Targeting microRNAs in neurons: Tools and perspectives

Experimental Neurology 235 (2012) 419–426

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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr

Review

Targeting microRNAs in neurons: Tools and perspectives Francesca Ruberti a,⁎, Christian Barbato a, b, Carlo Cogoni c a b c

IBCN—Institute of Cell Biology and Neurobiology, CNR—National Research Council, Via del Fosso di Fiorano, 64/65, 00143, Roma, Italy EBRI—European Brain Research Institute, Fondazione EBRI, Rita Levi-Montalcini, Via del Fosso di Fiorano, 64/65, 00143 Roma, Italy Dipartimento di Biotecnologie Cellulari ed Ematologia, Sezione di Genetica Molecolare, Università di Roma “La Sapienza” Viale Regina Elena, 324, 00161 Roma, Italy

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Article history: Received 5 August 2011 Revised 25 October 2011 Accepted 30 October 2011 Available online 6 November 2011 Keywords: MicroRNAs Neurons MicroRNA sponges Neurological diseases

a b s t r a c t In the past few years, the understanding of microRNA (miRNA) biogenesis, the molecular mechanisms by which miRNAs regulate gene expression, and the functional roles of miRNAs has been expanded. Interestingly, numerous miRNAs are expressed in a spatially and temporally controlled manner in the nervous system, suggesting that their post-transcriptional regulation may be particularly relevant in neural development and function. miRNA studies in neurobiology have shown their involvement in synaptic plasticity and brain diseases. Approaches for manipulating miRNA levels in neuronal cells in vitro and in vivo are described here. Recent applications of miRNA antisense oligonucleotides, miRNA gene knockout and miRNA sponges in neuronal cells are reviewed. Finally, miRNA-based therapies for neurological pathologies related to alterations in miRNA functions are discussed. © 2011 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . miRNA manipulation in neurons . . . . . . . . . . . . . . Strategies to inhibit miRNA function . . . . . . . . . . . . Gene knockout of miRNAs. . . . . . . . . . . . . . . . . Antisense oligonucleotides and tiny locked nucleic acids . . miRNA sponges and tough decoy RNAs. . . . . . . . . . . miRNA-masking ASOs. . . . . . . . . . . . . . . . . . . Strategies for microRNA-based therapeutics in CNS diseases . MicroRNA and pharmacogenomics . . . . . . . . . . . . . Conclusions and perspectives . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction MicroRNAs (miRNAs) are evolutionarily conserved short regulatory RNAs that are approximately 22 nucleotides long and generally modulate gene expression at the post-transcriptional level by both inducing mRNA degradation and inhibiting the translation of target mRNAs (Bartel, 2009). The biogenesis of canonical animal miRNAs starts with the production of an initial transcript called primary miRNA (pri-miRNA) that varies in length from hundreds to thousands of nucleotides and contains a 60- to 80-nucleotide stem–loop ⁎ Corresponding author at: CNR-Institute of Cell Biology and Neurobiology, Via del Fosso di Fiorano 64, 00143 Rome, Italy. E-mail address: [email protected] (F. Ruberti). 0014-4886/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2011.10.031

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structure (Lee et al., 2003). Pri-miRNAs are then further processed by a complex called Microprocessor, which contains the ribonuclease (RNAse) III-like Drosha enzyme and the RNA binding protein DGCR8/ Pasha; this process results in a hairpin intermediate called pre-miRNA (Gregory et al., 2004). The pre-miRNA is then exported to the cytoplasm by a protein heterodimer consisting of the transport factor Exportin-5 and its cofactor Ran (Bohnsack et al., 2004). In the cytoplasm, the pre-miRNA is processed by another RNase III enzyme called Dicer, which cuts out the loop region of the hairpin, releasing the mature miRNA:miRNA* duplex. After strand separation, the guide strand or mature miRNA is incorporated into an RNA-induced silencing complex (RISC), whereas the passenger strand, miRNA*, is usually degraded (Kim et al., 2009). RISC, which is the effector complex of the miRNA pathway, contains as a key component an

