Thinking small: towards microRNA-based therapeutics for anxiety disorders

Page 1

1. Introduction

2. Current therapies and

therapies in development

3. Factors contributing to the

development of anxiety

disorders

4. The emergence of miRNAs

5. miRNA function in animal

models of psychiatric illness

6. Conclusion

7. Expert opinion

Review

Thinking small: towardsmicroRNA-based therapeuticsfor anxiety disordersKaren A Scott, Alan E Hoban, Gerard Clarke, Gerard M Moloney,Timothy G Dinan & John F Cryan†

†University College Cork, Alimentary Pharmabiotic Centre, Department of Anatomy and

Neuroscience, Cork, Ireland

Introduction: Anxiety disorders are the most frequently diagnosed psychiatric

conditions, negatively affecting quality of life and creating a significant eco-

nomic burden. These complex disorders are extremely difficult to treat, and

there is a great need for novel therapeutics with greater efficacy and minimal

adverse side effects.

Areas covered: In this review, theauthorsdescribe the role thatmicroribonucleic

acids (microRNA or miRNA) play in the development of anxiety disorders and

their potential to serve as biomarkers of disease as well as targets for pharmaco-

logical treatment. Furthermore, the authors discuss the current state of miRNA

research, including both preclinical and clinical studies of anxiety disorders.

Expert opinion: There is mounting evidence that circulating miRNA may serve

as biomarkers of disease and play a role in the development of disease, includ-

ing psychiatric conditions such as anxiety disorders. Great strides have been

made in cancer research, with miRNA-based therapies already in use in clinical

studies. However, the use of miRNA for the treatment of neurological disor-

ders, and psychiatric disorders in particular, is still in its nascent stage. The

development of safe compounds that are able to cross the blood--brain barrier

and target specific cell populations, which are relevant to anxiety-related

neurocircuitry, is paramount for the emergence of novel, efficacious miRNA-

based therapies in clinical settings.

Keywords: anxiety disorders, clinical trials, drugs, microRNA, preclinical models

Expert Opin. Investig. Drugs [Early Online]

1. Introduction

Anxiety disorders are some of the most common illnesses experienced, affecting anestimated 16% of people according to the WHOWorld Mental Health studies [1,2].Anxiety disorders comprise several conditions, including general anxiety and socialanxiety disorders, separation anxiety, phobias and panic disorders [1,3]. Obsessivecompulsive disorder and post-traumatic stress disorder (PTSD) are considered bymany to be anxiety disorders and have been classified as such in the past, but thesehave been removed from the category in the most recent edition of the AmericanPsychological Association Diagnostic and Statistical Manual of Mental Disorders(DSM-5) and are now described in different chapters (Obsessive-Compulsive andRelated Disorders and Trauma- and Stressor-Related Disorders, respectively) [4].Anxiety disorders are notoriously difficult to successfully treat and a variety ofgenetic and environmental factors contribute to their development and severity [5].The perinatal and adolescent periods are particularly critical; early life adversity is asignificant risk factor for the development of anxiety disorders, estimated at 30% [6].It is also well-recognised that anxiety disorders have a strong heritability, althoughfrom a genetic standpoint anxiety has received less focus than other psychiatric

10.1517/13543784.2014.997873 © 2014 Informa UK, Ltd. ISSN 1354-3784, e-ISSN 1744-7658 1All rights reserved: reproduction in whole or in part not permitted

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conditions [7]. There has been a strong push for a betterunderstanding of the aetiology of anxiety disorders andPTSD, as the numbers of combat veterans presenting withanxiety-related disorders have risen dramatically in recentyears [8,9]. Genetic contribution to the development of anxietydisorders has been estimated to range from 30% to nearly70% [10,11]. To date, it appears that this heritability is a resultof numerous genetic and environmental interactions ratherthan a single factor [10-13].MicroRNAs (miRNAs) have gained much attention over

the past two decades, and like other epigenetic mechanisms,act as an interface between genes and the environment. Recentstudies implicate miRNAs in the development of pathologicalconditions, and may in turn serve as novel targets for theirtreatment. Indeed, targeting endogenous miRNA levels is cur-rently used in the clinical setting for the treatment of hepatitisC and certain liver cancers. In this review, we will discussrecent advances in the field of miRNA drug developmentand the potential of miRNA-based therapies for the treatmentof anxiety disorders.

2. Current therapies and therapiesin development

Current strategies for treatment of anxiety disorders centeron a combination of pharmacotherapy and psychotherapy.

Most often, selective serotonin reuptake inhibitors (SSRIs)and serotonin noradrenergic reuptake inhibitors (SNRIs)are prescribed for long-term treatment, whereas anxiolytics,such as benzodiazepines, targeting g-aminobutyric acid arewidely used for acute treatment of acute anxiety episodeswith polypharmacy common [14-16]. These treatments arefar from optimal for most individuals. A significant propor-tion of patients do not respond to SSRIs/SNRIs, and forthose that do, there is a delay of weeks prior to onset of ther-apeutic efficacy. In addition, some patients are not able totolerate associated side effects, such as sexual dysfunction,gastrointestinal issues and sleep disturbances that are fre-quently reported with SSRI and SNRI usage [3,16]. Sideeffects of benzodiazepines are well-known; there is a clearrisk of dependence, they can impair cognitive function andhave sedating effects [3,17,18].

