Review

Thinking small: towards microRNA-based therapeutics for anxiety disorders 1.

Introduction

2.

Current therapies and therapies in development

3.

Factors contributing to the

Karen 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

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development of anxiety disorders 4.

The emergence of miRNAs

5.

miRNA function in animal models of psychiatric illness

6.

Conclusion

7.

Expert opinion

Introduction: Anxiety disorders are the most frequently diagnosed psychiatric conditions, negatively affecting quality of life and creating a significant economic 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, the authors describe the role that microribonucleic 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 pharmacological 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, including 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 disorders, 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 miRNAbased 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 an estimated 16% of people according to the WHO World Mental Health studies [1,2]. Anxiety disorders comprise several conditions, including general anxiety and social anxiety disorders, separation anxiety, phobias and panic disorders [1,3]. Obsessive compulsive disorder and post-traumatic stress disorder (PTSD) are considered by many to be anxiety disorders and have been classified as such in the past, but these have been removed from the category in the most recent edition of the American Psychological Association Diagnostic and Statistical Manual of Mental Disorders (DSM-5) and are now described in different chapters (Obsessive-Compulsive and Related Disorders and Trauma- and Stressor-Related Disorders, respectively) [4]. Anxiety disorders are notoriously difficult to successfully treat and a variety of genetic and environmental factors contribute to their development and severity [5]. The perinatal and adolescent periods are particularly critical; early life adversity is a significant risk factor for the development of anxiety disorders, estimated at 30% [6]. It is also well-recognised that anxiety disorders have a strong heritability, although from 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 All rights reserved: reproduction in whole or in part not permitted

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K. A. Scott et al.

Article highlights. .

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.

.

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Anxiety disorders affect a significant proportion of the population, but the mechanisms underlying their development are largely unknown. Furthermore, many individuals are unable to manage symptoms with currently available therapies. Over the last two decades, significant interest in microribonucleic acids (microRNAs or miRNAs) has arisen. These short, endogenous, non-coding molecules influence gene expression and have the potential to serve as diagnostic and prognostic markers of disease. Additionally, there is great interest in targeting miRNA for the treatment of numerous conditions, including neuropsychiatric illnesses such as anxiety disorders. Preclinical models of anxiety-like disorders are associated with changes in miRNA levels in corticolimbic structures associated with stress and anxiety. Furthermore, targeted manipulation has been demonstrated to ameliorate or exacerbate the phenotype. However, to date, there is little concordance between studies in the specific miRNAs associated with anxiety-like phenotype. Targeting of miRNA expression holds much promise in the treatment of numerous illnesses and is currently in use for the treatment of some forms of cancer and liver disease. However, at this point, miRNA targeting for the treatment of neuropsychiatric conditions is still in its infancy and much more research is necessary to better understand the role that they play in the etiology, and potential treatment, of anxiety disorders.

This box summarises key points contained in the article.

conditions [7]. There has been a strong push for a better understanding of the aetiology of anxiety disorders and PTSD, as the numbers of combat veterans presenting with anxiety-related disorders have risen dramatically in recent years [8,9]. Genetic contribution to the development of anxiety disorders has been estimated to range from 30% to nearly 70% [10,11]. To date, it appears that this heritability is a result of numerous genetic and environmental interactions rather than 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. Recent studies implicate miRNAs in the development of pathological conditions, and may in turn serve as novel targets for their treatment. Indeed, targeting endogenous miRNA levels is currently used in the clinical setting for the treatment of hepatitis C and certain liver cancers. In this review, we will discuss recent advances in the field of miRNA drug development and the potential of miRNA-based therapies for the treatment of anxiety disorders. 2. Current therapies and therapies in development

Current strategies for treatment of anxiety disorders center on a combination of pharmacotherapy and psychotherapy. 2

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 are widely used for acute treatment of acute anxiety episodes with polypharmacy common [14-16]. These treatments are far from optimal for most individuals. A significant proportion of patients do not respond to SSRIs/SNRIs, and for those that do, there is a delay of weeks prior to onset of therapeutic efficacy. In addition, some patients are not able to tolerate associated side effects, such as sexual dysfunction, gastrointestinal issues and sleep disturbances that are frequently reported with SSRI and SNRI usage [3,16]. Side effects of benzodiazepines are well-known; there is a clear risk of dependence, they can impair cognitive function and have sedating effects [3,17,18]. Our current understanding of anxiety disorders at the molecular level comes from a wide variety of studies using samples from both living and deceased patients and animal models of anxiety disorders. There has been great interest in the glutamatergic and endocannabinoid systems and different neuropeptides, including melanin concentrating hormone, corticotropin releasing hormone, oxytocin, vasopressin, cholecystokininand neuropeptide Y [3,19,20]. Recent studies focused on these have indeed had modest success, although specificity is often a problem [3]. As these systems have wide projections and many of these neuropeptides and/or their receptors are ubiquitously expressed throughout the CNS, ensuring that effects are only produced in the regions involved in anxiety are necessary in order to prevent the off-target effects that often accompany pharmacotherapeutics. For an excellent review on the current status of studies investigating the potential of these neurotransmitters and peptides for treating anxiety disorders, please see Bukalo et al. [15].

