Dysregulated microRNAs in neurodegenerative disorders

Seminars in Cell & Developmental Biology 21 (2010) 768–773

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Seminars in Cell & Developmental Biology journal homepage: www.elsevier.com/locate/semcdb

Review

Dysregulated microRNAs in neurodegenerative disorders Pierre Lau a,b , Bart de Strooper a,b,∗ a b

Center for Human Genetics. K.U. Leuven. Herestraat 49 Bus 602 B-3000 Leuven, Belgium Department of Molecular and Developmental Genetics, Flanders Interuniversity Institute for Biotechnology, Herestraat 49 Bus 602 B-3000 Leuven, Belgium

a r t i c l e

i n f o

Article history: Available online 18 January 2010 Keywords: microRNA Non-coding RNA Neuron Nervous system Homeostasis Neurodegeneration

a b s t r a c t The complexity of the nervous system arises in part, from the large diversity of neural cell types that support the architecture of neuronal circuits. Recent studies have highlighted microRNAs as important players in regulating gene expression at the post-transcriptional level and therefore the phenotype of neural cells. A link between microRNAs and neurodegenerative diseases such as Alzheimer’s disease, Huntington’s disease and Parkinson’s disease is becoming increasingly evident. Here, we discuss microRNAs in neurodegeneration, from the fruit fly and mouse utilized as experimental models to dysregulated microRNAs in human neurodegenerative disorders. We propose that studying microRNAs and their mRNA targets in the context of neurodegeneration will significantly contribute to the identification of proteins important for neuronal function and might reveal underlying molecular networks that drive these diseases. © 2010 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4.

5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . miRNAs in neurodegeneration: three examples of studies using the fruit fly as a model of neurodegeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . miRNAs in neurodegeneration: strong evidence after inactivation of Dicer in mouse neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence for miRNAs going astray in human neurodegenerative diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. miRNAs in Alzheimer’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. miRNAs in Huntington’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. miRNAs in Parkinson’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. miRNAs in other neurodegenerative diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction microRNAs (miRNAs) are ∼21 nucleotide-long non-coding RNAs that act as post-transcriptional regulators of gene expression in plants and animals. miRNAs bind to the 3 untranslated region (3 UTR) of mRNA targets and recruit the RNA induced silencing complex (RISC) to inhibit the expression of these targets. The base pairing between miRNAs and their mRNA targets differs between plants and animals. There is perfect (or near perfect) complementarity base pairing in plants whereas in mammalian cells, only a

∗ Corresponding author at: Department of Molecular and Developmental Genetics, Flanders Interuniversity Institute for Biotechnology, Herestraat 49 Bus 602 B-3000 Leuven, Belgium. Tel.: +3216346227. E-mail address: [email protected] (B. de Strooper). 1084-9521/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2010.01.009

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pairing between the seed sequence of the miRNA and the 3 UTR of the mRNA target is thought to be important. Nucleotides between positions 2 and 8 of the miRNA are referred to as the seed sequence and current target prediction software rely on the seed sequence to predict up to a thousand of targets for each miRNA. It is yet unclear how RISC inhibits the expression of bound mRNA targets (reviewed in Ref. [1]). Several mechanisms are proposed and involve inhibition of translation at the initiation step [2] and/or deadenylation of targets followed by mRNA decay [3]. There are currently 721 human miRNAs and their sequences can be retrieved from the miRBase (http://microrna.sanger.ac.uk/cgi-bin/sequences/browse.pl). Primary miRNA transcripts (pri-miRNAs) are transcribed by RNA polymerase II and can be several thousand bases in length [4]. In the nucleus, pri-miRNAs are processed by the Drosha/DGCR8 complex to produce precursor miRNAs (pre-miRNAs) which are ∼70 nucleotides in length and characterized by a stem-loop structure.

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After nuclear export by Exportin 5/Ran, pre-miRNAs are cleaved in the cytoplasm by Dicer to generate mature miRNAs (reviewed in Ref. [5]). Two broad roles have been attributed to miRNAs to date. In the first model, miRNAs serve to limit the expression of mRNA targets in tissues where expression of such targets is normally low or absent and where leaky expression would be detrimental. In contrast, the second model involves fine-tuning of target mRNAs within cells where miRNAs and targets are normally co-expressed (reviewed in Ref. [6]). Based on the large number of predicted mRNA targets, miRNAs are anticipated to be implicated in a wide range of cellular processes such as proliferation, differentiation and apoptosis. Taking into account the high complexity of the mammalian brain and the large diversity of neural cell types, miRNAs are logically emerging as strong candidates for regulating neural proliferation, differentiation and function. Importantly, dysregulation of miRNAs has been reported in neurological disorders. This review summarizes recent findings showing aberrant expression of miRNAs in neurodegenerative disorders.

