Profiling olfactory stem cells from living patients identifies miRNAs relevant for autism pathophysiology

Abstract

Background: Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders caused by the interaction between genetic vulnerability and environmental factors. MicroRNAs (miRNAs) are key posttranscriptional regulators involved in multiple aspects of brain development and function. Previous studies have investigated miRNAs expression in ASD using non-neural cells like lymphoblastoid cell lines (LCL) or postmortem tissues. However, the relevance of LCLs is questionable in the context of a neurodevelopmental disorder, and the impact of the cause of death and/or post-death handling of tissue likely contributes to the variations observed between studies on brain samples.

Methods: miRNA profiling using TLDA high-throughput real-time qPCR was performed on miRNAs extracted from olfactory mucosal stem cells (OMSCs) biopsied from eight patients and six controls. This tissue is considered as a closer tissue to neural stem cells that could be sampled in living patients and was never investigated for such a purpose before. Real-time PCR was used to validate a set of differentially expressed miRNAs, and bioinformatics analysis determined common pathways and gene targets. Luciferase assays and real-time PCR analysis were used to evaluate the effect of miRNAs misregulation on the expression and translation of several autism-related transcripts. Viral vector-mediated expression was used to evaluate the impact of miRNAs deregulation on neuronal or glial cells functions.

Results: We identified a signature of four miRNAs (miR-146a, miR-221, miR-654-5p, and miR-656) commonly deregulated in ASD. This signature is conserved in primary skin fibroblasts and may allow discriminating between ASD and intellectual disability samples. Putative target genes of the differentially expressed miRNAs were enriched for pathways previously associated to ASD, and altered levels of neuronal transcripts targeted by miR-146a, miR-221, and miR-656 were observed in patients' cells. In the mouse brain, miR-146a, and miR-221 display strong neuronal expression in regions important for high cognitive functions, and we demonstrated that reproducing abnormal miR-146a expression in mouse primary cell cultures leads to impaired neuronal dendritic arborization and increased astrocyte glutamate uptake capacities.

Conclusions: While independent replication experiments are needed to clarify whether these four miRNAS could serve as early biomarkers of ASD, these findings may have important diagnostic implications. They also provide mechanistic connection between miRNA dysregulation and ASD pathophysiology and may open up new opportunities for therapeutic.

Keywords: Astrocyte; Autism spectrum disorders; MicroRNA; Neuron; Olfactory mucosa stem cells.

Fig. 1

Identification of a conserved miRNA signature in ASD. a Significantly deregulated miRNAs after validation in ASD OMSC after the validation round, representing the miRNA deregulation signature of ASD: miR-146a, miR-221, miR-654-5p, and miR-656 (±SD, controls—dark gray bar, patients—light gray bar). Dots represent average of technical triplicates for each biological repeat. *P < 0.05, **P < 0.01 by Wilcoxon rank sum test. b Tissue and disease specificity of miRNA signature. Expression (±SD) of miR-146a, miR-221, miR-654-5p, and miR-656 were assessed in primary skin fibroblasts of ASD patients (n = 5, light gray bar), ID patients (n = 12, white bar), and controls (n = 4, dark gray bar). Results were obtained from Taqman assays performed on Fluidigm array. Dots represent average of technical triplicates for each biological repeat. *P < 0.05, ***P < 0,001 by Student’s paired two-tailed t test. c Expression (±SD) of miRNA signature in peripheral blood mononuclear cells (PBMC). No significant difference in miR expression was detected between the controls (n = 20, dark gray box) and the ASD patients (n = 9, light gray box). Results were obtained from Taqman assays performed on Fluidigm array. Dots represent average of technical triplicates for each biological repeat