Factors contributing to the development of anxiety disorders

3.

Genetics Genetics are believed to play a moderate role in the development of anxiety disorders, but in comparison with other psychiatric conditions, it has received less attention [7]. Many of the candidate genes implicated in anxiety are related to monoaminergic and catecholaminergic signalling [10]. Some genes with variants that are commonly suggested to be involved in anxiety disorders are catechol-O-methyltransferase, solute carrier family 6, member 4 and brain-derived neurotrophic factor. However, a meta-analysis performed by McGrath et al. shows that there is little evidence suggesting an association with these variants and anxiety disorders, as well as a lack of repeatability in findings [21]. Similarly, linkage studies have also had problems as far as replication of results [10,21,22]. Using a multifaceted approach may have more success. There is increased emphasis on the use of imaging to look at patterns of activation within corticolimbic structures in response to 3.1

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Thinking small: towards microRNA-based therapeutics for anxiety disorders

anxiety-producing stimuli which can then be correlated with genetic information from subjects [10]. The above-mentioned genetic findings highlight the great variability in clinical findings pertaining to anxiety disorders. Indeed, it appears that multiple environmental and genetic factors contribute to the development, severity and duration of these disorders. Epigenetic mediators of anxiety One of the keys to developing better therapies for anxiety disorders is the need to understand the molecular basis of pathological anxiety and to understand the factors that contribute to its development. In addition to genetic and environmental factors, it is clear that epigenetics, changes in gene expression that are influenced by the environment and that do not change the actual sequence of the DNA, are also involved in susceptibility and resilience to pathological conditions [23,24]. Epigenetic modifications may explain some of the large variations that are seen in phenotype amongst individuals with anxiety and other psychiatric disorders. It is now understood that gene expression can be altered by environmental factors including stressors, environmental enrichment, chemical exposures, and so on. The most common epigenetic changes involve (de)methylation of DNA and modification of histone groups [23]. By altering the structure of the DNA, the ability of transcriptional machinery to bind is changed, altering expression of the gene. DNA methylation can inhibit gene expression, whereas modification of histone tails can promote or inhibit gene expression, depending on the groups added [24]. For example, it has recently been demonstrated that individuals with a single-nucleotide polymorphism in the FKBP5 gene, which regulates glucocorticoid receptor expression, are more susceptible to psychiatric conditions including PTSD and major depressive disorder (MDD) when exposed to childhood trauma. In this case, the variant of FKBP5 is preferentially demethylated in response to adversity [25,26]. miRNAs are another way that gene expression can be modified in the disease state. There has been much interest in these molecules and their potential use in the diagnostic, prognostic and therapeutic treatment of multiple pathologies, including CNS disorders. In this review, we focus on their potential for the treatment of anxiety disorders.

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3.2

4.

The emergence of miRNAs

Since their discovery two decades ago, miRNAs have garnered much interest for their potential use for a treatment of a number of medical disorders [23,27-31]. These short, endogenous, noncoding RNA sequences (~21--25 nucleotides in length) were originally thought of as ‘junk RNA,’ but it is now known that they can influence the expression of genes, primarily by inhibiting their translation to functional proteins. There is evidence that miRNAs can also increase gene expression, but this appears to be much less common [32]. Because multiple genes within a biological network are responsive to even small alterations in miRNA levels, they are particularly appealing as therapeutic

targets in complex heterogeneous disorders [33]. Figure 1 shows the miRNA pathway. Briefly, miRNAs can bind to complementary sequences on mRNA, altering (usually be preventing) translational machinery to bind and translate the mRNA to protein. For a more thorough review on the biogenesis and function of miRNA, please see reviews by Bartel et al. [30,34]. The entire miRNA sequence does not need to be precisely the same in order to bind to an mRNA; a subregion of the miRNA referred to as a ‘seed sequence’ binds with complementary sequences on the 3¢ untranslated region of the mRNA, repressing translation and/or marking the structure for degradation [34-37]. The greater the complementarity, the greater the effect upon gene expression (Figure 1). miRNAs have the potential to bind to a number of different mRNAs due to their short length; this means a higher likelihood of sharing complementary sequences with multiple mRNAs allowing one miRNA to influence the expression of numerous genes, often within the same signalling [38,39]. miRNAs in disease miRNAs have been implicated in a great number of diseases, but the majority of studies come from the field of cancer research. Much of the initial work has focused on the potential of miRNA to serve as biomarkers for the diagnosis and prognosis of various cancers. Indeed, differential miRNA expression in cancerous tissues has been widely reported and a number of miRNAs have been identified as biomarkers of malignancies [28,29,40,41]. Furthermore, some studies suggest that miRNA profiles may predict response to different types of chemotherapy [29]. Theoretically, these miRNA biomarkers may inform the best methods of treatment, facilitating the personalisation of medicine, tailoring treatments to the specific set of symptoms experienced [42,43]. Manipulating endogenous miRNAs also shows great potential for the treatment of pathological conditions. In some cancers, it may be possible to directly treat the cancerous tissue with compounds that alter miRNA expression. For example, administration of exogenous miRNAs has been used to halt the metastasis in several animal models of cancer [29,44]. The first clinical trials utilising miRNA mimetics have emerged from cancer research [45]. In the past year, a clinical trial has been started in which liver cancer is being treated with intravenous injections of synthetic miR-34, a miRNA with known tumour-suppressing properties. There is also a current clinical trial targeting mir-122, involved in hepatitis C virus (HCV). Miravirsen is an anti-miR-122 oligonucleotide, which inhibits viral replication and to date, preliminary results on its usage for treatment of HCV are quite promising [45-47]. 4.1