2. miRNAs in neurodegeneration: three examples of studies using the fruit fly as a model of neurodegeneration To understand how aging affects human neurons, it would be highly desirable to conduct studies using animal models such as non-human primates that are physiologically similar to humans. However, this strategy is expensive, time-consuming and consequently most of the current knowledge about miRNAs in neurodegeneration has come from studies of more accessible model organisms with shorter lifetime such as the mouse (∼3 years lifespan) and Drosophila Melanogaster (∼3 months lifespan). The fruit fly has been notably used as a successful model to study miRNAs in neurodegeneration associated with trinucleotide repeat expansion. The polyglutamine (PolyQ) diseases are caused by expansion of a CAG trinucleotide repeat within protein coding regions such that this expanded repeat is translated into a stretch of unstable polyglutamine. Among PolyQ diseases, Huntington’s disease and several types of spinocerebellar ataxia (SCA) such as SCA1, -2, -3, -7, -6 and -17 are characterized by repeat expansion in Huntingtin, Ataxin-1, -2, -3, -7, Brain calcium channel 1 (CACNA1A) and the TATA-binding protein (TBP) respectively. The spinal bulbar muscular atrophy (SBMA) and the dentatorubral-pallidoluysian atrophy (DRPLA) are also characterized by PolyQ expansion within the Androgen receptor (AR) and Atrophin-1 (ATN1) proteins. Recently, it has been shown that deletion of Drosophila miR-8 causes elevated levels of Atrophin, increased apoptosis in the central nervous system (CNS) and behavioral defects in a climbing test [7]. The mammalian miR200 family contains miR-8 related miRNAs and whether members of this family are dysregulated in DRPLA would be interested to address. In Drosophila, inactivation of Dicer1 in the eye dramatically enhances neurodegeneration caused by overexpression of the pathogenic Ataxin-3 protein, such that the eye is severely compromised with complete loss of pigmentation [8]. Genetic screens to identify modifiers of this SCA3-associated neurotoxicity model reveal Bantam (Ban) which is a Drosophila specific miRNA that simultaneously stimulates cell proliferation and prevents apoptosis [9]. The up-regulation of Ban suppresses neurotoxicity while loss of Ban enhances SCA3-associated neurodegeneration. This model may be relevant to SCA3 however none of the human or mouse miRNA sequences reported to date is identical to Ban. Nevertheless, the possibility that homologous human miRNAs differing slightly in their nucleotide sequence may be functionally equivalent to ban exists. More recently, it has been shown that miR-19, -101 and -130 cooperatively regulate Ataxin-1 [10], which may be relevant for SCA1 and consistent with a putative role of miRNAs in SCA diseases. Apart from these PolyQ diseases, miRNAs may

