, there has been little evidence gathereddemonstrating miRNAs involvement in pathologicalanxiety. As far as we are aware, there has been nopreclinical study that directly examines the rolemiRNAs have in pathological anxiety. However, basedon the ubiquitous role of miRNAs in CNS function itis very likely they are involved in the neurobiology,which underlies pathological anxiety and indeedthere is a limited amount of evidence suggesting so.As in section Overexpression, the F344 rat strain is astress-sensitive rat strain that displays anxiogenicbehaviour in pre-clinical behavioural models.161,187

The increased levels of miR-18a in the PVN of F344rats may contribute to the decreased levels of GR thatin turn likely leads to increased HPA activity andincreased anxiety. Further studies will need to beconducted on the influence miRNAs have in the F344rats levels increased anxiety. As mentioned above,miR-16 is a target of the selective serotonin-reuptakeinhibitor fluoxetine and has itself been shown tomediate its antidepressant effects.139 Selective seroto-nin-reuptake inhibitors are also first-line treatment forthe treatment of a number of anxiety disorders11,13,14

and modulation of miR-16 expression is a potentialroute by which these drugs mediate their therapeuticeffect. In addition to treating depression, directlytargeting miR-16 in relevant brain regions may alsoserve as a more direct route to the treatment ofanxiety.

Genetic-association studies have also been utilisedto examine the influence polymorphisms in miRNAsloci have in the development of anxiety. Previously ithas been shown that that single-nucleotide poly-morphisms in the 30 UTR of the neurotrophin-receptor 3 gene (NTRK3) might contribute towardsan obsessive compulsive disorder phenotype.188 Itwas found that a single-nucleotide polymorphism inthe functional target site of the miRNA, miR-485-3p,was significantly associated with the hoarding phe-notype of obsessive compulsive disorder. Further-

more, this particular study identified two new rarevariants in the 30 UTR of NTRK3 present in onepatient with panic disorder, one located in the targetsite for miR-765 and the other in the target site forboth miR-509 and miR-128. Using in vitro analysis,the authors show these two variants greatly influencemiRNA-mediated regulation of NTRK3. The samegroup went on to show different single-nucleotidepolymorphisms associated with panic disorders tagthe miRNA loci of several miRNAs, namely miR-22,miR339, miR-138-2, miR-488, miR-491 and miR-148a.189 The authors then prioritised potential mRNAtargets for functional analysis by cross referencingwith candidate genes for panic disorder190 and foundmiR-138-2, miR-148a and miR-488 repress GABRA6,CCKBR and POMC mRNA in vitro, respectively andmiR-22 regulates four other candidate genes namelyBDNF, HTR2C, MAOA and RGS2. In the context ofobsessive compulsive disorder, it is worth consider-ing studies on Tourette’s syndrome, a debilitatingpsychiatric disorder characterised by involuntarymotor and vocal tics,191,192 which displays a highlevel of comorbidity with obsessive compulsivedisorder and depression.193 It has been shown thatthe binding site for miR-189 located in the 30UTR ofSLITRK1 mRNA, a potential candidate gene influen-cing the onset of Tourette’s syndrome, was found tohave a substitution in two unrelated individuals withTourette’s syndrome that increased the level ofrepression on SLITRK1.194

Fear extinction is the gradual reduction of the fearresponse due to repeated exposure of non-reinforcedfear-related cues allowing for the adaptive control ofconditioned fear responses.195 This phenomenon hasgenerated a great deal of research due to its relevanceto human phobias and post-traumatic stress disor-der.195,196 Briefly outlined, the procedure involves therepeated pairing of an innocuous cue such as a brieftone with an unpleasant stimulus such as a shockdelivered via a metal grid in the floor. Followingnumerous presentations of these two stimuli together,the animal will eventually display a fear response,such as freezing, even when the innocuous tone isproduced alone. Repeated presentations of the tonealone will eventually lead to the disassociation of thetwo stimuli. A recent study demonstrated that miR-143 showed increased levels in the infralimbic pre-frontal cortex, a brain region critical to fear-learningprocesses, however, this miRNA showed no furtherchanges to expression following fear-extinction learn-ing,197 suggesting this miRNA is involved in theoriginal pairing of the cues but not in the extinction ofthis pairing. Interestingly, a separate miRNA, miR-128b showed no significant increase due to theoriginal fear learning but showed a dramatic change(B4-fold increase) in response to fear extinction.Furthermore, when fear extinction was retarded usingthe NMDA receptor antagonist MK-801, no changes tomiR-128b levels were seen. Moreover, when miR-128b function was disrupted via a lentiviral constructdelivered to the infralimbic pre-frontal cortex, fear


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extinction was impaired, whereas conversely, increas-ing levels of miR-128b in this region enhanced fearextinction. The authors also showed this effect wasspecific to the IFLPFC as infusion of miR-128 into theprelimbic pre-frontal cortex had no effect on fearextinction. Although these are early results, miRNAsmay be intimately involved in fear-extinction pro-cesses and further research may allow for miRNA-based therapeutic interventions for post-traumaticstress disorder and severe phobias to be considered.