miRNAs in CNS disorders Although much of the initial research concerning miRNA and disease focused on viral infections and cancers that affect peripheral tissues, recent research has focused on neurological conditions, such as CNS cancers, Huntington’s, Parkinson’s and Alzheimer’s diseases. Even more recently, there has been 4.2

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miRNA gene

miRNA: MicroRNA; RISC: RNA-induced silencing complex.

Figure 1. miRNA processing and function.

Cytoplasm

Nucleus

Pre-MiRNA hairpin

Processing

(A)n CAP Primary transcript

Transcription

Drosha Dicer

P miRNA Duplex

P

Matue miRNA

RISC silencing complex

Cap

Ribosomes

Cap

Ribosomes

2

Degradation

RISC

Perfect comlpementarity

Translational repression

RISC

8

3′ 5′

Seed sequence in microRNA at 2 – 8nt 5′ – 3′

Imperfect complementarity

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(A)n

A(n)

K. A. Scott et al.

Thinking small: towards microRNA-based therapeutics for anxiety disorders

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

Effect

Region

Population

Ref.

let-7d let-7e miR-16

Upregulation Upregulation Upregulation

Whole blood Whole blood Whole blood

[50] [50] [54,55]

miR-16 miR-26a miR-26b miR-34c miR-103 miR-128 miR-132 miR-135a miR-135a miR-144/144*

Downregulation Downregulation Upregulation Downregulation Upregulation Upregulation Upregulation Downregulation Downregulation Upregulation

Raph e nuclei Upregulation Whole blood Whole blood Whole blood Whole blood Whole blood Raph e nuclei Blood Whole blood

miR-183 miR-192 miR-335 miR-494 miR-770 miR-1202

Upregulation Upregulation Upregulation Upregulation Downregulation Downregulation

Whole blood Whole blood Whole blood Whole blood Whole blood Prefrontal Cortex

Depressed patients following SSRI treatment Depressed patients with SSRI treatment Healthy medical students leading up to and immediately following exams Depressed suicide completers Whole blood Depressed patients with SSRI treatment Depressed patients with SSRI treatment Depressed patients with SSRI treatment Depressed patients with SSRI treatment Depressed patients with SSRI treatment Depressed suicide completers (with comorbid anxiety) Currently depressed patients Healthy medical students leading up to and immediately following exams Depressed patients with SSRI treatment Depressed patients with SSRI treatment Depressed patients with SSRI treatment Depressed patients with SSRI treatment Depressed patients with SSRI treatment Depressed suicide completers with comorbid anxiety

[74] [50] [50] [50] [50] [50] [50] [74] [74] [55] [50] [50] [50] [50] [50] [75]

miRNA: microRNA; SSRI: Selective serotonin reuptake inhibitor.

an increased interest in the role that miRNAs may play in the development of neurological and psychiatric disorders [37,48-50]. Of particular interest is identifying miRNAs that may provide diagnostic and prognostic insights. Although anxiety disorders are the most common psychiatric conditions, there are far fewer studies of miRNAs and anxiety to date [1,21,51,52]. The paucity of studies is also reinforced by the fact that anxiety is often comorbid with other disorders. For example, many individuals with anxiety disorders also present with depressive disorders [11,21]. This is particularly true in the case of postmortem analyses, as many of these brains are acquired from suicide victims. Conditions such as PTSD that are often studied in veterans 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 disorders in humans.

students with higher anxiety scores, peaking immediately after the 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 may also suggest a mechanism by which miR-16 may influence perceived anxiety levels. Interestingly, miR-16 expression was not strongly correlated with salivary cortisol measurements in these studies. PTSD, although no longer categorised under the heading of anxiety disorders in the DSM-5, is also associated with profound fear and anxiety. PTSD is also associated with altered circulating miRNAs, and interestingly, these may reflect immune dysregulation that may be contributing to the neuropathological state. In particular, miR-125a is found to be downregulated in individuals with PTSD, which is associated with elevated PBMC levels, and elevated IFN-g. Blood levels of miRs-22, 138-2, 148a, 339, 488 and 491 have been correlated with panic disorder and phobic conditions, which also have strong anxiety components [56].