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also be implicated in other kinds of trinucleotide repeat expansion diseases. For example, the fragile X mental retardation (FraX) syndrome is characterized by an expansion of a CGG trinucleotide repeat in the 5 untranslated region (5 UTR) of the fragile X mental retardation 1 (FMR1) gene, which results in a failure to express the fragile X mental retardation protein (FMRP). Interestingly, FMRP controls neuronal translation and physically associates with specific miRNAs such as Ban [11], thus reinforcing a connection between miRNAs and translational control pathway in neurons. 3. miRNAs in neurodegeneration: strong evidence after inactivation of Dicer in mouse neurons A deep sequencing experiment found a total of ∼300 miRNAs expressed in different mouse brain regions [12], which corresponds to around half of all 579 known mouse miRNAs. The important role of miRNAs in the mammalian CNS was initially demonstrated by conditional inactivation of Dicer in neuronal cells. For instance, loss of Dicer in dopaminergic neurons leads to cell death in the substantia nigra and behavioral studies reveal reduced locomotion in an open-field assay, reminiscent of the phenotype observed in Parkinson’s disease [13]. Specific deletion of Dicer in the hippocampus results in pleiotropic phenotypes including microcephaly, reduced dendritic branches and increased dendritic spine length [14] while Purkinje cell-specific deletion of Dicer causes cell death and cerebellar degeneration [15]. Collectively, these three studies provide strong indication that global dysregulation of miRNAs may result or contribute to neurological disorders. Of note, deletion of Dicer in dopaminoceptive neurons within the striatum produces behavioral abnormalities such as ataxia, front and hind limb clasping [16]. In this mouse model, striatal neurons are smaller in size and increased astrogliosis is observed without any apparent signs of neurodegeneration, showing that absence of Dicer does not always cause neuronal cell death. Although targeting Dicer represents a first experimental step to assess the importance of miRNAs in nervous system function and homeostasis, it does not address the role of individual miRNA. A more systematic knock-out of individual miRNA genes is now needed. However, the existence of miRNAs that derive from several loci and the presence of miRNA families [17] presently challenge this kind of approach as miRNAs sharing highly similar nucleotide sequence can theoretically compensate each other. Alternative gene transfer approaches using viral vectors such as Adeno-associated virus (AAV) or lentiviruses (LV) encoding either miRNAs or miRNA inhibitors can also be considered when demonstrating the function of individual miRNA in neuronal cells. 4. Evidence for miRNAs going astray in human neurodegenerative diseases 4.1. miRNAs in Alzheimer’s disease Alzheimer’s disease (AD) is the most common form of neurodegenerative disease and accounts for 50–70% of dementia cases. AD is characterized by a loss of memory, lack of thinking and concentration, and the presence of other intellectual inabilities such as language disturbance and failure to recognize some objects or people. At the cellular level, there is formation of abnormal structures between brain nerve cells called ␤-amyloid plaques. The major component of these senile plaques is the amyloid ␤ (A␤) peptide which is derived from the proteolytic processing of the amyloid precursor protein (APP). The presence of intraneuronal neurofibrillary lesions is another major abnormality in the brain of AD patients. These lesions contain paired helical filaments (PHFs) that accumulate in the neuronal cell body (neurofibrillary tangles) and distal processes (dystrophic neuritis and neuropil threads). The Braak’s

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classification of AD consisting of six distinct anatomically defined pathological stages is usually utilized to analyze post-mortem the brains of AD patients [18]. At stage 1, patients are never demented whereas at stage 6, they are always demented with a widespread distribution of neurons containing neurofibrillary tangles (NFT) in the higher neocortex and occipital cortex regions. Less than 1% of AD results from genetic mutations in three genes: APP, presenilin 1 (PSEN1) and presenilin 2 (PSEN2) (reviewed in Ref. [19]), which cause accumulation of A␤ peptide. The production of A␤ is the result of two proteolytic cleavages: a ␤-secretase called ␤-amyloid cleavage enzyme 1 (BACE1) first cleaves APP to generate the C99 terminal fragment [20] and in a second step, C99 further serves as substrate for a ␥-secretase cleavage mediated by presenilins, generating the A␤ peptide [21]. There are several isoforms of A␤ peptide ranging from 37 to 43 amino acid residues. The A␤40 peptide is the most common form but the A␤42 peptide is the more fibrillogenic and likely neurotoxic form (reviewed in Ref. [22]). Most cases of AD are of the late onset form, developing after age 65 with several genes involved. The apolipoprotein E-␧4 (APOE-␧4) was the first allele to be incriminated as it occurs in about 40% of all people who develop late onset forms [23]. Another possible risk-factor gene, sortilin-related receptor L (DLR class) A repeats-containing (SORL1), directs transport of APP into recycling pathways within neuronal cells. In the absence of SORL1, APP is released into endosomal pathways where it is subjected to ␤- and ␥-secretase cleavages [24]. Several other genes have been associated with the occurrence of sporadic AD but their roles, if any, remain to be confirmed [25–27]. A number of studies reported dysregulation of miRNAs in AD and obvious transcripts such as BACE1 or APP have been demonstrated to be targets (Fig. 1). One study reported significant decreased miR-107 in AD patients, even at earliest stages of disease [28]. Remarkably, BACE1 transcripts are increasing as miR107 is decreasing during the progression of AD. In vitro reporter assays confirm that miR-107 directly interacts with the 3 UTR of BACE1. Another study examining the miRNA expression profiles of anterior temporal cortex from five sporadic AD patients finds down-regulation of 13 miRNAs among which miR-9, -29b, -181 can be classified as brain-enriched miRNAs [29]. Interestingly, the level of BACE1 during mouse brain aging is decreasing and inversely correlates with that of miR-29a and miR-29b1. In vitro luciferase assays in human cell culture confirm a direct interaction between