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Argonaute protein that directly binds the miRNA. In addition to the canonical miRNA biogenesis, a number of alternative miRNA biogenesis pathways have been described, including the “mirtron” pathway in which intron-derived miRNAs come out from their host RNAs after splicing directly as pre-miRNAs that do not require the activity of the Drosha–Pasha/DGCR8 complex (Winter et al., 2009). The target mRNAs are recognized depending on the complementarities between positions 2 to 8 from the 5′ of miRNA (the seed sequence), and an miRNA Responsive Element (MRE) usually located within the mRNA 3′ untranslated region (3′ UTR) (Bartel, 2009). In animals the interaction between miRNAs and MREs generally results in either the block of translation or the decay of the target mRNAs, which are deadenylated, decapped and eventually degraded (Fabian et al., 2010). In rare cases, however, it was reported that miRNAs may promote rather than repress translation (Niepmann, 2009; Vasudevan et al., 2007). The number of known miRNAs has sharply increased in recent years; the latest estimate is about 1000 different miRNAs in human cells. Each miRNA has the potential to target a large number of mRNAs (200–500 mRNAs for each miRNA), suggesting that a large fraction of the proteincoding genes may be somehow regulated by miRNAs (Friedman et al., 2009). Frequently, a single mRNA is regulated by different miRNAs that act on its 3′UTR, indicating that miRNAs may act in a cooperative and/ or combinatorial mode in the regulation of a target mRNA (Krek et al., 2005). Altogether, these features show that miRNAs are emerging as a class of master regulatory molecules that may add another level of complexity to the canonical regulatory networks necessary to govern complex cell processes. Given the complex architecture of the brain, it is not surprising that miRNAs are abundantly expressed in the brain, where they have been found to play important roles in the regulation of brain function (Saba and Schratt, 2010). It is noteworthy that neurons compartmentalize specific mRNAs in different subcellular compartments; in this regard, miRNAs may provide a unique system to spatially regulate gene expression, separate from transcriptional regulatory mechanisms (Vo et al., 2010). The importance of miRNA regulation in the central nervous system (CNS) is highlighted by mounting evidence of the role played by miRNAs in neurological disorders (Ceman and Saugstad, 2011). Changes in miRNA expression have been found in the brains of patients affected by various neurological diseases, such as Alzheimer's and Parkinson's, opening up the possibility that some of the misregulated miRNAs may cause or contribute to pathogenesis (Saugstad, 2010). Although there is clear evidence for the involvement of miRNAs in brain function, both in normal and in pathogenic conditions, the specific role of each miRNA in different conditions, as well as the identification of the target mRNAs that are relevant for a particular phenotype, are only beginning to be defined. For this task, two main experimental strategies are currently used: overexpression by adding exogenous miRNAs, and loss-of-function approaches. Overexpression strategies are frequently criticized because artificially increasing the intracellular concentration of miRNAs may result in the repression of mRNAs that are not physiological targets. By contrast, lossof-function approaches, if carefully designed to avoid off-target effects, may reveal miRNA functions that rely on physiological miRNA levels. Here, we discuss the different methods that are currently used for miRNA loss-of-function studies in the CNS. The principal strategies, including miRNA gene knockouts, miRNA antisense oligonucleotide inhibitors, miRNA sponges and miRNA decoys, are illustrated, and the limits and advantages of each approach are highlighted. Special attention is dedicated to miRNA-based therapies for neurological pathologies related to alterations in miRNA functions. miRNA manipulation in neurons The manipulation of miRNAs has elucidated the critical role of these genes in neuronal development and physiology, both in primary neuronal cultures and in the mouse brain in vivo, and may open the way to understanding the action of miRNAs in neurological disorders. Handling of

miRNAs in neurons shows experimental pitfalls similar to those addressed in the study of protein-coding genes. Indeed miRNA action may be specific for a neuronal histotype, and associated to dendritic or axonal domains of neuronal cells. Studies performed in neuronal cultures partially allow to solve the cell complexity present within different tissues of the mammalian brain. During recent years, the expression of exogenous small RNAs or miRNA antagonists in neuronal cells has been generally performed by transfection or viral transduction. The transfection of synthetic miRNA duplex (miRNA mimic) or miRNA antagonists in primary cultured neurons, in comparison with other cells, presents the serious disadvantages of toxicity and low efficiency. The high transfection rate reached with nucleofection and electroporation is limited to cells in suspension at the time of plating (Zeitelhofer et al., 2007). Therefore it is mainly used for developmental studies. On the other hand lipofection and magnetofection have been pursued for example to investigate the role of miRNAs in synaptic trasmission and plasticity. Methods like lipofection may achieve reasonable efficiency when used for very young neurons (4–7 days in culture) from specific neuronal tissues (Cohen et al., 2011; Fiore et al., 2009; Schratt et al., 2006). For example Schratt et al. were the first to identify a dendritically localized microRNA, miR-134, and by lipofection of primary cortical and hippocampal neurons in culture, demostrate that miR-134 regulates the expression of the synaptic localized Limk1 protein thereby modulating dendritic spine size (Schratt et al, 2006). Until now several studies have shown that miRNAs are localized in neuronal dendrites and concomitantly several miRNAs have been demonstrated to regulate the expression of dendritic mRNAs. More recently, magnetofection was succesfully used to transfect cultured hippocampal neurons at 10 days in vitro (Buerli et al., 2007; Muddashetty et al., 2011). Muddashetty et al. (2011), by magnetofection of primary cortical and hippocampal neurons, have shown that PSD-95 mRNA local translation can be dynamically regulated by miR125 through Ago2/RISC and the phosphorylation status of the RNA binding protein FMRP. This molecular mechanism is required for the response to mGluR activation. Note that FMRP protein level are associated to Fragile X syndrome chracterized by intelectual disability and autism like behavior. The delivery of miRNA mimic and miRNA antagonists oligonucleotides in vivo is possible, as discussed in the next sections, but there are limiting factors to overcome, including the low stability of RNA nucleotides in vivo, the lack of regulated expression, and the inefficient uptake of oligonucleotides by neurons. Tools based on recombinant viral vectors derived from lentivirus, adeno-associated virus (rAAV), and retrovirus (Papale et al., 2009) reach a high efficency rate in neuronal cells and have been used to study miRNA function in neurons in vitro and in vivo. For example overexpression or inhibition (see miRNA sponge section) of miR-92 in primary cerebellar granule neurons by lentiviral vectors showed that miR-92 may be involved, by regulating the K + Cl − cotransporter KCC2 abundance, in determining the gradual shift of GABA from depolarization to hyperpolarization (Barbato et al., 2010). Among in vivo studies using lentiviral-mediated miRNA overexpression, miR-134 was recently showed to regulate synaptic plasticity. Gao et al. (2010) demonstrated that lentiviral-mediated overexpression of miR-134, via post-transcriptional regulation of the cAMP-response element binding protein, CREB, abrogated LongTerm Potentiation in CA1 neurons of the hippocampus and also impaired long-term memory formation during contextual fear-conditioning. Interestingly miR-134 was shown to be regulated by NAD-dependent histone deacetylase sirtuin 1 (SIRT1) that is involved in several complex processes relevant to aging, neuronal survival and age-dependent neurodegenerative disorders (Gao et al., 2010). Retrovirus-mediated gene delivery makes it possible to specifically infect dividing cells, and because postnatal neurogenesis persists in the adult hippocampus, retrovirus-mediated gene delivery into newborn cells in the dentate gyrus enables detailed morphological and