Peripheral changes in miRNAs associated with psychiatric illness

4.3

Exposure to psychological stress is often associated with anxiety, and several recent studies have linked circulating miRNAs with perceived stress and anxiety. For example, peripheral miRNA levels have been tracked in the blood of students preparing for exams [54,55]. Anxiety levels of medical students leading up to a final, major exam were significantly correlated with whole blood levels of miR-16, which in turn correlated with downregulation of WNT4 [54]. This group previously found that miR-144/144* and miR-16 elevations correlated with TNF-a and IFN-g in male and female medical students prior to exams. These inflammatory markers were elevated in

Peripheral changes: what do they mean in relation to psychiatric illness?

4.4

Peripheral changes in miRNAs have been reported in many illnesses, but there is some debate as to what these findings mean, particularly in the case of brain related disorders. While in general, RNAs are very unstable, miRNAs are surprisingly stable within body fluids, including whole blood, plasma, serum and cerebrospinal fluid (CSF). This stability, particularly in the more accessible minimally invasive bodily fluids, is considered a major advantage to their utility as biomarkers [57-60]. It is now known that miRNAs within the

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K. A. Scott et al.

circulation can come from a number of sources, including cellular material within the fluid (e.g., lymphocytes), or microvesicles and exosomes that have been released from tissues (including the brain) into the circulation. In addition to protection by encapsulation in microvesicles and exosomes, miRNAs can be complexed with proteins that protect them from degradation [61,62]. The fact that miRNAs can exist in a functional capacity in these circulating microvesicles or exosomes that mediate organ--to-cell and cell-to-cell communication may explain both their stability and relevance as indicators of pathology [63]. Questions remain as to the origin and precise meaning of alterations in circulating miRNAs in the context of psychiatric disorders. For instance, miRNAs that are isolated from whole blood may not reflect what is going on in the CNS, but may instead correlate to specific changes within blood cells [41,64]. Even in the case of cancers located outside of the CNS, circulating miRNAs may be unrelated to the cancer itself [64]. Nevertheless and as indicated above, there is evidence to support the thesis that at least in some instances, circulating miRNA levels reflect tissue-specific pathologies, with, for example, serum miR-141 concentrations distinguishing patients with prostrate cancer from healthy controls, while miR-21 expression in sputum has shown potential utility in the diagnosis of lung cancer [33,65].

Central changes in miRNAs associated with psychiatric illness

4.5

The majority of research concerning miRNAs in pathological conditions has focused on diseases that affect the periphery and specific tissues, including cancers. While it is not currently clear whether systemic miRNA profiles reflect those within the CNS, there is good evidence to support a role for these RNA molecules in the pathology of CNS disorders [66]. Because biopsy of brain tissue is invasive with associated substantive risks for the patient, little research on central miRNAs involved in psychiatric conditions have been conducted. Studies in living patients are mostly limited to analyses of CSF. Changes in CSF levels of miRNAs have been noted in many brain disorders, including stroke, multiple sclerosis and Alzheimer’s disease [67-71]. There is less information regarding miRNA expression in brain tissue of living patients with neurological diseases except in the case of CNS malignancies, wherein biopsies and surgical resection of cancerous tissues are performed. Therefore, much clinical research is restricted to human tissues acquired postmortem. Many of the postmortem studies focus on neurological diseases that have an increased risk of mortality. These include disorders that directly increase mortality rates, such as neurodegenerative diseases like Huntington’s, Parkinson’s and Alzheimer’s diseases, brain malignancies like glioblastoma, and those that indirectly increase likelihood of death through increased risk-taking behaviours or suicidal ideation, as in the case with bipolar disorder, schizoaffective disorders and major depression. Less is known about the role of miRNAs in 6

anxiety disorders alone, although anxiety is often also present along with other psychiatric disorders [7,12]. To our knowledge, there are no studies of postmortem tissue of patients who had anxiety disorders in the absence of other psychopathological conditions. However, it is known that a large proportion of individuals with MDD also exhibit symptoms associated with anxiety disorders, and therefore, these miRNAs may also be involved in the development of these disorders [72,73]. A number of miRNAs have been reported to be altered in corticolimbic structures and the raphe nuclei of individuals with MDD that commit suicide. For example, miR-135 has been found to be downregulated within the raphe nuclei of suicide completers. This downregulation has also been observed in the blood of patients with MDD, as well as an upregulation of blood miR-135a following cognitive behavioural therapy [74]. A downregulation of miR-1202 has also been observed in the prefrontal cortex of depressed patients that committed suicide [74,75]. While postmortem tissue can provide valuable information regarding changes that occur within brains of those with neurological conditions, these findings are correlational; we cannot make a conclusion as to whether these changes are involved in the development of disease or are changes resulting from the illness itself. The limitations associated with the assessment of postmortem brain tissue in suicide completers, overlaid with long-term medication use and other confounding variables, have been well documented and make interpretation of these data difficult [5,12,76]. For these reasons, animal models play a vital role in our understanding the mechanistic role of miRNA in neuropathology.

miRNA function in animal models of psychiatric illness

5.