miR-29a, -29b1 and the 3 UTR of BACE1. A third study show inverse correlation between BACE1 protein and two miRNAs (miR-298 and miR-328) in a mouse model of AD [30] and a direct interaction between these miRNAs and the 3 UTR of BACE1 is demonstrated using mouse cell lines. APP is a second obvious mRNA target as it plays an important role in AD. In Caenorhabditis elegans, APL-1 is the homolog of APP and is developmentally regulated by let-7 [31]. In vitro validation is also provided for human APP which interacts with miR-106a, -520c [32] and members of the miR-106 family such as miR-17-5p, -20a and -106b [33]. Apart from BACE1 and APP, other transcripts with unknown function in neurons can also be identified when studying miRNAs and a clear example is given by complement factor H (CFH), a known repressor of the inflammatory response. In AD brains, miR-146a is up-regulated whereas CFH is down-regulated and in neuroglial co-cultures, miR-146a directly interacts with the 3 UTR of CFH [34]. Dysregulated miRNAs in AD patients have been revealed using post-mortem brain samples. To date, several biomarkers of AD have been obtained from cerebrospinal fluid (CSF) such as increased phosphorylated tau, total tau and decreased A␤42 peptide [35]. Profiling miRNAs from CSF in neurodegenerative diseases could also lead to the identification of miRNA markers. However, it should be noticed that it is far from being evident how cytoplasmic changes of miRNAs in neurons or other neural cells correlate with changes in the extracellular CSF. The fact that changes in Tau expression (which is a cytoplasmic protein) can also be measured in the CSF suggests a possible correlation and certainly merits further investigation. Of note, a recent study shows dysregulated miRNAs not only in the CNS but also in the CSF of AD patients [36]. Notably, downregulation of miR-9 and miR-132 is observed in the cerebellum, hippocampus and medial frontal gyrus of AD patients diagnosed at Braak stages 5 or 6. Interestingly, miR-146b is found as downregulated in the CSF. miR-146b negatively regulates the Toll-like receptor (TLR) and cytokine signaling pathways in human monocytes [37] and represents the strongest prediction for the prognosis of squamous cell lung cancer [38] but its function in neuronal cells still remains to be determined. In the CSF of AD patients, miR-146b is slightly down-regulated whereas miR-138 is the only brainenriched miRNA showing increased expression level. miR-138 is important for neurons as it negatively regulates dendritic spine size in rat hippocampal neurons [39]. Whether miR-138 expression is only altered in AD or in other neurodegenerative disorders as well would be highly interesting to address in the future. 4.2. miRNAs in Huntington’s disease

Fig. 1. Dysregulation of miRNAs might cause accumulation of A␤ peptide. The scheme shows simplified molecular pathway of APP processing by BACE1 to generate the C99 fragment and A␤ peptide after PSEN1 mediated cleavage of C99. In the depicted pathway, APP and BACE1 have been shown as mRNA targets. Dysregulation of such miRNAs may contribute to up-regulation of APP and BACE1, leading to accumulation of plaques and tangles in Alzheimer’s disease.

In Huntington’s disease (HD), a CAG trinucleotide repeat expansion within the huntingtin gene leads to abnormal accumulation of a misfolded Huntingtin (HTT) protein into intranuclear inclusions and causes progressive loss of striatal neurons (reviewed in Ref. [40]). The physical symptoms of HD are initially random and uncontrollable movements called chorea, followed by more obvious signs of motor dysfunction. The PolyQ expansion abrogates HTT binding to the neuronal repressor RE1-silencing transcription factor (REST), allowing translocation of REST to the nucleus of neuronal cells and abnormal repression of neuronal genes and miRNAs such as miR-124a, a brain-enriched miRNA [41]. In total, five miRNAs including miR-9, -9*, -29b, -124a and -132 are down-regulated in HD patients [42]. Another study independently confirms downregulation of miR-132 in human HD cortex and in a mouse model of HD [43]. The introduction of miR-132 into hippocampal neurons enhances dendrite morphogenesis by inhibiting p250GAP, a negative modulator of dendritic plasticity [44]. HTT also interacts with components of the RISC and mouse striatal cells with misfolded HTT show reduced reporter gene silencing activity [45]. Of note, HTT can be co-immunoprecipitated with Argonaute 2 (Ago2)