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phenotypic analyses of these newly generated neurons (Zhao et al., 2006). For example Smrt et al. (2010) overexpressed miR-137, a neuron-enriched miRNA, in newborn cells of the adult dentate gyrus using retrovirus-mediated gene delivery. The overexpression of miR-137 resulted in reduced dendritic complexity and spine density through the repression of the mouse homolog of Drosophila mindbomb 1 (Mib1), a ubiquitin ligase known to be important for neurogenesis and neurodevelopment (Smrt et al., 2010). Strategies to inhibit miRNA function Loss of function of miRNAs is the best way to reveal the biological function of physiological miRNA levels. Independent approaches, gene knockout, chemically modified antisense oligonucleotides (ASOs), and the expression of exogenous transcripts sequestering miRNAs, are discussed below. Gene knockout of miRNAs miRNA knockout allows for the generation of mouse lines with the genetic deletion or tissue-specific destruction of specific miRNA genes. Gene knockout is achieved through either homologous recombination or insertion of gene trap cassettes. Individual miRNA knockouts, first reported in 2007, revealed the importance of miRNAs in the cardiovascular and immune systems (Park et al., 2010). Conditional gene knockout is a powerful method to study the function of single genes in the CNS (Gavériaux-Ruff and Kieffer, 2007). The Cre–LoxP system enables the spatial and temporal control of gene inactivation. In this approach, mice with LoxP sites flanking the gene of interest (floxed gene) are bred with transgenic mice expressing Cre recombinase under the control of a selected promoter. In selected cell populations in which the promoter actively transcribes Cre recombinase, the enzyme recombines the floxed gene and produces a gene knockout. Difficulties in the application of gene knockout to the study of miRNAs derive from the fact that many miRNAs exist as duplicates or highly similar genes, thus raising the question of functional redundancy and cooperation. In addition, many miRNAs are located in clusters, making it difficult to delete one microRNA without affecting the expression of the others. Finally, the biological function of both strands of the microRNA may complicate the interpretation of the results deriving from gene knockout studies. For example, considering the miRNAs expressed in neurons, miR-124 is transcribed from multiple locations in the genome, miR-134 is located in a large cluster, and both strands of miR-9, miR-9 and miR9* play biological functions in neuronal precursors. The two examples reported here show the effect of specific miRNA knockouts in neuronal cells, and underline the limits and advantages of miRNA knockout studies. miR-182 is robustly espressed in the mammalian retina, suggesting that it may play an important role in retinal development and maintenance. The generation of a miR-182 knockout mouse line showed that miR-182 deficiency alone did not affect retinal development and maintenance (Jin et al., 2009). miR182 deficiency may be ineffective because miR-182, miR-183 and miR-96 have similar seed sequences and target genes. Therefore, these miRNAs may act in a coordinated manner and compensate each other in vivo. However, the absence of effects following miR182 knockout raises the question of whether specific miRNAs are necessary only under stressful and pathological states, as discussed below. miR-132 is transcribed at a single locus and is part of the miR-212/ 132 cluster. Four potential microRNAs are generated from this locus: miR-212, miR-212*, miR-132 and miR-132*. miR-212/132 floxed mice have been produced (Magill et al., 2010). The deletion of the miR-132/212 locus in newborn neurons in the adult hippocampus was achieved through the injection of a retrovirus expressing Cre recombinase (Magill et al., 2010). The knockout of miR-212/132 in