Our current understanding of anxiety disorders at themolecular level comes from a wide variety of studies usingsamples from both living and deceased patients and animalmodels of anxiety disorders. There has been great interest inthe glutamatergic and endocannabinoid systems and differentneuropeptides, including melanin concentrating hormone,corticotropin releasing hormone, oxytocin, vasopressin, chol-ecystokininand neuropeptide Y [3,19,20]. Recent studies foc-used on these have indeed had modest success, althoughspecificity is often a problem [3]. As these systems have wideprojections and many of these neuropeptides and/or theirreceptors are ubiquitously expressed throughout the CNS,ensuring that effects are only produced in the regions involvedin anxiety are necessary in order to prevent the off-targeteffects that often accompany pharmacotherapeutics. For anexcellent review on the current status of studies investigatingthe potential of these neurotransmitters and peptides for treat-ing anxiety disorders, please see Bukalo et al. [15].

3. Factors contributing to the developmentof anxiety disorders

3.1 GeneticsGenetics are believed to play a moderate role in the develop-ment of anxiety disorders, but in comparison with other psy-chiatric conditions, it has received less attention [7]. Many ofthe candidate genes implicated in anxiety are related to mono-aminergic and catecholaminergic signalling [10]. Some geneswith variants that are commonly suggested to be involved inanxiety disorders are catechol-O-methyltransferase, solute car-rier family 6, member 4 and brain-derived neurotrophic fac-tor. However, a meta-analysis performed by McGrath et al.shows that there is little evidence suggesting an associationwith these variants and anxiety disorders, as well as a lack ofrepeatability in findings [21]. Similarly, linkage studies havealso had problems as far as replication of results [10,21,22].Using a multifaceted approach may have more success. Thereis increased emphasis on the use of imaging to look at patternsof activation within corticolimbic structures in response to

Article highlights.

. Anxiety disorders affect a significant proportion of thepopulation, but the mechanisms underlying theirdevelopment are largely unknown. Furthermore, manyindividuals are unable to manage symptoms withcurrently available therapies.

. Over the last two decades, significant interest inmicroribonucleic acids (microRNAs or miRNAs) hasarisen. These short, endogenous, non-coding moleculesinfluence gene expression and have the potential toserve as diagnostic and prognostic markers of disease.Additionally, there is great interest in targeting miRNAfor the treatment of numerous conditions, includingneuropsychiatric illnesses such as anxiety disorders.

. Preclinical models of anxiety-like disorders are associatedwith changes in miRNA levels in corticolimbic structuresassociated with stress and anxiety. Furthermore,targeted manipulation has been demonstrated toameliorate or exacerbate the phenotype. However, todate, there is little concordance between studies in thespecific miRNAs associated with anxiety-like phenotype.

. Targeting of miRNA expression holds much promise inthe treatment of numerous illnesses and is currently inuse for the treatment of some forms of cancer and liverdisease. However, at this point, miRNA targeting forthe treatment of neuropsychiatric conditions is still in itsinfancy and much more research is necessary to betterunderstand the role that they play in the etiology, andpotential treatment, of anxiety disorders.

This box summarises key points contained in the article.

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anxiety-producing stimuli which can then be correlated withgenetic information from subjects [10]. The above-mentionedgenetic findings highlight the great variability in clinical find-ings pertaining to anxiety disorders. Indeed, it appears thatmultiple environmental and genetic factors contribute to thedevelopment, severity and duration of these disorders.

3.2 Epigenetic mediators of anxietyOne of the keys to developing better therapies for anxietydisorders is the need to understand the molecular basis ofpathological anxiety and to understand the factors that con-tribute to its development. In addition to genetic and environ-mental factors, it is clear that epigenetics, changes in geneexpression that are influenced by the environment and thatdo not change the actual sequence of the DNA, are alsoinvolved in susceptibility and resilience to pathological condi-tions [23,24]. Epigenetic modifications may explain some of thelarge variations that are seen in phenotype amongst individu-als with anxiety and other psychiatric disorders. It is nowunderstood that gene expression can be altered by environ-mental factors including stressors, environmental enrichment,chemical exposures, and so on. The most common epigeneticchanges involve (de)methylation of DNA and modification ofhistone groups [23]. By altering the structure of the DNA, theability of transcriptional machinery to bind is changed, alter-ing expression of the gene. DNA methylation can inhibit geneexpression, whereas modification of histone tails can promoteor inhibit gene expression, depending on the groups added [24].For example, it has recently been demonstrated that individu-als with a single-nucleotide polymorphism in the FKBP5gene, which regulates glucocorticoid receptor expression, aremore susceptible to psychiatric conditions including PTSDand major depressive disorder (MDD) when exposed to child-hood trauma. In this case, the variant of FKBP5 is preferen-tially demethylated in response to adversity [25,26].

miRNAs are another way that gene expression can be mod-ified in the disease state. There has been much interest in thesemolecules and their potential use in the diagnostic, prognosticand therapeutic treatment of multiple pathologies, includingCNS disorders. In this review, we focus on their potentialfor the treatment of anxiety disorders.