Because of the limitations associated with studying miRNA function in anxiety disorders in humans, animal models are often utilised. Although it is impossible to recreate the complete constellation of symptoms associated with anxiety disorders, animal models allow us to better understand the mechanisms that may underlie individual susceptibility to and the development of these conditions [76-79]. Many of these studies have been run in genetic and environmental rodent models of anxiety, using strains predisposed to anxiety-like behaviour or using the environment (particularly stress exposure) to generate anxious phenotypes. Behaviours are generally assessed using ethologically relevant tests of anxiety. For instance, the Light-Dark box, the elevated plus maze and the open field tests utilise the inherent avoidance of lighted areas by rats and mice. Marble burying, novel object and novel food tests utilise their neophobia, or fear of previously unexperienced objects and foods. Social interaction tests utilise the social nature of rats and mice and has clear correlational value to social anxiety disorders. Fear conditioning is also used to assess the development and perseverance of fearrelated behaviours. Models utilising foot and tail shock have

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Thinking small: towards microRNA-based therapeutics for anxiety disorders

are often used to assess anxiety-like behaviours and fear formation and memory, in particular those associated with PTSD [15,53,78,80]. Preclinical studies have identified a number of brain miRNAs that may play a role in the development of anxiety disorders [81-89]. The majority of studies have looked at the effects of genetic and environmental effects on brain regions associated with mood disorders, stress and fear, including corticolimbic structures (e.g., the frontal cortex, the paraventricular nucleus of the hypothalamus, the amygdala, hippocampus) and the serotonergic neurons of the raphe nuclei. In this section, we will review some of the recent notable findings regarding preclinical models of anxiety disorders. For the sake of brevity, we have selected studies in this section that we feel show a clear effect of manipulation; these studies not only identified changes associated with anxiety-like behaviour, but also demonstrated that experimental manipulation of these miRNAs could alter behavioural phenotypes. Table 2 includes a more thorough list of miRNAs implicated in preclinical models of anxiety disorders. Recently, Ressler’s group has demonstrated that miRNAs can influence the development of fear memories in a set of elegant experiments [90]. Normally, mice that have been exposed to sessions where a tone was paired with footshock will freeze on subsequent exposures to the tone, anticipating the associated footshock. Shortly following fear conditioning, miR-34a is elevated within the basolateral amygdala (BLA) of these mice. Ressler’s group decided to test the role of this miRNA in the formation of fear memories by decreasing its levels prior to fear conditioning by using a lentiviral-mediated miR-34a ‘sponge.’ This virus induces the production of mRNAs that bind with miR-34a, essentially acting as a sponge and preventing miR-34a within the BLA from binding with its endogenous mRNA targets. They found that while the sponge group was able to develop a fear response (freezing in response to the tone paired with footshock) on the day of fear conditioning, they did not freeze when presented with the tone on the following day, suggesting impaired memory consolidation [90]. Chronic social defeat is a model of chronic stress that has been shown to induce a phenotype with characteristics of anxiety and depressive disorders [91-94]. Recently, this paradigm has also been used to explore changes in miRNA associated with anxiety-like behaviours. Issler et al. recently demonstrated that altering miR-135 expression in serotonergic neurons within the raphe nuclei of mice can have significant effects upon anxiety-related behaviours [74]. In these studies, they first demonstrated that miR-135 is expressed in mouse serotonergic neurons and mediates Htr1a (serotonin 1a receptor, 5-HT1A) and Slc6a4 (serotonin transporter, SERT or 5-HTT) gene expression, and that miR-135a is upregulated following antidepressant treatment. Furthermore, chronic social defeat was associated with a downregulation of miR-135a, and lentiviral-mediated downregulation of miR-135 in the raphe nuclei of naı¨ve mice recapitulated the anxious phenotype. Anxiety-like (and depressive-like) behaviours that develop