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newborn hippocampal neurons caused a dramatic decrease in dendrite length and spine density (Magill et al., 2010). To evaluate which of the four potential miRNAs of the miR-212/132 locus are functionally important for newborn hippocampal neurons, microRNA sensor transcripts encoding fluorescent proteins with a 3′UTR incorporating MRE for the miRNA of interest were espressed in immature granule neurons of the hippocampus in vivo. Quantitative analysis of miRNA sensors for miR-132, miR-132*, miR-212 and miR-212* suggested that only miR-132 is a functional miRNA in immature granule neurons (Magill et al., 2010). Alternative methods to gene knockout may overcome miRNA family complexity, and the redundancy and cooperation of microRNAs. Antisense oligonucleotides and tiny locked nucleic acids Antisense oligonucletides (ASOs) have been widely used to target specific mRNAs to study gene function. ASOs complementary to mature miRNA sequences are the backbone of miRNA silencing. Several independent chemical modifications of ASOs that improve affinity and stability have been used to inhibit miRNA function both in vitro and in vivo. In some cases, miRNA ASO inhibition leads to target miRNA degradation. Indeed, recent studies have shown that the stability of miRNAs is defined by the Argonaute protein with which it binds, and the degree of complementarity between the miRNA and its target (Ameres et al., 2010). “AntagomiRs” were the first miRNA inhibitors demonstrated to work in mammals (Krutzfeldt et al., 2005). These ASOs harbor various modifications for RNAse protection and pharmacological properties, such as enhanced tissue and cellular uptake. They contain 2′-O-Methyl-modified ribose sugars (2′-OMe), a terminal phosphorothioate linkage instead of a natural phosphate linkage (Fig. 1), and a cholesterol group at the 3′ end. ASOs likely target mature miRNAs without affecting pre-miRNA levels. Krützfeldt et al. (2007) have shown that AntagomiRs efficiently target miRNAs when injected locally into the mouse cortex, while they are ineffective in the brain when they are systemically delivered. The intracerebroventricular (ICV) infusion of miR-219 or miR-132 AntagomiR into the cerebrospinal fluid in mice strongly suppressed the expression of endogenous miR-219 and miR-132 in the suprachiasmatic nucleus, a periventricular brain region, for up to 2 weeks (Cheng et al., 2007). These studies showed that miR-219 inhibition extends the circadian period, while miR132 inactivation potentiates light-induced clock resetting (Cheng et al., 2007). In addition, ICV injection of miR-155 or miR-802 antagomir in Ts65Dn mice, a model of Down syndrome (DS), led to the attenuation (30–40%) of the endogenous expression of mature miR-155 or miR-802, respectively, in the hippocampus (Kuhn et al., 2010). Methyl-CPG-binding protein (MECP2), a miR-155 and miR-802 target, is underexpressed in DS brain specimens from either humans or mice (Kuhn et al., 2010). The in vivo silencing of miR-155 or miR802 resulted in the normalization of MECP2 levels, which in turn normalized the expression of MECP2-activated and -silenced target genes (Kuhn et al., 2010). Although AntagomiRs have been found to inhibit a specific miRNA in several tissues, they require higher doses to achieve the same efficacy as other ASO strategies. Other chemical modifications of ASOs have been found to be more effective at inhibiting microRNAs in vivo than 2′-OMe (Fig. 1). 2′Omethoxyethyl anti-microRNA oligonucleotides contain a methoxyethyl group bound to the 2′ oxygen of the ribose and bind with more affinity to the miRNA (Davis et al., 2006; Esau et al., 2006). Locked Nucleic Acid (LNA) antisense nucleotides introduce a 2′, 4′ methylene bridge in the ribose to form a bicyclic nucleotide (Fig. 1). LNA modification increases the RNA:RNA melting temperature by 2–4 °C per modification and confers resistance to many endonucleases. These features make LNA-antimiR more specific and permit the use lower levels of antisense nucleotides. LNA ASOs have been used

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Fig. 1. Chemical modifications of antisense oligonucleotide anti-miRNA. 2′-O-methyl RNA contains a methyl group bound to the 2′ oxygen of the ribose; 2′-O-methoxyethyl RNA contains a methoxy group bound to the 2′ oxygen of the ribose; locked nucleic acid introduces a 2′,4′ methylene bridge in the ribose to form a bicyclic nucleotide. S indicates sulfur substitution of a non-bridging oxygen to make a phosphorothioate linkage between nucleotides.