4. The emergence of miRNAs

Since their discovery two decades ago, miRNAs have garneredmuch interest for their potential use for a treatment of a numberof medical disorders [23,27-31]. These short, endogenous, non-coding RNA sequences (~21--25 nucleotides in length) wereoriginally thought of as ‘junk RNA,’ but it is now known thatthey can influence the expression of genes, primarily by inhibit-ing their translation to functional proteins. There is evidencethat miRNAs can also increase gene expression, but this appearsto be much less common [32]. Because multiple genes within abiological network are responsive to even small alterations inmiRNA levels, they are particularly appealing as therapeutic

targets in complex heterogeneous disorders [33]. Figure 1 showsthe miRNA pathway. Briefly, miRNAs can bind to comple-mentary sequences on mRNA, altering (usually be preventing)translational machinery to bind and translate the mRNA toprotein. For a more thorough review on the biogenesis andfunction of miRNA, please see reviews by Bartel et al. [30,34].

The entire miRNA sequence does not need to be preciselythe same in order to bind to an mRNA; a subregion of themiRNA referred to as a ‘seed sequence’ binds with comple-mentary sequences on the 3¢ untranslated region of themRNA, repressing translation and/or marking the structurefor degradation [34-37]. The greater the complementarity, thegreater the effect upon gene expression (Figure 1). miRNAshave the potential to bind to a number of different mRNAsdue to their short length; this means a higher likelihood ofsharing complementary sequences with multiple mRNAsallowing one miRNA to influence the expression of numerousgenes, often within the same signalling [38,39].

4.1 miRNAs in diseasemiRNAs have been implicated in a great number of diseases,but the majority of studies come from the field of cancerresearch. Much of the initial work has focused on the poten-tial of miRNA to serve as biomarkers for the diagnosis andprognosis of various cancers. Indeed, differential miRNAexpression in cancerous tissues has been widely reported anda number of miRNAs have been identified as biomarkers ofmalignancies [28,29,40,41]. Furthermore, some studies suggestthat miRNA profiles may predict response to different typesof chemotherapy [29]. Theoretically, these miRNA biomarkersmay inform the best methods of treatment, facilitating thepersonalisation of medicine, tailoring treatments to the spe-cific set of symptoms experienced [42,43].

Manipulating endogenous miRNAs also shows great poten-tial for the treatment of pathological conditions. In some can-cers, it may be possible to directly treat the cancerous tissuewith compounds that alter miRNA expression. For example,administration of exogenous miRNAs has been used to haltthe metastasis in several animal models of cancer [29,44]. Thefirst clinical trials utilising miRNA mimetics have emergedfrom cancer research [45]. In the past year, a clinical trial hasbeen started in which liver cancer is being treated with intrave-nous injections of synthetic miR-34, a miRNA with knowntumour-suppressing properties. There is also a current clinicaltrial targeting mir-122, involved in hepatitis C virus (HCV).Miravirsen is an anti-miR-122 oligonucleotide, which inhibitsviral replication and to date, preliminary results on its usagefor treatment of HCV are quite promising [45-47].

4.2 miRNAs in CNS disordersAlthough much of the initial research concerning miRNA anddisease focused on viral infections and cancers that affectperipheral tissues, recent research has focused on neurologicalconditions, such as CNS cancers, Huntington’s, Parkinson’sand Alzheimer’s diseases. Even more recently, there has been

Thinking small: towards microRNA-based therapeutics for anxiety disorders

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miRNA:MicroRNA;RISC:RNA-inducedsilencingcomplex.

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an increased interest in the role that miRNAs may play in thedevelopment of neurological and psychiatric disorders [37,48-50].Of particular interest is identifying miRNAs that may providediagnostic and prognostic insights. Although anxiety disordersare the most common psychiatric conditions, there are farfewer studies of miRNAs and anxiety to date [1,21,51,52]. Thepaucity of studies is also reinforced by the fact that anxiety isoften comorbid with other disorders. For example, many indi-viduals with anxiety disorders also present with depressive dis-orders [11,21]. This is particularly true in the case of postmortemanalyses, as many of these brains are acquired from suicide vic-tims. Conditions such as PTSD that are often studied in veter-ans often are confounded by mood disorders including MDD,as well as by traumatic brain injury acquired in combat [53].See Table 1 for a listing of miRNAs associated with anxiety dis-orders in humans.

4.3 Peripheral changes in miRNAs associated with

psychiatric illnessExposure to psychological stress is often associated with anxi-ety, and several recent studies have linked circulating miRNAswith perceived stress and anxiety. For example, peripheralmiRNA levels have been tracked in the blood of students pre-paring for exams [54,55]. Anxiety levels of medical studentsleading up to a final, major exam were significantly correlatedwith whole blood levels of miR-16, which in turn correlatedwith downregulation of WNT4 [54]. This group previouslyfound that miR-144/144* and miR-16 elevations correlatedwith TNF-a and IFN-g in male and female medical studentsprior to exams. These inflammatory markers were elevated in

students with higher anxiety scores, peaking immediately afterthe exam, and returning to lower levels 1-week post-exam [55].miR-16 has also been linked with serotonin transporter(SERT, also referred to as 5-HTT) expression, which mayalso suggest a mechanism by which miR-16 may influenceperceived anxiety levels. Interestingly, miR-16 expressionwas not strongly correlated with salivary cortisol measure-ments in these studies. PTSD, although no longer categorisedunder the heading of anxiety disorders in the DSM-5, is alsoassociated with profound fear and anxiety. PTSD is also asso-ciated with altered circulating miRNAs, and interestingly,these may reflect immune dysregulation that may be contrib-uting to the neuropathological state. In particular, miR-125ais found to be downregulated in individuals with PTSD,which is associated with elevated PBMC levels, and elevatedIFN-g . Blood levels of miRs-22, 138-2, 148a, 339, 488 and491 have been correlated with panic disorder and phobic con-ditions, which also have strong anxiety components [56].

