Background
Autism spectrum disorders (ASD) represent a group of childhood neurodevelopmental disorders characterized by deficits in verbal communication and social interaction and restricted and repetitive patterns of interests and behaviors. These conditions are often considered as “connectopathies” or “synaptopathies,” whereby neuronal circuits are mis-wired or dysfunctional. Much progress has been made in unravelling the genetics of ASD with large copy number variation [
1] and de novo mutations in a subset of genes [
2] as well as inherited mutations being associated with ASD [
3]. Yet, the current consensus agrees that majority of ASD cases are caused by complex interaction of multiple genetic and environmental risk factors. Orchestrated by environmental factors, epigenetic modifications, such as DNA methylation, covalent modification of histones, or the activation or silencing of genes by microRNAs (miRNAs), may drive genetically predisposed individuals to develop autism and represent the crossroads between the environment and genetics in ASD. Accordingly, gene expression profiling of monozygotic twins discordant in diagnosis of autism has identified differentially expressed, neurologically relevant genes [
4]. Similarly, abnormal DNA methylation patterns have been detected at several candidate genes in ASD patients [
5].
miRNAs play key roles in neuronal development, synapse formation, and fine-tuning of genes underlying synaptic plasticity and memory formation [
6]. Several studies have identified aberrant miRNA expression in a wide range of neurological diseases [
7,
8], but a comprehensive understanding of miRNA networks deregulated in ASD has not been achieved. The expression of miRNAs in lymphoblastoid cell cultures of ASD patients has already been explored [
9‐
12], but the relevance of this cell type is questionable in the context of a neurodevelopmental disorder. One study has identified deregulated miRNAs in the cerebellum of ASD patients [
12] and another investigated the superior temporal sulcus and auditory cortex [
13], but it raises the issue of the investigated brain regions and the impact of cause of death and/or post-death handling of tissue on gene expression.
To overcome these limitations, we used olfactory mucosal stem cells (OMSCs) biopsied from living ASD patients and controls. OMSCs exhibit characteristics of ecto-mesenchymal stem cell and maintain neurogenic ability [
14,
15]; they could be considered as precursor of neuronal and glia-like cells. These characteristics make OMSC a very relevant model for transcriptomic studies with great potential to identify genes and pathways relevant for neurodevelopmental disorders. Indeed, OMSC has been used successfully to study the pathology of schizophrenia [
16], Rett syndrome [
17], and Parkinson’s disease [
18] in which they displayed disease-specific alterations in gene expression and cellular functions (for review, see [
19]).
Here, using OMSC, we report a signature of miRNA commonly deregulated in ASD. We show that this signature is conserved in primary skin fibroblast. We demonstrate that these miRNAs have essential roles in brain function through their regulation of neuronal specific genes and provide evidence that deregulation of their expression alter the development of primary neurons and the function of glial cells. Our results demonstrate the importance of the miRNA circuitry in ASD, opening up opportunities for the development of early diagnostic test and potential treatment.
Methods
Ethic statements
Human samples were obtained with informed consent of the patients, and studies were carried out under a protocol that was approved by the ethic committees of the Hôpital Necker, Paris, and the Montperrin Hôpital, Aix en Provence (CPP Marseille2). The experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. All the biological collections (blood, nucleic acid, tissues) are kept securely in one place, and their management and the quality of their preservation follow the French regulations and ethical recommendations. Primary cortical neurons were extracted from mouse embryos (E15.5) following protocols approved by the local ethical committee of the Interdisciplinary Institute of Neuroscience of Bordeaux accordingly to the European Communities Council Directive (86/809/EEC).
The patients included in this study have been clinically diagnosed as having ASD according to the criteria of DSM-V. Full clinical description of these patients has already been reported [
20]. We include here a brief clinical description of the patients (see Additional file
1: Table S1). To investigate the genetic etiology, we extracted DNAs from their OMSCs and subjected to array comparative genomic hybridization and whole exome sequencing according to published protocol. One patient (A3) carries two variants leading to premature translation termination in
SCN2A and
FAT1, which are both recurrent non-fully penetrant ASD genes (see Additional file
1: Tables S2 and S3); two patients (A1 and A10) were found to carry deletions at genomic regions 22q13.3 and 15q13.2–q13.3, respectively, which have been found to be associated with ASD (see Additional file
1: Table S3). The genetic etiology of the remaining patients remained inconclusive.