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and both proteins co-localize inside the P bodies (also known as GW or Dcp bodies), which are dynamic cellular structures within the cytoplasm and consisting of many enzymes involved in mRNA turnover. 4.3. miRNAs in Parkinson’s disease Parkinson’s disease (PD) is a movement disorder characterized by rigidity, tremor, bradykinesia (slowing of physical movement) and postural instability (reviewed in Ref. [46]). Loss of dopaminergic neurons in the substantia nigra of PD patients causes insufficient released dopamine in the striatum and decreased stimulation of the motor cortex by the basal ganglia. In neurons, there is abnormal accumulation of cytoplasmic inclusions called Lewy bodies which consist mainly of ␣-synuclein (SNCA), a protein of unknown function whose aggregation forms insoluble fibrils in pathophysiological conditions such as PD, but also in dementia with Lewy bodies, multiple system atrophy (MSA) and in both sporadic and familial cases of AD. Genetic studies show that Parkin (PARK2), PTEN induced putative kinase 1 (PINK1), DJ-1 (PARK7) and Leucine-rich repeat kinase 2 (LRRK2) are genes associated with PD (reviewed in Ref. [47]). The function of these genes is mostly unknown and only a small number of familial forms of PD are explained by these mutations (reviewed in Ref. [48]). To identify dysregulated miRNAs in PD, miRNA expression of midbrain from PD patients was determined, revealing changes in the expression of miR-133b [13]. This study also shows that the transcription factor Pituitary homeobox 3 (PITX3) transcriptionally activates miR-133b, which in turn inhibits PITX3 thereby creating a negative feedback loop, consistent with the presence of high number of positive and negative feedback loops/interactions when analyzing networks of miRNAs and predicted mRNA targets [49], [50]. Overexpression of miR-133b in primary midbrain cultures prevents dopaminergic differentiation whereas its inhibition results in increased tyrosine hydroxylase (TH) positive cells. Although miR-133b plays an important role in dopaminergic neurons, the neurodegenerative aspect was not addressed and therefore other miRNAs/mRNA targets may account for the neurodegenerative phenotype of dopaminergic neurons lacking Dicer expression. Of note, Fibroblast Growth Factor 20 (FGF20) is highly enriched in the substantia nigra and a polymorphic variation associated with the risk of developing PD is located in the 3 UTR of FGF20 [51]. This small nucleotide polymorphism (SNP) disrupts a binding site for miR-433, which results in increased FGF20. Overexpression of SNCA appears to be a common feature in PD and other ␣-synucleinopathies [52] and recently, it has been demonstrated that miR-7 interacts in vitro with the 3 UTR of SNCA [53]. Of note, miR-7 inhibits cellular susceptibility of neuroblastoma cells to oxidative stress induced by a mutant form of SNCA, providing evidence that miRNAs protect neuronal cells

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against cellular stress. The presence of miR-7 in the substantia nigra is also verified thus supporting a physiological role in dopaminergic neurons. However, miR-7 was initially reported to be highly expressed in the mouse pituitary gland [54] and its expression is ∼15 fold lower in the substantia nigra when compared to the pituitary gland, leaving open the possibility that other miRNAs regulate dopaminergic neurons in a more dynamic and efficient manner. Of note, another human SNP (rs10024743) located in the SNCA gene lies in a potential binding site for miR-34b. As for other miRNAs, determining whether miR-34b normally protects neurons against neurotoxicity is a necessary question to address. Specific targeting of miRNAs in the mouse brain will also be necessary to address the physiological relevance of these in vitro findings. 4.4. miRNAs in other neurodegenerative diseases Several other neurodegenerative disorders are also characterized by dysregulation of miRNAs. For example, in frontotemporal dementia (FTD) with neuronal cytoplasmic inclusions positive for the ubiquitin and TAR DNA protein (TDP-43) (FTLD-U), mutations in the progranulin (GRN) gene are found in the familial form. A SNP located in the 3 UTR of GRN corresponds to a binding site for miR-659, a human specific miRNA. The binding of miR-659 to the high risk allele is more efficient, resulting in translational inhibition of GRN [55]. In a mouse model of prion diseases, 15 miRNAs such as miR-342-3p, -146a and -128 are found up-regulated [56] and higher expression of miR-342-3p is further confirmed in a nonhuman primate model of prion diseases [57]. 5. Discussion and conclusions It is now clear that miRNAs are dysregulated in neurodegenerative diseases (Table 1) and several challenges need to be addressed. First, linking miRNAs to their mRNA targets, as it has been shown for obvious targets such as BACE1, APP and SNCA is important as it could reveal novel pathways of neurodegeneration. However, the identification of mRNA targets currently relies on prediction software [58] and although in silico approaches can give important clues when obvious targets are predicted, their interpretation is often not as evident and further complicated by the small overlap set of predictions when comparing different algorithms. One approach to circumvent this problem is to overexpress or inhibit individual miRNA and analyze protein levels by using proteomics approaches such as a mass spectrometric method called stableisotope labeling with amino acid in cell culture (SILAC) [59], [60]. The co-immunoprecipitation of mRNA targets with Ago2 combined with high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) represents an alternative option as it was successfully applied to identify mRNA targets in the mouse