succesfully in several in vitro studies to inhibit specific miRNAs. Studies in mice have demonstrated that the delivery of LNA-antimiR with phosphorothioate modifications may inhibit miR-122, a miRNA that binds the hepatitis C virus (HCV) and stimulates its replication. Studies in non-human primates have shown that the delivery of LNAantimiR122 effectively depletes miR-122 in the liver (Elmén et al., 2008) and improves HCV-induced liver pathology (Lanford et al., 2010). LNAantimiR has also been employed in loss-of-function studies of the mouse brain. For example, to study the role of miR-219 in NMDA receptor signaling in the mouse forebrain, LNA-antimiR, synthesized as unconjugated oligonucleotides with a phosphodiester backbone, complementary to nucleotides 2–16 in the mature miR-219 sequence, was used. In this study, LNA-antimiR-219 was delivered continuously for 7 days in the third ventricle of the forebrain (Kocerha et al., 2009). The silencing of miR-219 caused a significant increase in the levels of a protein target of miR-219 in the prefrontal cortex and modulated NMDA-R-mediated neurobehavioral dysfunction (Kocerha et al., 2009). Beyond silencing individual miRNAs with modified ASOs, tools to inhibit miRNA families are necessary to understand the biological role of animal miRNAs. Recently, a new method to silence an entire family of miRNAs was developed (Obad et al., 2011). This approach uses fully LNA-modified phosphorothioate oligonucleotides, termed tiny LNAs (Fig. 2), complementary to the miRNA seed region (Obad et al., 2011). Tiny LNAs may inhibit individual miRNAs and miRNA families in cultured cells and in several tissues in adult mice (Obad et al., 2011). However, tiny LNAs do not reach the brain through systemic delivery (Obad et al., 2011). An advantage of using ASOs as well as seed-targeting tiny LNAs, in contrast to miRNA knockout, is the possibility of studying the effects of both acute and partial inibition of microRNAs. miRNA sponges and tough decoy RNAs The expression of miRNA-binding transcripts designed to sequester and inhibit miRNA function is another approach used in miRNA loss-offunction studies. This method offers several advantages, such as the neutralization of a family of miRNAs and the delivery of transgenes by

viral vectors for cells that are difficult to transfect both in vitro and in vivo. Ebert et al. introduced microRNA sponges in 2007. These molecules are RNAs containing complementary multiple binding sites for an miRNA of interest and produced from a transgene (Fig. 2). Sponge constructs with miRNA-binding sites that perfectly complement the miRNA may inhibit miRNA function (Ebert et al., 2007; Gentner et al., 2009), but sponge mRNAs containing bulged sites that are mispaired opposite miRNA positions 9–12 seem to be more effective (Ebert et al., 2007; Gentner et al., 2009). Strong promoters that increase the concentration of sponge RNAs with respect to the miRNA concentration are essential to obtain effective miRNA sponges. Four to 10 tandem binding sites separated by a few nucleotides each have been used to inhibit miRNAs (Ebert and Sharp, 2010). Pol II-generated sponge RNAs may consist of a fluorescent reporter placed upstream of the miRNA-binding sites and enable the quantitative analysis and sorting of individual cells (Ebert et al., 2007). Pol III-generated sponges may include terminal stem loops as stabilizing elements (Ebert et al., 2007). More recently, tough decoy RNAs (TUDs) were designed to efficiently expose indigestible complementary RNAs to specific miRNA molecules (Haraguchi et al., 2009). TUDs are transcribed by RNA polymerase III U6 promoter and form a stem–loop structure (Fig. 2), which contains multiple miRNA-binding sites (MBS) for mature miRNAs, in addition to three linker bases at both ends (Haraguchi et al., 2009). A stem length of 18 base pairs was shown to be sufficent for binding to Exportin-5 and for the transfer of TUD RNA from the nucleus to the cytoplasm (Haraguchi et al., 2009). Two stemflanking MBS provide some resistance to cellular Rnase, as well as to degradation, through the RISC complex containing the corresponding miRNA. In addition, four nucleotides are inserted between positions 10 and 11 at the end of the perfectly complementary MBS, where the RISC cleaves the target RNAs, which improves the decoy activity (Haraguchi et al., 2009). Although the design and use of miRNA sponges are fast and not very laborious, the evaluation of miRNA sponge efficacy in inhibiting

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Fig. 2. Strategies to inhibit miRNA function. AntagomiR LNA-oligos, tiny LNAs, miRNA sponges and TUD RNAs (decoy) contain sequences that bind miRNAs and act as competitive inhibitors. Binding of these inhibitors prevents the interaction of miRNAs with their primary targets and leads to derepression of target mRNAs.