4.4 Peripheral changes: what do they mean in

relation to psychiatric illness?Peripheral changes in miRNAs have been reported in manyillnesses, but there is some debate as to what these findingsmean, particularly in the case of brain related disorders.While in general, RNAs are very unstable, miRNAs are sur-prisingly stable within body fluids, including whole blood,plasma, serum and cerebrospinal fluid (CSF). This stability,particularly in the more accessible minimally invasive bodilyfluids, is considered a major advantage to their utility asbiomarkers [57-60]. It is now known that miRNAs within the

Table 1. miRNAs implicated in anxiety and depressive disorders: clinical findings.

miRNA Effect Region Population Ref.

let-7d Upregulation Whole blood Depressed patients following SSRI treatment [50]

let-7e Upregulation Whole blood Depressed patients with SSRI treatment [50]

miR-16 Upregulation Whole blood Healthy medical students leading up to and immediatelyfollowing exams

[54,55]

miR-16 Downregulation Raph�e nuclei Depressed suicide completers [74]

miR-26a Downregulation Upregulation Whole blood [50]

miR-26b Upregulation Whole blood Depressed patients with SSRI treatment [50]

miR-34c Downregulation Whole blood Depressed patients with SSRI treatment [50]

miR-103 Upregulation Whole blood Depressed patients with SSRI treatment [50]

miR-128 Upregulation Whole blood Depressed patients with SSRI treatment [50]

miR-132 Upregulation Whole blood Depressed patients with SSRI treatment [50]

miR-135a Downregulation Raph�e nuclei Depressed suicide completers (with comorbid anxiety) [74]

miR-135a Downregulation Blood Currently depressed patients [74]

miR-144/144* Upregulation Whole blood Healthy medical students leading up to and immediatelyfollowing exams

[55]

miR-183 Upregulation Whole blood Depressed patients with SSRI treatment [50]

miR-192 Upregulation Whole blood Depressed patients with SSRI treatment [50]

miR-335 Upregulation Whole blood Depressed patients with SSRI treatment [50]

miR-494 Upregulation Whole blood Depressed patients with SSRI treatment [50]

miR-770 Downregulation Whole blood Depressed patients with SSRI treatment [50]

miR-1202 Downregulation Prefrontal Cortex Depressed suicide completers with comorbid anxiety [75]

miRNA: microRNA; SSRI: Selective serotonin reuptake inhibitor.

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circulation can come from a number of sources, includingcellular material within the fluid (e.g., lymphocytes), ormicrovesicles and exosomes that have been released from tis-sues (including the brain) into the circulation. In addition toprotection by encapsulation in microvesicles and exosomes,miRNAs can be complexed with proteins that protect themfrom degradation [61,62]. The fact that miRNAs can exist ina functional capacity in these circulating microvesicles or exo-somes that mediate organ--to-cell and cell-to-cell communi-cation may explain both their stability and relevance asindicators of pathology [63]. Questions remain as to the originand precise meaning of alterations in circulating miRNAsin the context of psychiatric disorders. For instance, miRNAsthat are isolated from whole blood may not reflect what isgoing on in the CNS, but may instead correlate to specificchanges within blood cells [41,64]. Even in the case of cancerslocated outside of the CNS, circulating miRNAs may be unre-lated to the cancer itself [64]. Nevertheless and as indicatedabove, there is evidence to support the thesis that at least insome instances, circulating miRNA levels reflect tissue-specificpathologies, with, for example, serum miR-141 concentrationsdistinguishing patients with prostrate cancer from healthy con-trols, while miR-21 expression in sputum has shown potentialutility in the diagnosis of lung cancer [33,65].

4.5 Central changes in miRNAs associated with

psychiatric illnessThe majority of research concerning miRNAs in pathologicalconditions has focused on diseases that affect the peripheryand specific tissues, including cancers. While it is not cur-rently clear whether systemic miRNA profiles reflect thosewithin the CNS, there is good evidence to support a role forthese RNA molecules in the pathology of CNS disorders [66].Because biopsy of brain tissue is invasive with associated sub-stantive risks for the patient, little research on central miRNAsinvolved in psychiatric conditions have been conducted. Stud-ies in living patients are mostly limited to analyses of CSF.Changes in CSF levels of miRNAs have been noted in manybrain disorders, including stroke, multiple sclerosis andAlzheimer’s disease [67-71]. There is less information regardingmiRNA expression in brain tissue of living patients with neu-rological diseases except in the case of CNS malignancies,wherein biopsies and surgical resection of cancerous tissuesare performed. Therefore, much clinical research is restrictedto human tissues acquired postmortem. Many of the post-mortem studies focus on neurological diseases that have anincreased risk of mortality. These include disorders thatdirectly increase mortality rates, such as neurodegenerativediseases like Huntington’s, Parkinson’s and Alzheimer’s dis-eases, brain malignancies like glioblastoma, and those thatindirectly increase likelihood of death through increasedrisk-taking behaviours or suicidal ideation, as in the casewith bipolar disorder, schizoaffective disorders and majordepression. Less is known about the role of miRNAs in

anxiety disorders alone, although anxiety is often also presentalong with other psychiatric disorders [7,12].