Although only limited studies have looked at thepossibility of miRNA expression influencing theonset of a pathological anxiety phenotype in clinicalpopulations, there is some preliminary evidence tosupport this. If altered miRNA levels demonstrateto influence the onset of anxiety it may be possible totreat anxiety by changing miRNA-expression levels.

Conclusions and perspectives

Since their discovery and original characterisation,the ubiquitous nature of miRNA involvement invarious biological processes has become clearer. Theyhave key roles in governing virtually all processes inthe CNS, making it very likely that altering theirfunction will produce many downstream changes at aphysiological and behavioural level.

It is clear that drug discovery efforts for thetreatment of mood disorders need to move from thetraditional focus on monoaminergic systems towardsnovel targets based on a rational drug discoveryapproach. As ubiquitous regulators of so manyphysiological processes including in the CNS,miRNAs represent a promising class of targets whosetherapeutic potential to date remains almost comple-tely unexplored.

Although research efforts to characterise the rolethese small RNAs have in depression and anxietydisorders is still in its infancy, a wealth of evidencehas already been collected showing aberrant miRNAexpression can influence the onset of both anxietyand depression (Table 1). It is through their sequence-specific regulatory effects on mRNAs that miRNAsmediate their ultimate cellular effects, and in vitro,in vivo and in silico work has shown miRNAs have thecapacity to regulate an enormous number of mRNAsthat have important roles in a host of processes, whichhave been linked to the onset of depression of anxiety,including regulation of monoamine transporters,circadian rhythm and synaptic plasticity.

miRNA expression has been shown to be respon-sive to a psychological stressor and early researchsuggests endogenous genetic differences in miRNA-expression levels contribute to the onset of anxietyand depression. Some early work to identify themRNA targets of these miRNAs revealed many ofthem have been previously highlighted as possiblycontributing to the onset of anxiety and depression.Targeting these miRNAs could prove a beneficialtherapeutic avenue. Some of the most strikingevidence supporting the concept of targeting miRNAs

to treat mood disorders is the fact that currentlyavailable medications indicated for the treatment ofthese disorders already serve to alter miRNA expres-sion. Directly targeting miRNAs may be a morebeneficial route that may lead to a more rapidtherapeutic effect with fewer side effects. Further-more, targeting miRNAs holds promise as a treatmentstrategy not just in situations where a clear aberrantmiRNA-expression profile has been detected but alsoin cases where altered mRNA or indeed aberrantprotein levels are seen as the underlying molecularpathophysiology. Given a single miRNAs ability toregulate many different mRNAs, targeting a miRNAmay prove a very useful strategy where changes tolevels in many different proteins have been identifiedas contributing to the molecular pathophysiology.However, it is worth noting that the amplificationability of targeting a single miRNAs to affect multipledownstream targets could also increase the suscept-ibility to side effects and the safety profile of any newmedication needs to be rigorously tested. Nonethe-less, although being early days in miRNA-basedtherapeutic development, a miRNA-based therapyhas already been shown to be safe in primates andhas entered phase-II clinical trials. Although thistreatment is not specific to the brain, it highlights thepotential safety and therapeutic benefits of usingmiRNAs.

However, with the application of any new technol-ogy to the treatment of human disorders there arenumerous issues that will need to be considered toensure the therapeutic potential of this technologyreaches fruition. One of the main issues will be thetranslating of data from pre-clinical models to thehuman situation, this is especially difficult withpsychiatric conditions owing to the complex natureof these disorders, with many aspects being exclusiveto the human condition.198 It is imperative thatresearch using animal models is conducted in tandemwith clinical research efforts to confirm the involve-ment of a miRNA in a pathological condition.Additionally, despite the huge leaps that are beingmade in understanding the biological significanceof individual miRNAs, this will not translate toadvances in therapies without parallel advances inpharmaceutical biotechnology to ensure that anypotential therapy is formulated appropriately withouttoxicological issues. Duration of treatment is anotherimportant issue that will have to be carefullyconsidered and will likely depend on the individualmiRNA and disease in question. Furthermore, due tothe enormous and almost completely untappedtherapeutic potential of miRNAs, there is an ever-expanding commercial interest in the application ofthis technology. It is imperative that investors beaware of the patent landscape that exists surroundingthis technology.199 Concomitant with being granted apatent for the therapeutic use of a particular miRNA isthe proof of novelty and utility, which are ambiguousconcepts likely to generate much debate in thecoming years. To promote the exploitation of this