the miRNA or miRNAs of interest may be more challenging. The validation of sponge constructs requires the use of a reporter assay or assays for the expression of known target genes. It is also difficult to prove whether the degree of inhibition of several miRNAs belonging to the same family is different. miRNA sponge technology has been applied in studies in primary neuronal cultures and specific neuronal populations in vivo. In most studies described below, the miRNA sponges contained multiple concatenated miRNA-binding sites with central mismatches. Edbauer et al. have shown the effect of different miRNAs on the morphogenesis of spines and dendrites in dissociated hippocampal neurons by transfecting individual miRNA sponges expressed in the 3′ UTR of mCherry (Edbauer et al., 2010). Barbato et al. used a lentiviral sponge in postmitotic neurons to evaluate the effect of miR-92, which is developmentally downregulated during the maturation of cerebellar granule neurons in vitro (Barbato et al., 2010). At 6 days in vitro, miR-92-sponge-expressing neurons showed derepression of the potassium chloride cotransporter KCC2 and a negative shift in the GABA reversal potential (Barbato et al., 2010). Krol et al. used an adeno-associated vector (AAV) to deliver a triple sponge containing four sites specific for each of three lightregulated microRNAs (miR-182, -96 and -183) into photoreceptors in the mouse retina in vivo (Krol et al., 2010). The eGFP sponge was driven by the rhodopsin promoter to allow for specific expression in photoreceptor cells. At 3 weeks postinjection, eGFP-expressing photoreceptors were dissected using Laser Capture Microscopy. Strong derepression for the target glutamate transporter SLC1A1 was demonstrated by western blotting (Krol et al., 2010). Remarkably, the efficacy of the triple sponge was not associated with changes in the expression levels of target miRNAs (Krol et al., 2010). Recently, a transgenic mouse model expressing a miR-182/96/ 183 cluster sponge was generated (Zhu et al., 2011). The sponge was composed of 10 copies of sites specific for each of the three miRNAs of the miR-183/96/183 cluster and was inserted into the 3′UTR of GFP and regulated by the opsin promoter. In this case, the expression of the miRNA sponge produced only a marginal decrease in miR-182, miR-96 and miR-183 in three sponge transgenic lines

(Zhu et al., 2011). This study demonstrated an important role for the miR182/96/183 cluster in preventing bright light-induced retinal degeneration in vivo and identified Casp2 as a target of the miRNA cluster and mediator of retinal degeneration. However, under normal conditions, the impairment of the miR-182/96/183 cluster showed no differences between wild-type and transgenic mice (Zhu et al., 2011). Also, miR-182 knockout mice did not reveal any apparent structural abnormalities in the retinas (Jin et al., 2009). Finally, Luikart et al. used a retrovirus to express a “U6 sponge” containing four perfect miR-132 target sites downstream of the U6 promoter in newborn neurons in the adult dentate gyrus (Luikart et al., 2011). Several assays were used to demonstrate the efficacy and specificity of the miR-132 sponge. The coexpression of a fluorescent reporter carrying specific miR-132 target sequences with the miR132 sponge indicated the inhibition of endogenous miR-132 (Luikart et al., 2011). Studies in PC12 cells infected with the miR132 sponge showed a 32% reduction in miR-132 expression, but unaltered expression of other microRNAs, including miR-212, which has a similar sequence to miR-132. Retroviral-mediated miR-132 sponge expression decreased dendritic spine density and the functional synaptic activity of newborn neurons in the adult dentate gyrus (Luikart et al., 2011). Of note in this study, the dendritic arborization of newborn neurons was not affected by the miR-132 sponge, while it was significantly impaired in knockout miR-132 newborn neurons (see above), suggesting the partial inhibition of endogenous miR-132 by the U6 sponge. It should be mentioned that natural miRNA decoys (i.e., endogenous miRNA-binding transcripts) that reduce the activity of specific miRNAs exist in plants (Franco-Zorrilla et al., 2007) and also in human tumor cells (Poliseno et al., 2010). miRNA-masking ASOs The miRNA-masking method allows for the inhibiton of the specific interaction between an miRNA and one of its mRNA targets. This strategy, developed by Xiao et al., is based on miRNA-masking ASOs that base pair with a miRNAbinding sequence in the 3′UTR of a

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specific mRNA (Xiao et al., 2007). In this case, single-stranded 2′-Omethyl-modified ASOs that are fully complementary to miR-1 and miR-133 target sites in the 3′UTR of pacemaker channel mRNAs HCN2 and HCN4 blocked the access of the miRNA to its binding sites and enhanced HCN2/HCN4 expression and function. This method has also been used in zebrafish to prevent the repressive action of miR-430 in transforming the growth factor-β signaling pathway (Choi et al., 2007).