following chronic social stress were prevented in transgenic mice overexpressing miR-135 within the raphe nuclei. Interestingly, Issler et al., were also able to demonstrate that similar changes may occur in depressed humans, as changes in blood and the postmortem samples from raphe nuclei also revealed lower levels of miR-135 expression in comparison with controls [74]. Chronic social defeat is also associated with upregulation of miRNAs within the corticolimbic structures. Haramati et al. observed an increase in expression of miR-34c within the central nucleus of the amygdala (CeA) in response to acute stress and chronic social defeat. They hypothesised that this expression following stress exposure may be a mechanism associated with stress coping. Using a lentiviral construct, they overexpressed miR-34c within the CeA and found that it did indeed have anxiolytic effects when naı¨ve mice were exposed to tests of anxiety-like behaviour including the light-dark box, open field and elevated plus maze. In addition, the enhancing effects of acute stress exposure on the anxiety behaviour tests were blocked in miR-34c overexpressing mice [95]. In addition to stress models of anxiety, preclinical research has also focused on genetic/strain differences in behaviour. Brain miRNA expression varies amongst mouse strains and differences in stress sensitivity between strains of rats may be mediated by underlying differences in miRNA expression [96-98]. Rats that have been bred for generations to display high or low stress responsivity also have differential miRNA expression within the prelimbic cortex, and these differences are suggested to underly behavioural phenotypes [99]. However, more research is needed to show a direct correlation between these changes in miRNA expression and inherent stress susceptibility, perhaps through experimental manipulations of miRNA levels. 6.

Conclusion

Although numerous candidate miRNAs have been implicated in the development of anxiety disorders (Table 2), there are some caveats that accompany these findings. Firstly, there is little overlap or replicability of findings between studies, even when similar preclinical models are utilised. There are many reasons that may explain the disparity. Some may be related to the animal strains used. As previously noted, miRNA expression can vary between the strains of mouse or rat used [96-99]. Furthermore, few studies look at the temporal expression of miRNAs and the time points selected often vary by study. For example, Haramati et al. examined changes in expression 2 weeks following the last chronic social defeat exposure, whereas others examined miRNA expression within hours of the final stress exposure. Some studies have noted that although changes in miRNA expression may be transient, there is the potential for long-lasting changes in protein expression [100]. There are few, if any, papers that have used multiple groups in order to assess brain miRNA over periods of 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 let-7b

Upregulation Upregulation

Frontal cortex Hippocampus

Mouse Rat

[100] [88]

let-7c

Downregulation

Hippocampus

Rat

[88]

miR-1

Upregulation

Amygdala (CeA)

Rat

[83]

miR-1 miR-9 miR-9 miR-19b

Downregulation Upregulation Upregulation Upregulation

Hippocampus Frontal cortex (mPFC) Frontal cortex Serum, amygdala

Rat Rat Mouse Rat

[83] [98] [100] [103]

miR-24a

Downregulation

Hippocampus

Rat

[88]

miR-26a/b miR-29a miR-30c

Upregulation Upregulation Downregulation

Frontal cortex Frontal cortex (mPFC) Hippocampus

Mouse Rat Rat

[100] [98] [88]

miR-34a miR-34a

Upregulation Downregulation

Amygdala Hippocampus

Mouse Rat

[90] [88]

miR-34c

Upregulation

Amygdala (CeA)

Mouse

[95]

miR-34c

Amygdala (CeA)

Mouse

[95]

miR-34c miR-124 miR-124 miR-124a

Lentiviral overexpression has anxiolytic effect Upregulation Downregulation Upregulation Upregulation

Acute stress (1-h restraint) Chronic mood stabiliser treatment (4 weeks) Chronic mood stabiliser treatment (4 weeks) Chronic stress (repeated immobilisation) Acute stress (4-h immobilisation) Maternal separation Acute stress (1-h restraint) Restraint + tail shock (PTSD model) Chronic mood stabiliser treatment (4 weeks) Acute stress (1-h restraint) Maternal separation Chronic mood stabiliser treatment (4 weeks) Fear conditioning Chronic mood stabiliser treatment (4 weeks) Acute stress (30-min restraint) and Chronic social stress (10 days) Chronic social stress

Hippocampus Amygdala Frontal cortex (mPFC) Amygdala

Fear conditioning Acute stress (2-h restraint) Maternal separation Environmental enrichment

[89] [82] [98] [81,86]

miR-128a

Downregulation

Hippocampus

miR-128a

Downregulation

Frontal cortex (prelimbic)

Rat

[99]

miR-128b miR-132 miR-132 miR-134 miR-134

Upregulation Upregulation Upregulation Upregulation Downregulation

Frontal cortex (infralimbic) Hippocampus Frontal cortex (mPFC) Amygdala (CeA) Amygdala (CeA)

Mouse Mouse Rat Rat Rat

[85] [87] [98] [83] [83]

miR-134 miR-134

Upregulation Downregulation

Hippocampus Hippocampus

Rat Rat

[83] [83]

miR-134 miR-135

Upregulation Upregulation

Frontal cortex (mPFC) Raphe nuclei

Rat Mouse

[98] [74]

miR-135a miR-142

Downregulation Upregulation

Amygdala Serum, Amygdala

Mouse Rat

[82] [103]

miR-144

Downregulation

Hippocampus

Rat

[88]

miR-182 miR-183 miR-183

Downregulation Upregulation Unchanged

Amygdala (LA) Amygdala (CeA) Amygdala (CeA)