To our knowledge, there are no studies of postmortem tissueof patients who had anxiety disorders in the absence of otherpsychopathological conditions. However, it is known that alarge proportion of individuals with MDD also exhibit symp-toms associated with anxiety disorders, and therefore, thesemiRNAs may also be involved in the development of these dis-orders [72,73]. A number of miRNAs have been reported to bealtered in corticolimbic structures and the raph�e nuclei of indi-viduals with MDD that commit suicide. For example,miR-135 has been found to be downregulated within the raph�enuclei of suicide completers. This downregulation has alsobeen observed in the blood of patients with MDD, as well asan upregulation of blood miR-135a following cognitive behav-ioural therapy [74]. A downregulation of miR-1202 has alsobeen observed in the prefrontal cortex of depressed patientsthat committed suicide [74,75].

While postmortem tissue can provide valuable informationregarding changes that occur within brains of those with neu-rological conditions, these findings are correlational; we can-not make a conclusion as to whether these changes areinvolved in the development of disease or are changes result-ing from the illness itself. The limitations associated withthe assessment of postmortem brain tissue in suicide com-pleters, overlaid with long-term medication use and otherconfounding variables, have been well documented andmake interpretation of these data difficult [5,12,76]. For thesereasons, animal models play a vital role in our understandingthe mechanistic role of miRNA in neuropathology.

5. miRNA function in animal models ofpsychiatric illness

Because of the limitations associated with studying miRNAfunction in anxiety disorders in humans, animal models areoften utilised. Although it is impossible to recreate the com-plete constellation of symptoms associated with anxiety disor-ders, animal models allow us to better understand themechanisms that may underlie individual susceptibility toand the development of these conditions [76-79]. Many of thesestudies have been run in genetic and environmental rodentmodels of anxiety, using strains predisposed to anxiety-likebehaviour or using the environment (particularly stress expo-sure) to generate anxious phenotypes. Behaviours are generallyassessed using ethologically relevant tests of anxiety. Forinstance, the Light-Dark box, the elevated plus maze andthe open field tests utilise the inherent avoidance of lightedareas by rats and mice. Marble burying, novel object andnovel food tests utilise their neophobia, or fear of previouslyunexperienced objects and foods. Social interaction tests uti-lise the social nature of rats and mice and has clear correla-tional value to social anxiety disorders. Fear conditioning isalso used to assess the development and perseverance of fear-related behaviours. Models utilising foot and tail shock have

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are often used to assess anxiety-like behaviours and fear for-mation and memory, in particular those associated withPTSD [15,53,78,80].

Preclinical studies have identified a number of brain miR-NAs that may play a role in the development of anxietydisorders [81-89]. The majority of studies have looked at theeffects of genetic and environmental effects on brain regionsassociated with mood disorders, stress and fear, includingcorticolimbic structures (e.g., the frontal cortex, the paraven-tricular nucleus of the hypothalamus, the amygdala, hippo-campus) and the serotonergic neurons of the raph�e nuclei.In this section, we will review some of the recent notable find-ings regarding preclinical models of anxiety disorders. For thesake of brevity, we have selected studies in this section that wefeel show a clear effect of manipulation; these studies not onlyidentified changes associated with anxiety-like behaviour, butalso demonstrated that experimental manipulation of thesemiRNAs could alter behavioural phenotypes. Table 2 includesa more thorough list of miRNAs implicated in preclinicalmodels of anxiety disorders.

Recently, Ressler’s group has demonstrated that miRNAscan influence the development of fear memories in a set of ele-gant experiments [90]. Normally, mice that have been exposedto sessions where a tone was paired with footshock will freezeon subsequent exposures to the tone, anticipating the associ-ated footshock. Shortly following fear conditioning, miR-34ais elevated within the basolateral amygdala (BLA) of thesemice. Ressler’s group decided to test the role of this miRNAin the formation of fear memories by decreasing its levels priorto fear conditioning by using a lentiviral-mediated miR-34a‘sponge.’ This virus induces the production of mRNAs thatbind with miR-34a, essentially acting as a sponge and prevent-ing miR-34a within the BLA from binding with its endoge-nous mRNA targets. They found that while the spongegroup was able to develop a fear response (freezing in responseto the tone paired with footshock) on the day of fear condition-ing, they did not freeze when presented with the tone on thefollowing day, suggesting impaired memory consolidation [90].

Chronic social defeat is a model of chronic stress that hasbeen shown to induce a phenotype with characteristics of anx-iety and depressive disorders [91-94]. Recently, this paradigmhas also been used to explore changes in miRNA associatedwith anxiety-like behaviours. Issler et al. recently demonstratedthat altering miR-135 expression in serotonergic neuronswithin the raph�e nuclei of mice can have significant effectsupon anxiety-related behaviours [74]. In these studies, they firstdemonstrated that miR-135 is expressed in mouse serotonergicneurons and mediates Htr1a (serotonin 1a receptor, 5-HT1A)and Slc6a4 (serotonin transporter, SERT or 5-HTT) geneexpression, and that miR-135a is upregulated followingantidepressant treatment. Furthermore, chronic social defeatwas associated with a downregulation of miR-135a, andlentiviral-mediated downregulation of miR-135 in the raph�enuclei of naı̈ve mice recapitulated the anxious phenotype.Anxiety-like (and depressive-like) behaviours that develop

following chronic social stress were prevented in transgenicmice overexpressing miR-135 within the raph�e nuclei. Inter-estingly, Issler et al., were also able to demonstrate that sim-ilar changes may occur in depressed humans, as changes inblood and the postmortem samples from raph�e nuclei alsorevealed lower levels of miR-135 expression in comparisonwith controls [74].