Maintaining of OMSC, human primary fibroblast, and HEK293T
OMSC was biopsied and cultivated according to published protocol [
20]. OMSC was maintained using Dulbecco’s modified Eagle’s medium (DMEM)/F-12 GlutaMAX™ (Life Technologies) supplemented with Penicillin-Streptomycin (100 U/ml) (Life Technologies) and 10 % fetal bovine serum (FBS). Human primary fibroblasts were extracted from skin punches with informed consents and maintained in RPMI 1640 Medium GlutaMAX™ (Life Technologies) supplemented with Penicillin-Streptomycin (100 U/ml) (Life Technologies) and 10 % FBS. HEK293T cells were maintained in DMEM High-Glucose Medium GlutaMAX™ (Life Technologies) supplemented with Penicillin-Streptomycin (100 U/ml) (Life Technologies) and 10 % FBS. Both cell types were passaged first by detaching using 0.05 % Trypsin-EDTA (1×) Phenol Red (Life Technologies) and replated in desired concentration.
Expression profiling of miRNA
miRNAs were extracted from frozen pellets using mirVanaTM miRNA isolation kit (Life Technologies) according to manufacturer’s instructions. Concentration was measured using Nanodrop 2000 (Thermo Scientific). During the discovery phase, miRNA profiling was performed across a two-card set of TaqMan® MicroRNA Arrays (arrays A and B) (Life Technologies) for a total of 667 unique assays specific to human miRNAs (Sanger miRBase v10). Analyses were done in technical triplicates and used two different miRNA extractions from two pellets. For validation, 24 miRNAs that were found either significantly deregulated in the first round or whose assays failed and the 7 reference miRNAs were assessed (see Additional file
1: Figure S1). Raw data (see Additional file
2) were analyzed using both GenEX and RealTime StatMiner® software. Both integrate quality controls, selection of best endogenous controls, clustering, differential expression, and two-way ANOVA analysis. The samples included in miRNA profiling were A2, A3, A5, A6, A7, A9, A10, and A11 vs. C1, C4, C5, C6, C7, and C10. The miRNA profiling and analyses were performed as paid service by the IMAGIF platform for high-throughput quantitative PCR, ICSN CNRS, Gif-sur-Yvette, France. Expression profiles of
miR-146a,
miR-221,
miR-654-5p, and
miR-656 were assessed using Taqman assays on Fluidigm 48.48 array in technical triplicates; for fibroblast samples, two different miRNA extractions from consecutive passages were analyzed and six miRNAs were used as reference miRNAs (see Additional file
1: Figure S2 and Additional file
2 for raw data); for peripheral blood mononuclear cells (PBMC), only one extraction was analyzed and seven miRNAs were used as reference miRNAs (see Additional file
1: Figure S3 and Additional file
2 for raw data). Sample preparation was done according to the manufacturer’s Protocol for Creating Custom RT and Preamplification Pools using Taqman® MicroRNA Assays (Life Technologies). This step was performed as paid service by Platform qPCR-HD-GPC, Ecole Normale Supérieure, Paris, France.
miRNA target prediction and pathway analysis
Target prediction was performed using mir-DIP which integrates predictions from multiple software. Only targets predicted by at least three different programs were included in pathway analysis. Gene ontology enrichment of predicted miRNA targets (~1000 targets per miRNA) was performed using ingenuity pathway analysis (IPA).
Quantitative real-time polymerase chain reaction
Total RNA was extracted using TRIzol reagent (Life Technologies) and RNeasy Mini Kit (QIAGEN) with DNase I treatment step (QIAGEN). cDNA was reversed transcribed from extracted RNA using SuperScript® II Reverse Transcriptase (Life Technologies). Gene expression was measured using SYBR Green Power Mix (Life Technologies) using primers specific for each gene. Additional file
1: Table S4 contains the sequences of all primers used in this study. Relative standard curve method was employed to calculate relative gene expression. Reactions were performed using the Step One Plus Real-Time PCR system (Applied Biosystems). Expression values were taken from mean of triplicates.
Cloning the 3′UTR of GRIA3, KCNK2, and MAP2
The entire or parts of the 3′ untranslated region (UTR) of
GRIA3 (~2000 bp),
KCNK2 (~1300 bp), and
MAP2 (~1300 bp) were amplified using specific primers (Additional file
1: Table S4), subcloned into TOPO 3.1 vector using TOPO® TA Cloning® Kit (Life Technologies) and transformed into One Shot® TOP10 Chemically Competent E. coli (Life Technologies) by heat shock method. Plasmid was extracted using PureYieldTM Plasmid MiniPrep System (Promega) and digested with XhoI and NotI-HF (New England Biolabs). Digested fragment was gel purified once more then cloned into cut psiCheck2 plasmid (Promega) using T4 DNA Ligase (New England Biolabs). For higher yield plasmid extraction, 100 ml of bacterial culture was subjected to Plasmid Midi Kit (QIAGEN). Cloned plasmids were sequenced using psiCheck2_hLucF and psiCheck2_hLucR for screening.