Chronic mood stabiliser treatment (4 weeks) Genetic model of anxiety (High responders) Fear-extinction learning Foot shock Maternal separation Acute stress (4-h immobilisation) Chronic stress (repeated immobilisation) Acute stress (4-h immobilisation) Chronic stress (repeated immobilisation) Maternal separation Chronic social defeat followed by antidepressant treatment (imipramine) Acute stress (2 h restraint) Restraint+tail shock (PTSD model) Chronic mood stabiliser treatment (4 weeks) Fear conditioning Acute stress (4 h immobilisation) Chronic stress (repeated immobilisation)

Mouse Mouse Rat Indian field mouse Rat

Rat Rat Rat

[84] [83] [83]

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

8

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

[88]

Thinking small: towards microRNA-based therapeutics for anxiety disorders

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

Effect

Region

Model

Species

Ref.

miR-183 miR-192

Upregulation Downregulation

Acute stress (4 h immobilizaion) Genetic model of anxiety (high responders)

Rat Rat

[83] [99]

miR-221

Downregulation

Hippocampus Frontal cortex (prelimbic), nucleus accumbens (core and shell) Hippocampus

Rat

[88]

miR-322

Upregulation

Serum, amygdala

Rat

[103]

miR-324

Upregulation

Serum, amygdala

Rat

[103]

miR-421

Upregulation

Serum, amygdala

Rat

[103]

miR-429 miR-463

Downregulation Upregulation

Frontal cortex Serum, amygdala

Rat Rat

[113] [103]

miR-484

Downregulation

Rat

[99]

miR-544

Downregulation

Rat

[99]

miR-598

Downregulation

Rat

[99]

miR-674

Upregulation

Frontal cortex (prelimbic), nucleus accumbens (shell) Nucleus accumbens (core and shell) Nucleus accumbens (core and shell) Serum, amygdala

Rat

[103]

miR-1971

Downregulation

Frontal cortex (PFC)

Chronic mood stabiliser treatment (4 weeks) Restraint+tail shock (PTSD model) Restraint+tail shock (PTSD model) Restraint+tail shock (PTSD model) Inescapable shock Restraint + tail shock (PTSD model) Genetic model of anxiety (high responders) Genetic model of anxiety (high responders) Genetic model of anxiety (high responders) Restraint + tail shock (PTSD model) Inescapable shock + fluoxetine treatment

Mouse

[80]

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

chronic stress). This is important to note, as it is known that miRNA can follow circadian patterns of expression [101]. Subtle differences in paradigms may also cause differential findings in miRNA expression. It is also important to note that although many candidate miRNAs have been identified, there is a great variation in miRNAs implicated in preclinical models of anxiety and in clinical studies of humans and even less overlap between preclinical and human postmortem findings. There are many potential reasons for this heterogeneity. First of all, it is clear that there are many different types of anxiety and many different contributing factors. Most notably, the postmortem tissue assessments come from suicide completers with MDD; a clear comorbidty of psychiatric disorders is present in these individuals. Again, these may be related to the timing of miRNA assessment. It is also important to note that miRNA research is still in its relative infancy. New and improved methods of miRNA sequencing are readily becoming available and new miRNAs are being discovered. Newer systems are yielding more accurate and verifiable results, reducing the number of false positives [102]. In order to effectively treat anxiety disorders using miRNA-based techniques, a multifaceted approach is necessary (Figure 2). Improvements in screening technologies, greater clinical focus on alterations in miRNA expression and continued research using preclinical models may indeed lead to the development

of novel and effective miRNA-based therapies for anxiety disorders. 7.

Expert opinion

The potential use of blood and CSF to screen for disease markers would be a great breakthrough. However, it is unlikely that we will identify a single biomarker for disorders related to anxiety, due to the numerous factors that contribute to their development. Some have proposed that a more likely development will be the discovery of miRNA ‘signatures’, clusters of miRNA that act as biomarkers for illness. Indeed, recent studies suggest that such signatures exist for cancers and in some preclinical models of stress-related disorders [103-105]. The potential of miRNA-directed therapeutics is very exciting and encouraging. Current clinical trials utilising miRNA for the treatment of cancers and hepatitis are showing much promise and we remain hopeful that miRNA-mediated therapies will also be capable of treating neuropsychiatric disorders. Different methods that may be used to treat these conditions may involve molecules that replace necessary miRNAs with mimics or viral vectors that lead to an upregulation of the targeted miRNA. Obviously, viral-mediated treatments are not without risk and work must be done to ensure that it does not cause off-target effects as has been observed in prior gene therapy studies. Other therapies may inhibit the effects