Strategies for microRNA-based therapeutics in CNS diseases The altered expression of some miRNAs has been described in several disorders of the CNS. To date, there have been no examples of a direct link between the altered expression or function of miRNAs and human neurological disorders, such as traumatic CNS injuries and neurodegenerative diseases. The collection of data on the association between human brain diseases and miRNAs has focused on expression profiles of miRNAs and their quantitative modulation (i.e., upregulation versus downregulation), according to age, gender, phase of the disease and specific brain area. For example, in the Alzheimer's disease (AD) brain, expression profiles have revealed alterations in many individual miRNAs, as reported for over 300 miRNAs, which were isolated from the hippocampus, medial frontal gyrus and cerebellum of patients with earlyand late-stage AD, compared to normal age-matched controls (Cogswell et al., 2008). Analyses of the expression of different miRNAs in the cerebral cortical white matter and grey matter in human patients with normal and early AD using different experimental techniques have been performed (Wang et al., 2011). Several in vitro and in vivo neurobiological studies aiming to explore the functional role of miRNAs in neurological disease pathogenesis should address open questions about neuronal gene expression regulation and depict a novel class of therapeutic targets for nervous system diseases. Depending on the role of the miRNA, the goal of the treatment will be to either increase or reduce miRNA function. Given the importance that miRNAs might play in neuropathology, several strategies to manipulate miRNA activity and expression are being pursued. Two main strategies may be applied to target miRNA expression in the brain: directly, by using oligonucleotides or virusbased constructs, and indirectly, by using drugs to modulate miRNA expression at the transcriptional and/or processing level. To date, all delivery strategies have been important for identifying a suitable way to generate microRNA-based therapies for neurological diseases related to the perturbation of miRNAs. The main challenge for miRNA therapeutics in neurology, beyond stability and safety, is the delivery to the appropriate tissue and neurons. AntagomiRs, tough decoy RNAs and miRNA sponges must reach the cells and must function at the site of the disease (Ebert et al., 2007; Haraguchi et al., 2009; Kim et al., 2009). Mainly, there are three paths for how the ribonucleic acid can be delivered: systemic, local, and targeted; through systemic delivery, the therapeutic molecules are designed to reach the intended neuronal tissue. The main obstacle related to brain anatomy is the blood–brain barrier (BBB). The BBB represents a unique problem for any therapy involving the systemic delivery of oligonucleotides or other molecules. For example, it was recently reported that a new strategy could bypass the BBB using low-molecular-weight molecules up to 1 kDa; this strategy focuses on the tight junction protein claudin5, a structural element of the micro-neurovasculature that regulates the entry of small molecules through the BBB (Campbell et al., 2011). Briefly, transient silencing of claudin-5 caused the tight junctions between vascular endothelial cells to become slack and permit small molecules to pass through the BBB. Since the size of

miRNA mimics is 15 kDa and miRNA antagonists is approximately 7 kDa, other strategies to bypass the BBB are required. Nanoparticles , such as cationic lipids, polyethyleneimine (PEI), and dendrite and carbon nanotubes (Pérez-Martínez et al., 2011), are a powerful tool to efficiently deliver their cargo to neuronal cells in vitro, and when used in vivo, they should efficiently cross the BBB to reach neurons. For example, carbon nanotubemediated siRNA silencing of caspase-3 after focal ischemic damage of motor cortex was recently used (Al-Jamal et al., 2011). The perilesional stereotactic administration of caspase-3 siRNAs using carbon nanotubes in an endothelin-1-induced stroke rat model reduced neurodegeneration (Al-Jamal et al., 2011). Again, the most relevant unsolved problem remains designing nanoparticles to efficiently cross the BBB. Recently, a rabies virus glycoprotein (RVG)-mediated SSPEI (disulfide/polyethyleneimine) nanomaterial was used for the delivery of miRNAs in the brain in vivo (Hwang do et al., 2011). The RVG peptide is a neurotropic virus-originated 29-amino acid residue fragment that can cross the BBB, and it specifically binds to nicotinic acetylcholine receptors (AchRs) (Kumar et al., 2007; Lafon, 2005). After interactions between the RVG peptide and AchRs, the RVG-labeled specific molecules are internalized by endocytosis, making the RVG-mediated SSPEI polymeric particle an ideal tool for the intracellular delivery of miRNAs (Cantin and Rossi, 2007). The nanosized spherical RVG–SSPEI–miRNA124 complex system accumulated in the brain when injected into the blood. Higher amounts of miRNA were found in neuronal cells of the brain when the BBB was disrupted with mannitol infusion (Hwang do et al., 2011). Despite the tremendous potential for gene therapy, several brain cells may be a target of the RVG peptide, because microglia, astrocytes and endothelial cells all express AchRs. A promising tool to transfer small RNAs to the neurons consists in exosome nanoparticles. Recent studies evidenced the utilization of exosome vescicles to transfer mRNAs and microRNAs to the cells (Valadi et al., 2007). These authors previously demonstrated that the exosomes contain mRNAs from about a thousand genes and several small non-coding RNAs including specific microRNAs. All these RNA molecules were vehicled to target cells and were functionally active (Valadi et al., 2007). More recently, exosome nanoparticles loaded with siRNA were used to target mouse brain (Alvarez-Erviti et al., 2011). In particular, purified exosomes expressing Lamp2b, an exosomal membrane protein, fused to the neuron-specific RVG peptide, were electroporated with a siRNA against beta site APP-cleaving enzyme (BACE1), an enzyme associated with the formation of peptide that forms beta-amyloid plaques in AD. Intravenously injected RVG-targeted exosomes delivered siRNAs specifically to the brain, obtaining a 60% and 62% reduction of BACE1 mRNA and protein respectively. To date, the siRNA delivery with exosome nanoparticles, represent an efficient, tissuespecific and non-immunogenic tool aimed to develop small RNA molecules as therapeutic drugs to neurological diseases. However, even if we bypass the problem of overcoming the BBB, the toxicity of therapeutic small oligonucleotides would remain a problem. Viral delivery of miRNA-based molecules, as reported in viral-shRNA studies, might lead to toxicity and tissue damage (Boudreau et al., 2009; McBride et al., 2008). Moreover, it could overwhelm the components of the RISC complex and generate an impairment in RNA-mediated gene silencing. Studying shRNAmediated silencing of the disease protein torsinA as a therapeutic treatment for the dominantly inherited neurological disease DYT1 dystonia, Gonzalez-Alegre et al. observed a lethal toxicity caused by the intrastriatal injection of AAV2/1 expressing different shRNAs (Martin et al., 2011). Neurodegeneration observed in treated animals was not observed in mice injected with AAV2/1 encoding no shRNAs. Moreover, a different response to neurotoxicity was observed depending on the genetic background of the rodents (Martin et al., 2011).