Dual luciferase assay
Approximately 2 × 105 HEK293T cells were plated in each well of 12-well plate the day before transfection. Cells were transfected with either plasmids overexpressing miRNAs, LentimiRa-GFP-mmu-mir-146a/221/656 (mm10082/mh10296/mh10968, ABM Good) or empty vector LentimiRa-GFP-empty (m001, ABM Good), together with psiCheck2_GRIA3_UTR, psiCheck2_KCNK2_UTR, or psiCheck2_MAP2_UTR plasmids (ratio 1:3 to ensure good expression of luciferase). Transfection was performed using JetPRIME® Polyplus Transfection Reagent (Ozyme) following manufacturer’s instruction. Assay was performed using Dual-Luciferase® Reporter Assay System (Promega) 24 h after transfection. Ratio of Renilla luciferase to firefly luciferase was taken as mean of technical triplicates.
In situ hybridization analysis
Tissue preparation and processing
Male, 2-month-old wild type mice were deeply anesthetized using pentobarbital and fixed by intracardiac perfusion with 4 % paraformaldehyde in phosphate-buffered saline (PBS). Brains were removed, post-fixed overnight at 4 °C, and cryo-protected using two baths of Tris-HCl buffered saline (TBS) with 0.5 M sucrose at 4 °C during 48 h. Brains were then frozen in bath of isopentane at −35–40 °C and stored at −80 °C. Serial sections of 20 μm were done in a cryomold (Tissue-Tek) with a cryostat (Leica), mounted on Superfrost Plus glass slides (Thermo Scientific), and preserved at −80 °C. For embryos and early postnatal animals, intracardiac perfusion was replaced by direct 4 % paraformaldehyde fixation overnight after decapitation.
In situ hybridization procedure
Tissue sections mounted on glass slides were thawed and air dried for 1 h at room temperature, incubated in a solution containing 20 μg/ml proteinase K (Sigma) in TBS, pH 7.4 for 3 min, and then washed for 5 min two times in TBS. Samples were fixed in 4 % paraformaldehyde (PFA) for 10 min and washed with 2 mg/ml glycine in TBS and twice for 5 min in TBS. To remove residual phosphate from the TBS washes, slides processed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) fixation were incubated twice for 10 min in freshly prepared solution containing 0.13 M 1-methylimidazole, 300 mM NaCl, pH 8.0 adjusted with HCL. In the meantime, a solution of 0.16 M EDC (Sigma) is prepared by adding EDC into 1-methylimidazole and 300 mM NaCl (pH 8.0) solution. The pH of the EDC solution is readjusted by adding 12 M HCl to pH 8.0. Slides are maintained in a humidified chamber for 1 h at room temperature incubated in 500 μl of EDC solution to each slide. The slides are then washed in 2 mg/ml glycine/TBS solution and then twice for 5 min in TBS. For enzymatic inactivation, slides were acetylated by incubating for 30 min in a solution of freshly prepared 0.1 M triethanolamine and 0.5 % (
v/
v) acetic anhydride. Slides were then rinsed twice for 5 min in TBS. For pre-hybridization, the tissue sections were covered with 500 μl of hybridization buffer containing 50 % formamide, 5× SSC, 5× Denhardt’s solution, 0.25 mg/ml yeast transfer ribonucleic acid (tRNA), 0.5 mg/ml salmon sperm DNA, 20 mg/ml blocking reagent (Roche), 1 mg/ml 3-((3-cholaminodopropyl) dimethylammonio)-1-propanesulfate (CHAPs, Sigma), and 0.5 % tween at room temperature for 2 h in a humidified chamber. The hybridization buffer was removed by tilting the slide. Sequences of the different probes are described in Additional file
1: Table S4. For hybridization, 40 nM of DIG-labeled locked nucleic acids (LNA) probe (mmu-miR-146a, mmu-miR-scramble) diluted in hybridization buffer were applied per section and covered with coverslips. The slides were incubated in a sealed humidified chamber for 16 h at 42 °C.