Table 1 miRNAs and validated targets with potential relevance to human neurodegenerative disorders. miRNAs

Targets

Method of validation

Human pathology

miR-8 Bantam miR-19, -101, -130 miR-298, -328 miR-107 miR-29a, -b1 let-7 miR-17-5p, -20a, -106b miR-106a, -520c miR-146a miR-132 miR-9, -9* miR-133b miR-7

Atrophin Ataxin-3 Ataxin-1 BACE1 BACE1 BACE1 APL APP APP CFH p250GAP coREST, REST PITX3 SNCA

Fruit flies (Luciferase in S2 cells) Fruit flies (Screen of modifiers) Luciferase in HeLa cells Luciferase in N2a and NIH 3T3 cells Luciferase in HeLa cells Luciferase in HeLa cells C. elegans larvae (Loss of function) Luciferase in HeLa cells Luciferase in HEK293 cells mRNA level in HN cells Luciferase in Hippocampal neurons Luciferase in HEK293 cells Viral infection of midbrain cultures Luciferase in HEK293T cells

DRPLA SCA3 SCA1 Alzheimer’s disease Alzheimer’s disease Alzheimer’s disease Alzheimer’s disease Alzheimer’s disease Alzheimer’s disease Alzheimer’s disease Huntington’s disease Huntington’s disease Parkinson’s disease Parkinson’s disease

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P13 brain [61]. After covalent crosslinking of native Ago2 proteins to mRNAs, several thousands of miRNA–mRNA target interactions are detected not only in the 3 UTR but also more surprisingly in the 5 UTR and coding sequence of transcripts. The range of action for miRNAs is still under active debate as others showed that miRNAs are only active onto 3 UTR [62]. Determining how miRNAs bind to their targets will be necessary to address to better understand future maps of interactions obtained from experimental conditions more relevant to neurodegeneration. Second, it will be important to determine whether dysregulation of miRNAs results from transcriptional or post-transcriptional mechanisms. On one hand, the support for transcriptional changes is strongly provided by the abnormal activity of REST in PD, resulting in a globally dysregulated transcriptional network. On the other hand, dysregulation of post-transcriptional regulation of miRNAs may also exist since tissue-specific expression of several mammalian miRNAs relies on post-transcriptional control. For example, miR-138 is restricted to the CNS whereas its precursor form is ubiquitously expressed throughout all adult tissues [63]. The mechanisms of miRNA processing at the post-transcriptional level are only beginning to be elucidated. For example, hnRNP A1 binds to the loop of pre-miR18a, inducing a relaxation of the stem structure and allowing a more efficient processing by the Drosha/DGCR8 complex [64]. Similarly, the KH-type splicing regulatory protein (KSRP) binds to the terminal loop of let-7 precursors and promotes their maturation [65]. It has also been shown that Lin28, a developmentally regulated RNA-binding protein involved in pluripotency, down-regulates let7 expression by binding to the loop of pre-let-7 [66]. Of importance, other miRNAs potentially implicated in neurodegeneration such as miR-107 and miR-200c are also controlled by the same mechanism. Since the discovery of miRNAs in mammalian cells nearly 10 years ago, numerous papers and reviews have described how miRNAs are generated and how they regulate specific mRNA targets. It is presently estimated that each miRNA can target up to a thousand transcripts, which sharply contrasts with the few number of validated targets in vivo. The manipulation of miRNA expression in different animal models including the fruit fly, C. elegans, the zebrafish and the mouse, using either viral vectors or genome based modifications such as the Zinc Finger Nuclease (ZFN) technology will quickly allow neuroscientists to confirm the growing importance of miRNAs in neuronal function and open promising and fruitful approaches to the understanding of complexity in neurodegenerative disorders.

Acknowledgements We thank Hal Gainer (NINDS) for critical reading of the manuscript. This work was supported by stichting Alzheimer onderzoek (SAO, Belgie), the Fund for Scientific Research, Flanders, K.U. Leuven (Geconcerteerde onderzoeksactie and Methusalem grant), Federal Office for Scientific Affairs, Belgium (IUAP P6/43/).

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