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MicroRNA and pharmacogenomics An important player in the molecular medicine landscape is pharmacogenomics, which includes investigations of the associations between individual patient genomes and transcriptomes and the efficacy and toxicity of a drug. Drug efficacy could be modified by altering miRNA function through several approaches, as described above. From the literature, it is evident that pharmacogenomically relevant genes are also regulated by miRNAs (Rukov and Shomron, 2011). For instance, chronic treatment with the selective serotonin reuptake inhibitor fluoxetine (Prozac) increases the expression of miR-16 in serotonergic neurons of the raphe nuclei, which in turn decreases the density of serotonin transporters (Baudry et al., 2010). Moreover, the efficacy of Prozac regulation of miR-16 in two mouse models of depression was demonstrated (Baudry et al., 2010). Another example of the relationship between pharmacogenomically relevant genes and miRNA regulation was demonstrated by treating the rat hippocampus with both lithium and valproate (VPA) (Zhou et al., 2009). This work showed for the first time that selected miRNAs can contribute to the effects of the mood stabilizers lithium and VPA by targeting specific proteins, as demonstrated by miR-34's ability to regulate the metabotropic glutamate receptor 7 (GRM7), which reinforces the effects of lithium and VPA on GRM7 (Zhou et al., 2009). Conclusions and perspectives The dissection of miRNA pathways is only beginning, and a detailed brain atlas of microRNAs has not been completed. A novel sequencing era is going to dramatically change our view of studying gene expression, post-transcriptional modifications, DNA copy number variations, and single nucleotide polymorphisms. Novel highthroughput sequencing techniques are emerging at an impressive rate on the market and in the scientific community. In the near future, these novel approaches will surely help to elucidate the function of miRNAs and their role as fine regulators of neuronal physiology. Here, we highlighted several methodologies applied to the study of the role of miRNAs in neuroscience research, showing the strengths and weaknesses of these approaches. miRNA overexpression and inhibition studies will demand the development of viral vectors as well as transgenic and knockout mice, which should primarily consider the complexity of the brain and take advantage of selective promoters to dissect miRNA function in specific neuronal populations. Furthermore, inducibile promoters may elucidate the function of miRNAs in synaptic plasticity processes. Recent studies have shown that microRNAs may also be involved in local protein synthesis at synapses (Banerjee et al., 2009; Muddashetty et al., 2011; Schratt et al., 2006; Siegel et al., 2009). Novel tools to manipulate miRNAs in neuronal subcellular compartments are needed to study the role of miRNAs in local protein synthesis. The discovery of endogenous transcripts acting as miRNA sponges could be the basis for expanding ways to inhibit miRNA function. Regarding the strategies important for developing microRNAbased therapies for neurological diseases related to the perturbation of miRNAs, the finding that tiny LNAs may inhibit whole families of miRNAs opens new avenues for drug development. Effort to achieve precise spatial and temporal control of LNA activity will be required. However, we are faced with the problem of crossing the BBB. Paradoxically, the limiting factor for treating neuronal tissues is not the tools for modulating miRNA expression, but the endogenous filter of the BBB. Acknowledgments This work was supported by Compagnia di Sanpaolo, Programma Neuroscienze Grant ‘MicroRNAs in Neurodegenerative Diseases’ (to C.B.), and by CNR grant DG.RSTL.059.012 (to F.R).

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We thank Studio 2CV idee e paesaggi (www.studio2cv.it) for graphical assistance.

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