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

9

K. A. Scott et al.

Rx

Experimental groups and clinical samples

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miRNA expression assays

Potential therapeutics

qRT-PCR validation

In silico target analysis

In vitro models Preclinical models

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

of miRNAs, or act as miRNA sponges -- designed to bind endogenous miRNAs to reduce their effects in target areas. Antagomirs, locked nucleic acids (LNAs) and antisense oligonucleotides have been used to inhibit miRNAs in preclinical studies. However, there is still much work that must be done before they are able to be used in a clinical setting for the treatment of anxiety. One challenge that miRNA therapies pose is stability. RNAs are typically unstable, but endogenous miRNAs tend to be stable as they are often contained within exosomes or microvesicles or bound to proteins that have protective properties. Some of the current developments in administering exogenous miRNAs and mimics include nanoparticle encapsulation, LNAs and conjugation with cholesterol [28,106,107]. As previously noted, it is unlikely that many psychiatric conditions can be effectively treated by a single miRNA manipulation, as multiple miRNAs are often differentially expressed in pathological conditions. In a preclinical model of cancer, multiple miRNAs have been targeted using an antisense miRNA oligodeoxyribonucleotide [107,108]. Another necessity is to ensure that these methods of delivery do not cause issues as far as toxicity. Recent nonhuman primate studies examining the effects of LNA anti-miR therapies for the treatment of cholesterol and HCV were successful and well-tolerated when delivered intravenously [38,109]. Similarly, trials of LNA anti-miRNA therapies in humans with liver cancer and HCV are also promising. Recent clinical trials of miraversin, an anti- miRNA oligonucleotide targeting 10

miR-122 that is administered subcutaneously, has been used to treat HCV and preliminary results suggest it is both safe and effective [47,110]. An miRNA mimic is currently in clinical trials for the treatment of liver cancer. MRX34 utilises liposomes for delivery and is administered intravenously for treatment of metastatic liver cancer, but results from these trials are yet to be published [45]. An additional challenge as far as miRNA-mediated therapies for psychiatric disorders is the need for minimally invasive methods of delivery. The majority of preclinical studies have explored the effects of miRNA manipulation through nuclei-specific manipulations using microinjections. Ideally, miRNA therapies could be administered peripherally, and could be taken orally or through intravenous injection. Recently, groups have had success in developing methods for crossing the blood--brain barrier (BBB). Yang et al. developed recombinant adeno-associated viruses (rAAVs) that, when administered intravenously, are capable of crossing the BBB in both mouse and nonhuman primate preclinical models [111]. Similarly, Iida et al. developed an rAAV that was able to cross the BBB of mice. They noted that use of a neuronspecific promoter may reduce or eliminate the immune response that is often observed in response to CNS gene therapies that also transduce astrocytes [112]. Because miRNAs have the potential to modulate the expression of so many genes, it is also imperative to develop therapies that are specific and that exert their effects in specific

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Thinking small: towards microRNA-based therapeutics for anxiety disorders

regions related to anxiety disorders. Many of the miRNAs that are altered in neuropsychiatric disorders are also implicated in cancers and are related to tumorigenesis, whereas others are involved in general cell signalling pathways [28]. This highlights the need for specificity, as there are serious implications as far as off-target effects are concerned. Preclinical models have demonstrated that peripherally administered miRNA therapeutics can specifically target neurons, but targeting specific neural populations has yet to be demonstrated [112]. Development of systemically administered compounds that target miRNAs in specific brain regions involved in anxietyrelated pathologies would be ideal but remains a challenge. In conclusion, the field of miRNA research holds much promise and may yield tangible benefits for the clinical management of anxiety disorders, but progress has been modest, with their use as biomarkers providing the most promise at this point. It is clear that a multidisciplinary approach, utilising both clinical and preclinical approaches, is necessary to identify candidate miRNAs with therapeutic potential. Recent advances in miRNA delivery and the continued exponential improvements in miRNA sequencing technology can be harnessed to build on recent advances. Taken together, these Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

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Declaration of interest All of the authors are employed by University College, Cork. JF Cryan and TG Dinan were supported in part by Science Foundation Ireland in the form of a Centre Grant (grant nos. 02/CE/B124, 07/CE/B1368 and SFI/12/RC/2273). The Alimentary Pharmabiotic Centre is a research centre funded by Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan. JF Cryan, TG Dinan and KA Scott are also supported by HRB Grant HRA_POR/2012/32. G Clarke is supported by a NARSAD Young Investigator Grant from the Brain and Behavior Research Foundation (Grant Number 20771). The authors have no other relevant affiliations or financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. J Neural Transm 2014. [Epub ahead of print]

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

Affiliation Karen 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 correspondence 1 Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland; E-mail: [email protected] 2 Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland 3 Department of Psychiatry, University College Cork, Cork, Ireland

Thinking small: towards microRNA-based therapeutics for anxiety disorders.

Anxiety disorders are the most frequently diagnosed psychiatric conditions, negatively affecting quality of life and creating a significant economic b...
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