Slides were immersed for 3 min in 2× SSC at 52 °C and then washed for 5 min twice in 1× SSC at 52 °C and once in 1× SSC at room temperature. Slides were washed again for 5 min twice in 0.2× SSC at room temperature and once in TBS. In preparation for probe detection, 500 μl of blocking solution containing 5 mg/ml blocking reagent (Roche), 10 % (v/v) normal goat serum, and 0.1 % (v/v) tween in TBS was applied to each slide for 1 h at room temperature in a humidified chamber. Antibody anti-DIG-FAB peroxidase (POD) (Roche) diluted 1:500 in blocking solution was incubated overnight. Slides were then washed three times in TBS. Slides were incubated in freshly prepared NBT/BCIP substrate reagent containing 50 mM MgCl2, 100 mM NaCl, 0.2 mM Levamisole, 0.5 mg/ml NBT, and 0.1875 mg/ml BCIP in 100 mM TBS pH 9.5 and protected from light during the exposure time (approximately 6 h). Slides were then washed twice in water, then twice in TBS and incubated for 10 min in 4 % PFA solution. Finally, slides were washed in water and mounted using two drops of mounting medium.
Immunohistochemistry and miRNA ISH co-staining
To assess cellular localization of miR-146a, brain sections were processed for both immunofluorescence staining and in situ hybridization (ISH) staining. Slides were washed three times in PBS and then incubated 1 h at room temperature in humidified chamber, with 500 μl of blocking solution containing 0.1 % (v/v) triton-100× and 2 % (v/v) normal donkey serum in PBS. The blocking solution was removed by tilting the slide. Slides were incubated overnight at room temperature with the same blocking solution containing polyclonal antibody against Fox3/NeuN (1:1000; Abcam, AB104225) for specific neuronal staining and antibody against GFAP (1:500; Abcam, ab7260). Slides were rinsed three times in PBS and incubated for 1 h 30 min at room temperature with donkey Alexa fluor 488-conjugated anti-rabbit (for NeuN), secondary antibody diluted in blocking solution. After being rinsed in PBS, then water, slides were mounted in mounting medium.
Microscopy and image processing
Images were captured on an upright epifluorescence microscope, Nikon Eclipse Ni-U (Nikon France S.A) using 10× objective CFI Plan Fluor NA 0.30 and 40× objective CFI Plan Fluor NA 0.75. For fluorescent imaging filters, sets for GFP were used, and pictures were acquired using a Zyla SCMOS camera (Andor Technology Ltd., Belfast, UK).
Primary neuronal cultures
Cultured mouse hippocampal neurons were prepared from E16.5 embryos, grown on 18-mm glass coverslips coated with laminin/polylysine and maintained in Neurobasal B27-supplemented medium. Neurons were transfected using Lipofectamine 2000 on days in vitro 11 (DIV11) with 800 ng of plenti-III-miR-GFP-Blank or pLenti-III-miR146a-GFP, and experiments were performed at DIV18.
Sholl analysis
Analysis of the dendritic morphology was done blind to the genotype. Sholl analysis was done using a Sholl analysis pluggin on ImageJ and determined the number of dendritic branch intersections with concentric circles of increasing radii (interval of 10 μm) from the soma. All neurons were imaged at the same magnification, and images were threshold, and to avoid false intersections because of noise, each threshold images were checked and compare to the original. The BI (branching index) was shown: The BI compares the difference in the number of intersections made in pairs of circles relative to the distance from the neuronal soma, following the following equation: BI = ∑(intersections circle n − Intersections circle n − 1).rn according to “A new mathematical function to evaluate neuronal morphology using the Sholl analysis: Luis Miguel Garcia-Segura, Julio Perez-Marquez”.
Virus production
Lentivirus was produced using lentiviral compatibility vectors as mentioned above by the Plateforme Vecterus Viraux et Transfert de Gènes from Hospital Necker as paid service. Viral titre was determined by FACS of GFP signal. For all experiments, virus infection was performed at the multiplicity of infection (MOI) of 2 in normal media; virus media was replaced after at least 4 h of incubation.