Chronic social defeat is also associated with upregulation ofmiRNAs within the corticolimbic structures. Haramati et al.observed an increase in expression of miR-34c within the cen-tral nucleus of the amygdala (CeA) in response to acute stressand chronic social defeat. They hypothesised that this expres-sion following stress exposure may be a mechanism associatedwith stress coping. Using a lentiviral construct, they overex-pressed miR-34c within the CeA and found that it did indeedhave anxiolytic effects when naı̈ve mice were exposed to testsof anxiety-like behaviour including the light-dark box, openfield and elevated plus maze. In addition, the enhancingeffects of acute stress exposure on the anxiety behaviour testswere blocked in miR-34c overexpressing mice [95].

In addition to stress models of anxiety, preclinical researchhas also focused on genetic/strain differences in behaviour.Brain miRNA expression varies amongst mouse strains anddifferences in stress sensitivity between strains of rats may bemediated by underlying differences inmiRNA expression [96-98].Rats that have been bred for generations to display high or lowstress responsivity also have differential miRNA expressionwithin the prelimbic cortex, and these differences are suggestedto underly behavioural phenotypes [99]. However, moreresearch is needed to show a direct correlation between thesechanges in miRNA expression and inherent stress susceptibil-ity, perhaps through experimental manipulations ofmiRNA levels.

6. Conclusion

Although numerous candidate miRNAs have been implicatedin the development of anxiety disorders (Table 2), there aresome caveats that accompany these findings. Firstly, there islittle overlap or replicability of findings between studies,even when similar preclinical models are utilised. There aremany reasons that may explain the disparity. Some may berelated to the animal strains used. As previously noted,miRNA expression can vary between the strains of mouse orrat used [96-99]. Furthermore, few studies look at the temporalexpression of miRNAs and the time points selected often varyby study. For example, Haramati et al. examined changes inexpression 2 weeks following the last chronic social defeatexposure, whereas others examined miRNA expression withinhours of the final stress exposure. Some studies have notedthat although changes in miRNA expression may be transient,there is the potential for long-lasting changes in proteinexpression [100]. There are few, if any, papers that have usedmultiple groups in order to assess brain miRNA over periodsof time (for instance, at different times during recovery from

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Table 2. miRNAs implicated in anxiety disorders: preclinical findings.

miRNA Effect Region Model Species Ref.

let-7a Upregulation Frontal cortex Acute stress (1-h restraint) Mouse [100]

let-7b Upregulation Hippocampus Chronic mood stabilisertreatment (4 weeks)

Rat [88]

let-7c Downregulation Hippocampus Chronic mood stabilisertreatment (4 weeks)

Rat [88]

miR-1 Upregulation Amygdala (CeA) Chronic stress (repeatedimmobilisation)

Rat [83]

miR-1 Downregulation Hippocampus Acute stress (4-h immobilisation) Rat [83]

miR-9 Upregulation Frontal cortex (mPFC) Maternal separation Rat [98]

miR-9 Upregulation Frontal cortex Acute stress (1-h restraint) Mouse [100]

miR-19b Upregulation Serum, amygdala Restraint + tail shock (PTSDmodel)

Rat [103]

miR-24a Downregulation Hippocampus Chronic mood stabilisertreatment (4 weeks)

Rat [88]

miR-26a/b Upregulation Frontal cortex Acute stress (1-h restraint) Mouse [100]

miR-29a Upregulation Frontal cortex (mPFC) Maternal separation Rat [98]

miR-30c Downregulation Hippocampus Chronic mood stabilisertreatment (4 weeks)

Rat [88]

miR-34a Upregulation Amygdala Fear conditioning Mouse [90]

miR-34a Downregulation Hippocampus Chronic mood stabilisertreatment (4 weeks)

Rat [88]

miR-34c Upregulation Amygdala (CeA) Acute stress (30-min restraint)and Chronic socialstress (10 days)

Mouse [95]

miR-34c Lentiviraloverexpressionhas anxiolyticeffect

Amygdala (CeA) Chronic social stress Mouse [95]

miR-34c Upregulation Hippocampus Fear conditioning Mouse [89]

miR-124 Downregulation Amygdala Acute stress (2-h restraint) Mouse [82]

miR-124 Upregulation Frontal cortex (mPFC) Maternal separation Rat [98]

miR-124a Upregulation Amygdala Environmental enrichment Indian fieldmouse

[81,86]

miR-128a Downregulation Hippocampus Chronic mood stabilisertreatment (4 weeks)

Rat [88]

miR-128a Downregulation Frontal cortex (prelimbic) Genetic model of anxiety (Highresponders)

Rat [99]

miR-128b Upregulation Frontal cortex (infralimbic) Fear-extinction learning Mouse [85]

miR-132 Upregulation Hippocampus Foot shock Mouse [87]

miR-132 Upregulation Frontal cortex (mPFC) Maternal separation Rat [98]

miR-134 Upregulation Amygdala (CeA) Acute stress (4-h immobilisation) Rat [83]

miR-134 Downregulation Amygdala (CeA) Chronic stress (repeatedimmobilisation)

Rat [83]

miR-134 Upregulation Hippocampus Acute stress (4-h immobilisation) Rat [83]

miR-134 Downregulation Hippocampus Chronic stress (repeatedimmobilisation)