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Table 1 Evidence collected demonstrating the potential of targeting miRNAs for the treatment of depression and anxietydisorders

MicroRNA Effect Reference

Let-7a Upregulated expression in the frontal cortex following acute stress Rinaldi et al.147

Let-7b Increased expression in the hippocampus due to treatment with lithium and sodiumvalproate

Zhou et al.71

Let-7c Decreased expression in the hippocampus due to treatment with lithium and sodium valproate Zhou et al.71

miR-124 Upregulated expression in the medial pre-frontal cortex following maternal separation Uchida et al.156

miR-124-1 Pre-miRNA upregulated expression in the medial pre-frontal cortex following maternalseparation

Uchida et al.156

miR-128 SNP found in a patient with panic disorders and shown to regulate NTRK3 (which hasbeen implicated in contributing to panic disorder)

Muinos-Gimeno et al.188

miR-128a Decreased expression in the hippocampus due to treatment with lithium and sodiumvalproate

Zhou et al.71

miR-128b Regulates formation of fear-extinction memory in the infralimbic pre-frontal cortex Lin et al.197

miR-132 Upregulated expression in the medial pre-frontal cortex following maternal separation Uchida et al.156

Pre-miRNA upregulated expression in the medial pre-frontal cortex following maternalseparation

miR-134 Upregulated expression in the amygdala following acute stress, Downregulatedexpression in the amygdala following chronic stress

Meerson et al.146

miR-138-2 SNP associated with Panic disorder Muinos-Gimeno et al.189

miR-144 Decreased expression in the hippocampus due to treatment with lithium and sodium valproate Zhou et al.71

miR-148a SNP associated with Panic disorder Muinos-Gimeno et al.189

miR-16 Increased expression in the raphe nucleus due to administration of the SSRI fluoxetine Baudry et al.139

Decreased expression in the locus coeruleus due to administration of the SSRI fluoxetine

Increasing expression in the raphe nucleus led to an antidepressant-like effect

Decreasing expression in the locus coeruleus led to an antidepressant-like effect

miR-182 A polymorphism in the miR-182 gene is associated with major depressive disorder Saus et al.181

miR-183 Upregulated expression in the amygdala following acute stress Meerson et al.146

miR-189 Variation in binding site of one of its target mRNA, SLITRK1, found in patients withTourette0s Syndrome

Abelson et al.194

miR-18a Increased in the paraventricular nucleus of the stress sensitive Fischer 344 rat strain Uchida et al.156

miR-212 Pre-miRNA upregulated expression in the medial pre-frontal cortex following maternalseparation

Uchida et al.156

miR-22 SNP associated with Panic disorder Muinos-Gimeno et al.189

miR-221 Decreased expression in the hippocampus due to treatment with lithium and sodiumvalproate

Zhou et al.71


Zhou et al.71

miR-26a/b Upregulated expression in the frontal cortex following acute stress Rinaldi et al.147

miR-29a Upregulated expression in the medial pre-frontal cortex following maternal separation Uchida et al.156

Pre-miRNA upregulated expression in the medial pre-frontal cortex following maternalseparation

miR-30c Decreased expression in the hippocampus due to treatment with lithium and sodiumvalproate

Zhou et al.71

miR-30e A polymorphism in the miR-30e gene is associated with major depressive disorder Wu et al.176



Zhou et al.71

miR-485-3p Significantly associated with hoarding subtype of OCD Muinos-Gimeno et al.188







miR-9 Upregulated expression in the frontal cortex following acute stress Rinaldi et al.147

Upregulated expression in the medial pre-frontal cortex following maternal separation Uchida et al.156


Uchida et al.156


Uchida et al.156


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technology to its maximum benefit, the conditionsdictating the success of patent applications will needto be broad enough to allow commercial entities thathave sufficiently contributed enough knowledge tosatisfy both novelty and utility to recoup initialinvestments, while at the same time preventing thegranting of patents that act as place holders until areal-world utility can be established as this wouldstagnate research efforts.199 (For a more extensivereview on miRNA patents to date, please see ref. 199).

Overall, targeting miRNAs clearly represent a novelapproach that holds great promise in neuropsychiatry.Research into these small molecules should beprioritised so that someday they may represent afully realised, truly novel therapeutic strategy for thetreatment of mood and anxiety disorders. Time willtell whether these, like the red rose in Wilde’s shortstory,200 hold the promise of supplanting all otherstrategies for happiness.

Conflict of interest

The authors declare no conflict of interest.

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Little things on which happiness depends: microRNAs as novel therapeutic targets for the treatment of anxiety and depression