Primary astrocyte culture
Astrocytes were extracted from cortexes of P1 mice and grown on uncoated flask in DMEM Low Glucose Medium GlutaMAX™ (Life Technologies) supplemented with Penicillin-Streptomycin (100 U/ml) (Life Technologies) and 10 % FBS. When cell reach 70–80 % confluent, they were detached with 0.05 % Trypsin-EDTA (1×) Phenol Red (Life Technologies) and replated in desired cell density; this passaging step was only performed once for each culture. For cell growth assay, 20,000 cells were replated into E-Plate 96 for continuous monitoring on the Xcelligence RTCA-MP instrument (ACEA Biosciences) for 5 h before being infected with virus; growth rate was measured over 72 h during which cell index was analyzed every 15 min. Growth rate was measured as slope of normalized cell index from at least three technical triplicates over a set period of time; this analysis was performed using RTCA Software V2.0 (ACEA Biosciences).
For glutamate uptake assay, 200,000 cells were replated into a 24-well plate and allowed to reach 100 % confluent. Cells were then treated with a mitotic blocker 8 μM cytosine β-d-arabinofuranoside (Sigma) for 5–6 days followed by a 90-min incubation with 75 mM l-leucine methyl ester (Sigma) to completely diminish the culture of microglia; viral infection was performed overnight. The next day, cells were serum starved for 2 h before being incubated in media containing 1 μCi/ml (H3)-glutamic acid (Perkin Elmer) and 50 μM of unlabeled glutamate (Sigma); uptake was allowed in the incubator for 30 min before cells were washed three times with ice cold Hanks’ balanced salt solution (Life Technologies) supplemented with HEPES 15 mM (Life Technologies) and lysed in 1 M NaOH 0.1 % Triton-X solution at 42 °C for 10 min. For negative control, the transporters were blocked using 250 μM L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) (Sigma) (maintained even during the serum starved step). Radioactivity was measured as average count per minute during 10 min. Rate of uptake was measured from duplicates, normalized to samples treated with PDC, and the protein concentration was measured by Bradford assay.
Discussion
We report here a comprehensive analysis of miRNA expression profiles in human stem cells from ASD patients. We identified a signature of four miRNAs (
miR-146a,
miR-221,
miR-654-5p, and
miR-
656) commonly deregulated in ASD. This signature is not conserved in PBMCs, a result consistent with the two previously reported miRNA expression studies performed in serum from children with autism [
30,
31], but is also observed in primary skin fibroblasts from unrelated patients. While this observation may restrict the ease to rapidly validate our data in a larger cohort, it highlights the relevance of OMSCs, a neurologically relevant tissue, for understanding the pathophysiological mechanisms underlying neurodevelopmental disorders and supports the use of primary fibroblasts for routine testing. Notably, comparable miRNA profiling in OMSCs was recently performed in the context of schizophrenia [
32], and similar results were obtained with increased
miR-382 expression observed in OMSCs samples from patients but not in peripheral blood-derived/non-neuronal samples. Despite the limited number of samples tested, potentially restricting the statistical power, the observed conservation of the signature in primary skin fibroblasts from unrelated patients strongly supports its biological relevance. Lastly, our finding that ID patients do not share the same signature as ASD may have diagnostic implications and raises the possibility of using miRNA signature as a biomarker for ASD, providing a means for detecting early signs of the disease and allowing early intervention.
Target prediction analysis suggests that the transcripts regulated by these miRNAs code for proteins involved in neurodevelopmental processes as well as immune response and inflammation, both of which are relevant for the pathology of ASD. Previous studies have demonstrated that immune imbalance impairs higher order brain functioning and altered inflammatory responses have been reported in ASD patients [
33,
34]. Moreover, DNA methylation analysis of the autistic brains has identified a very significant enrichment of differentially methylated region in genomic areas responsible for immune functions [
5]. Along these lines, it is noteworthy that
miR-146a was first identified as an immune system regulator and possibly plays an important role in neuroinflammation [
28]. Our results support the hypothesis that altered
miR146a expression contributes to the neuroinflammatory processes reported in the autistic brain.
miR-146a and
miR-
221 have also established roles in neurodevelopment. Validated targets of
miR-146a include
MAP1B, which regulates AMPA receptor endocytosis [
35], while direct targets of
miR-
221 include
FMR1, the fragile X gene which is the most frequent cause of syndromic intellectual disability (ID) and ASD [
36]. Using target prediction tools combined to dual luciferase assays and Western Blot analysis, we identified additional neuronal transcripts regulated by these miRNAs. We showed that
GRIA3 is a target transcript for both
miR-146a and
miR-221. GRIA3 encodes a core subunit of the AMPA receptor, and findings from various experimental systems implicate ionotropic GluR dysfunction in ASD [
37].
miR-146a upregulation in ASD neurons might thus alter AMPA receptor biology through both impaired MAP1B-mediated endocytosis and decreased amount of
GRIA3. We also demonstrated that miRNA expression deregulation correlates with a reduced level of KCNK2 protein in patients’ OMSCs and that both
miR-146a and
miR-221 directly target
KCNK2 3′UTR.
