Abstract
MicroRNAs (miRs) are non-coding gene transcripts abundantly expressed in both the developing and adult mammalian brain. They act as important modulators of complex gene regulatory networks during neuronal development and plasticity. miR-181c is highly abundant in cerebellar cortex and its expression is increased in autism patients as well as in an animal model of autism. To systematically identify putative targets of miR-181c, we repressed this miR in growing cortical neurons and found over 70 differentially expressed target genes using transcriptome profiling. Pathway analysis showed that the miR-181c-modulated genes converge on signaling cascades relevant to neurite and synapse developmental processes. To experimentally examine the significance of these data, we inhibited miR-181c during rat cortical neuronal maturation in vitro; this loss-of miR-181c function resulted in enhanced neurite sprouting and reduced synaptogenesis. Collectively, our findings suggest that miR-181c is a modulator of gene networks associated with cortical neuronal maturation.
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Supplement Figure 1: MiR-181c transduction efficiency into primary cortical neurons. A. Schematic overview of the lentiviral miR-181c-sponge construct designed to continuously and specifically sequester miR-181c. A concatemer of four complementary miR-181c binding sites is placed within the 3′UTR of the eGFP gene. As example, one binding site of the sponge with the mature miR-181c is shown, the central mismatch is included to enhance efficiency [56,63,64]. B. Micrographs of primary cortical neurons infected with the miR-181c sponge lentivirus. The left image shows cell nuclei visualized with DAPI (bleu), the middle panel shows GFP from the miR-181c sponge (green) and on the right is the DAPI and GFP overlay. C. Relative expression of miR-181c in DIV 9-old primary cortical neurons expressing the GFP control or miR-181c sponge construct normalized to control. D. Representative images of primary cortical neurons transfected with siGLO transfection indicator. The micrographs from left to right show the nuclear DAPI staining (blue), siGLO (red) and a DAPI with siGLO overlay. E. Relative expression of miR-181c in DIV 6 primary cortical neurons transfected with NT control or miR-181c mimic, levels were normalized to control. F. Relative expression of miR-181c in DIV 6 primary cortical neurons transfected with NT control or anti-miR-181c, levels were normalized to control. Data are shown as mean ± SEM; p values are determined by two-tailed unpaired Student’s t test. *p ≤ 0.01 and ** p ≤ 0.001 (JPEG 1349 kb)
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Supplement Figure 2: Growing pure axonal fractions in microfluidic chambers. A. Schematic representation of a microfluidic chamber with the soma side (gray) and the axon side (green) connected with microgrooves in the middle. Neurons are shown in red, which grow their axons through the microgrooves to the axonal side. B. An example immunostaining of cortical neurons cultured in microfluidic devices. The DIC image shows the microgrooves of the microfluidic chamber, MAP2 highlights the dendrites in green and Tau1 for visualizing axons in red. The image on the bottom right corner shows an overlay of DIC, MAP2 and Tau1 (JPEG 403 kb)
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Supplement Figure 3: Mature cortical neurons transduced with miR-181c mimic have a necrotic phenotype. Representative micrographs of DIV16 primary cortical neurons transfected with 30 nM NT control (upper panels) or miR-181c mimic (lower panels) and 20 nM SiGlo fluorescent transfection indicator at DIV 3. The panels depict micrographs of DIV16 neurons stained with DAPI (blue), PSD-95 (green) and the SiGlo transfection indicator (red). The necrotic phenotype (condensed nuclei) in miR-181c transfected neurons was observed for around 90% of the cells; this experiment was repeated three times, with similar outcomes (JPEG 853 kb)
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Supplement Table 1: List of genes functioning in behavior, cell-to-cell signaling and interaction, nervous system development and function (PDF 100 kb)
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Kos, A., Olde Loohuis, N., Meinhardt, J. et al. MicroRNA-181 promotes synaptogenesis and attenuates axonal outgrowth in cortical neurons. Cell. Mol. Life Sci. 73, 3555–3567 (2016). https://doi.org/10.1007/s00018-016-2179-0
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DOI: https://doi.org/10.1007/s00018-016-2179-0