Fig. 2

miRNAs directly regulate neuronal relevant genes. a Gene ontology enrichment of predicted miRNA targets by ingenuity pathway analysis (IPA). About 1000 genes were predicted to be regulated by each miRNA by at least three different prediction programs and were included in the analyses. Only the top 5 enriched pathways are shown. P values were calculated by Fisher’s exact test; red line represents correction threshold by Bonferroni Correction. b Mean expression (±SD) of known (in bold) and predicted targets of miRNAs in ASD (n = 9, light gray bar) and control OMSCs (n = 8, dark gray bar). Gene expression was measured using relative standard curve method, normalized against GPBP1 as reference gene. Results shown represent one of two independent repeats showing the same results. c Western blot showing down regulation of KCNK2 in ASD (n = 3) with respect to control OMSCs (n = 3). ACTB was used as loading control. Bottom panel displays densitometry of the bands using two images taken at low exposures. *P < 0.05 by Student’s paired two-tailed t test. d The 3′UTRs of GRIA3, KCNK2, and MAP2 are targeted by miRNAs. The 3′UTRs of GRIA3, KCNK2, and MAP2 were subcloned into the 3′UTR of Renilla luciferase in the psiCheck2 plasmid and co-transfected into HEK293T with either plasmid overexpressing miR-146a, miR-221, and miR-656 or empty plasmid. Ratio of Renilla/firefly luciferase (±SD) indicates the repression activity of miRNAs directly on the 3′UTR. Results are represented from one repeat of two showing the same results. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s paired two-tailed t test

Fig. 3

Expression of miR-146a and miR-221 in the brain. a ISH of miR-146a and miR-221 in the adult mouse brain. Typical coronal slices showed elevated miR-146a in the hippocampal CA1 and dentate gyrus (DG), the amygdala nuclei (amyg), and the entorhinal cortex (ctx). b Hybridization of scramble or miR-146a and miR-221 was performed onto mice at different developmental from embryonic (E11 and E16) and postnatal (P5 and P30) stages. Arrows indicate hippocampal formation or territories at the different age, allowing appreciation of the specific labeling of different cellular layers in the postnatal brains. c Neuronal expression of both miRNAs was confirmed by combining miR-ISH with fluorescent labeling of neuronal (NeuN) and glial (GFAP) cells in the CA1 region

Fig. 4

miR-146a overexpression alter neuronal and glial cells biology. a Efficacy of viral induction 24 h post-infection with lentivirus carrying the empty vector (control) or miR-146a. Top panel shows primary mouse astrocyte expressing GFP; DAPI was used to counterstain the nucleus. Bottom left panel shows miR-146a level (±SD) induced by the virus; expression was measured by Taqman assay and normalized against miR-221 in technical triplicates; bottom right panel shows the expression of Nlng1 and Traf6 (±SD) measured by RT-qPCR in technical triplicates, normalized against Actb; **P < 0.01, ***P value <0.001 by Student’s paired two-tailed t test. b Growth of primary astrocyte is significantly impaired by miR-146a upregulation (red line) compared to the control (green line). Cell growth, displayed as normalized cell index (±SD), was continuously monitored using the Xcelligence system over 4 days. Statistic analysis was performed using data collected from technical quadruplicates at 72 h post-infection. The graph is from one repeat of three showing the same results. ***P value <0.001 by Student’s paired two-tailed t test. c Glutamate uptake is significantly increased in astrocytes overexpressing miR-146a. Following a published protocol [53], uptake of radioactive glutamate (±SD) was counted as count per minute (CPM), normalized against total protein and CPM values of samples treated with the glutamate transporter blocker PDC. The graph is from one repeat of three showing the same results. *P value <0.05 by Student’s paired two-tailed t test. d miR-146a upregulation inhibits neurite outgrowth. Sholl analysis of dendritic arborisation of control and miR-146a-overexpressing neurons. *P < 0.05; **P < 0.01. To test for differences in intersections between blank and miR-146a neurons, each distance (10 μm steps) was tested by one-way ANOVA statistical test followed by the Holm-Sidak post hoc test. When data where not following a normal distribution, we used the one-way ANOVA on ranks and Dunn’s method for post hoc test