KCNK2 is a member of the potassium leak channel family, a group of proteins that are critical determinants of neuronal excitability in the cortex [
38], and
KCNK2 knockdown impairs neuronal migration in the developing mouse cerebral cortex [
39]. Reduced amount of KCNK2 protein may thus contribute to the failure or delay in neuronal migration that has been observed in some cases of autism and ID.
One prevalent theory for the pathogenesis of autism relates to a deficiency in the plasticity of axonal sprouting and synaptic connectivity. There have been reports in autism cases of enlarged or abnormally oriented neurons, densely packed neuronal regions, and isolated regions of atrophic neurons with reduced dendritic arborisation [
40,
41]. Increased spine density has also been reported in fragile X syndrome [
42]. Conversely, neuronal atrophy and reduced dendritic spines is mentioned in Rett syndrome [
43]. Our observation that
miR-146a overexpression disturbs neuronal dendritic arborization is thus consistent with the defective neural connectivity found in ASD.
miR-146a has also been linked to the processes underlying glial cells differentiation, and previous reports demonstrated that it inhibits the expression of neuron-specific targets
Nlgn1 and
Syt1, preventing glial cells from mistakenly adopting neuron-specific phenotypes [
27]. Using in vitro assays, we demonstrated that
miR-146a overexpression alters astrocyte glutamate uptake capacity. Interestingly, these findings coincides with our growing knowledge for the role of glial cells in every aspects of the development and function of neuronal cells, with the recent evidence that astrocytes play a major role in neurodevelopmental disorders and the demonstration of altered glutamatergic synaptic transmission in ASD [
44,
45]. We propose that abnormal
miR-146a expression impairs astrocyte differentiation and function in ASD brain and that altered synaptic clearance of glutamate may contribute to the excitatory-inhibitory activity imbalance underlying autism.
Remarkably,
miR-146a overexpression is not restricted to ASD patients. First, we demonstrated that
miR-146a overexpression is also observed in cells from patients with ID. Second, increased amount of
miR-146a has been found in epilepsy-associated glioneuronal lesions [
46], in serum of epilepsy patients [
47] as well as in the hippocampus in a rat model of temporal lobe epilepsy [
48].
miR-146a upregulation seems thus to be a common hallmark of different neurodevelopmental disorders and may mediate common pathways underlying these conditions. Understanding how this miRNA contributes to the interactions between synaptic function and immune molecules and cells will therefore have broad implications.
Lastly, our findings may have important consequences for the development of future therapies, as several strategies have been established to restore miRNA levels or block miRNA function. The expression levels of particular miRNAs can be restored either with miRNA mimics or with miRNAs encoded in expression vectors. Conversely, current strategies for inhibitory targeting of microRNAs are mainly based on antisense oligonucleotides (so-called anti-miRNAs), comprised of locked nucleic acids (LNA) along with tiny LNA anti-miRNA constructs, antagomirs, and miRNA sponges [
49‐
51]. Indeed, it was recently shown that intracerebroventricular injections of
antagomir-155 and
antagomir-802 in the Ts65Dn mice, a Down syndrome mouse model that overexpress such miRNAs, resulted in the attenuation of 30–40 % of endogenous
miRNA-155 and
miR-802 and increased
Mecp2 target gene expression 7 days after administration [
52]. While manipulating miRNAs expression to achieve biological levels still requires optimization, our results support the idea that it might be a promising strategy in the field of ASD. Further studies are needed to address the precise molecular mechanisms underlying miRNA expression deregulation, and whether this is the consequence of a transcriptional defect or of a miRNA maturation processing defect need further clarification. Investigation in animal models will also be necessary to determine the exact impact of
miR-146a and
miR-221 deregulation on synaptogenesis and synaptic plasticity.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LC, CR, YH, LSN, and MM designed the study; YH, ML, and CM conducted the in situ hybridization experiments, primary neuronal cultures, and subsequent Scholl analysis; LSN and JF conducted qRT-PCR, Western blots, luciferase assays, and functional assays in astrocytes; FF and BG collected the OMSCs samples; AP and KSP collected the fibroblast and PBMCs samples; LC and LSN wrote the manuscript, and all the authors contributed to the editing. All authors read and approved the manuscript.