Rat [83]

miR-134 Upregulation Frontal cortex (mPFC) Maternal separation Rat [98]

miR-135 Upregulation Raphe nuclei Chronic social defeat followedby antidepressanttreatment (imipramine)

Mouse [74]

miR-135a Downregulation Amygdala Acute stress (2 h restraint) Mouse [82]

miR-142 Upregulation Serum, Amygdala Restraint+tail shock (PTSDmodel)

Rat [103]

miR-144 Downregulation Hippocampus Chronic mood stabilisertreatment (4 weeks)

Rat [88]

miR-182 Downregulation Amygdala (LA) Fear conditioning Rat [84]

miR-183 Upregulation Amygdala (CeA) Acute stress (4 h immobilisation) Rat [83]

miR-183 Unchanged Amygdala (CeA) Chronic stress (repeatedimmobilisation)

Rat [83]

miRNA: MicroRNA; PTSD: Post-traumatic stress disorder.

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chronic stress). This is important to note, as it is known thatmiRNA can follow circadian patterns of expression [101]. Sub-tle differences in paradigms may also cause differential find-ings in miRNA expression.

It is also important to note that although many candidatemiRNAs have been identified, there is a great variation inmiRNAs implicated in preclinical models of anxiety and inclinical studies of humans and even less overlap between pre-clinical and human postmortem findings. There are manypotential reasons for this heterogeneity. First of all, it is clearthat there are many different types of anxiety and many dif-ferent contributing factors. Most notably, the postmortemtissue assessments come from suicide completers withMDD; a clear comorbidty of psychiatric disorders is presentin these individuals. Again, these may be related to the timingof miRNA assessment. It is also important to note thatmiRNA research is still in its relative infancy. New andimproved methods of miRNA sequencing are readily becom-ing available and new miRNAs are being discovered. Newersystems are yielding more accurate and verifiable results,reducing the number of false positives [102]. In order to effec-tively treat anxiety disorders using miRNA-based techniques,a multifaceted approach is necessary (Figure 2). Improve-ments in screening technologies, greater clinical focus onalterations in miRNA expression and continued researchusing preclinical models may indeed lead to the development

of novel and effective miRNA-based therapies for anxietydisorders.

7. Expert opinion

The potential use of blood and CSF to screen for diseasemarkers would be a great breakthrough. However, it isunlikely that we will identify a single biomarker for disordersrelated to anxiety, due to the numerous factors that contributeto their development. Some have proposed that a more likelydevelopment will be the discovery of miRNA ‘signatures’, clus-ters of miRNA that act as biomarkers for illness. Indeed, recentstudies suggest that such signatures exist for cancers and insome preclinical models of stress-related disorders [103-105].

The potential of miRNA-directed therapeutics is very excit-ing and encouraging. Current clinical trials utilising miRNAfor the treatment of cancers and hepatitis are showing muchpromise and we remain hopeful that miRNA-mediated thera-pies will also be capable of treating neuropsychiatric disorders.Different methods that may be used to treat these conditionsmay involve molecules that replace necessary miRNAs withmimics or viral vectors that lead to an upregulation of thetargeted miRNA. Obviously, viral-mediated treatments arenot without risk and work must be done to ensure that itdoes not cause off-target effects as has been observed in priorgene therapy studies. Other therapies may inhibit the effects

Table 2. miRNAs implicated in anxiety disorders: preclinical findings (continued).

miRNA Effect Region Model Species Ref.

miR-183 Upregulation Hippocampus Acute stress (4 h immobilizaion) Rat [83]

miR-192 Downregulation Frontal cortex (prelimbic),nucleus accumbens(core and shell)

Genetic model of anxiety (highresponders)

Rat [99]

miR-221 Downregulation Hippocampus Chronic mood stabilisertreatment (4 weeks)

Rat [88]

miR-322 Upregulation Serum, amygdala Restraint+tail shock (PTSDmodel)

Rat [103]

miR-324 Upregulation Serum, amygdala Restraint+tail shock (PTSDmodel)

Rat [103]

miR-421 Upregulation Serum, amygdala Restraint+tail shock (PTSDmodel)

Rat [103]

miR-429 Downregulation Frontal cortex Inescapable shock Rat [113]

miR-463 Upregulation Serum, amygdala Restraint + tail shock (PTSDmodel)

Rat [103]

miR-484 Downregulation Frontal cortex (prelimbic),nucleus accumbens (shell)

Genetic model of anxiety (highresponders)

Rat [99]

miR-544 Downregulation Nucleus accumbens (coreand shell)

Genetic model of anxiety (highresponders)

Rat [99]

miR-598 Downregulation Nucleus accumbens (coreand shell)

Genetic model of anxiety (highresponders)

Rat [99]

miR-674 Upregulation Serum, amygdala Restraint + tail shock (PTSDmodel)

Rat [103]

miR-1971 Downregulation Frontal cortex (PFC) Inescapable shock + fluoxetinetreatment

Mouse [80]

miRNA: MicroRNA; PTSD: Post-traumatic stress disorder.

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of miRNAs, or act as miRNA sponges -- designed to bindendogenous miRNAs to reduce their effects in target areas.Antagomirs, locked nucleic acids (LNAs) and antisense oligo-nucleotides have been used to inhibit miRNAs in preclinicalstudies. However, there is still much work that must bedone before they are able to be used in a clinical setting forthe treatment of anxiety.One challenge that miRNA therapies pose is stability. RNAs

are typically unstable, but endogenous miRNAs tend to be sta-ble as they are often contained within exosomes or microvesiclesor bound to proteins that have protective properties. Some ofthe current developments in administering exogenous miRNAsand mimics include nanoparticle encapsulation, LNAs andconjugation with cholesterol [28,106,107]. As previously noted, itis unlikely that many psychiatric conditions can be effectivelytreated by a single miRNA manipulation, as multiple miRNAsare often differentially expressed in pathological conditions. Ina preclinical model of cancer, multiple miRNAs have been tar-geted using an antisense miRNA oligodeoxyribonucleo-tide [107,108]. Another necessity is to ensure that these methodsof delivery do not cause issues as far as toxicity. Recent nonhu-man primate studies examining the effects of LNA anti-miRtherapies for the treatment of cholesterol and HCV were suc-cessful and well-tolerated when delivered intravenously [38,109].Similarly, trials of LNA anti-miRNA therapies in humanswith liver cancer and HCV are also promising. Recent clinicaltrials of miraversin, an anti- miRNA oligonucleotide targeting

miR-122 that is administered subcutaneously, has been usedto treat HCV and preliminary results suggest it is both safeand effective [47,110]. An miRNA mimic is currently in clinicaltrials for the treatment of liver cancer. MRX34 utilises lipo-somes for delivery and is administered intravenously for treat-ment of metastatic liver cancer, but results from these trialsare yet to be published [45].

An additional challenge as far as miRNA-mediated thera-pies for psychiatric disorders is the need for minimally inva-sive methods of delivery. The majority of preclinical studieshave explored the effects of miRNA manipulation throughnuclei-specific manipulations using microinjections. Ideally,miRNA therapies could be administered peripherally, andcould be taken orally or through intravenous injection.Recently, groups have had success in developing methodsfor crossing the blood--brain barrier (BBB). Yang et al. devel-oped recombinant adeno-associated viruses (rAAVs) that,when administered intravenously, are capable of crossing theBBB in both mouse and nonhuman primate preclinical mod-els [111]. Similarly, Iida et al. developed an rAAV that was ableto cross the BBB of mice. They noted that use of a neuron-specific promoter may reduce or eliminate the immuneresponse that is often observed in response to CNS gene ther-apies that also transduce astrocytes [112].

Because miRNAs have the potential to modulate theexpression of so many genes, it is also imperative to developtherapies that are specific and that exert their effects in specific

Experimental groupsand clinical samples

Preclinical models

miRNA expressionassays

In vitro models

qRT-PCR validation

Potential therapeutics

In silico target analysis

Rx

Figure 2. miRNAs in drug discovery.miRNA: MicroRNA.

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regions related to anxiety disorders. Many of the miRNAs thatare altered in neuropsychiatric disorders are also implicated incancers and are related to tumorigenesis, whereas others areinvolved in general cell signalling pathways [28]. This high-lights the need for specificity, as there are serious implicationsas far as off-target effects are concerned. Preclinical modelshave demonstrated that peripherally administered miRNAtherapeutics can specifically target neurons, but targeting spe-cific neural populations has yet to be demonstrated [112].Development of systemically administered compounds thattarget miRNAs in specific brain regions involved in anxiety-related pathologies would be ideal but remains a challenge.

In conclusion, the field of miRNA research holds muchpromise and may yield tangible benefits for the clinical man-agement of anxiety disorders, but progress has been modest,with their use as biomarkers providing the most promise atthis point. It is clear that a multidisciplinary approach, utilis-ing both clinical and preclinical approaches, is necessary toidentify candidate miRNAs with therapeutic potential. Recentadvances in miRNA delivery and the continued exponentialimprovements in miRNA sequencing technology can be har-nessed to build on recent advances. Taken together, these

approaches hold the potential to yield miRNA-based thera-peutics for anxiety disorders. Only time will tell if this prom-ise satisfies the demand for faster acting and more efficaciousagents.

Declaration of interest

All of the authors are employed by University College, Cork.JF Cryan and TG Dinan were supported in part by ScienceFoundation Ireland in the form of a Centre Grant (grantnos. 02/CE/B124, 07/CE/B1368 and SFI/12/RC/2273).The Alimentary Pharmabiotic Centre is a research centrefunded by Science Foundation Ireland (SFI), through the IrishGovernment’s National Development Plan. JF Cryan,TG Dinan and KA Scott are also supported by HRB GrantHRA_POR/2012/32. G Clarke is supported by a NARSADYoung Investigator Grant from the Brain and BehaviorResearch Foundation (Grant Number 20771). The authorshave no other relevant affiliations or financial involvementwith any organisation or entity with a financial interest in orfinancial conflict with the subject matter or materials discussedin the manuscript apart from those disclosed.

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AffiliationKaren A Scott1,2, Alan E Hoban1,2,

Gerard Clarke2,3, Gerard M Moloney1,2,

Timothy G Dinan2,3 & John F Cryan†1,2

†Author for correspondence1Department of Anatomy and Neuroscience,

University College Cork, Cork, Ireland;

E-mail: [email protected] Pharmabiotic Centre, University

College Cork, Cork, Ireland3Department of Psychiatry, University College

Cork, Cork, Ireland

K. A. Scott et al.

14 Expert Opin. Investig. Drugs (2014) 24(3)

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