High content image analysis reveals function of miR-124 upstream of Vimentin in regulating motor neuron mitochondria

Abstract

microRNAs (miRNAs) are critical for neuronal function and their dysregulation is repeatedly observed in neurodegenerative diseases. Here, we implemented high content image analysis for investigating the impact of several miRNAs in mouse primary motor neurons. This survey directed our attention to the neuron-specific miR-124, which controls axonal morphology. By performing next generation sequencing analysis and molecular studies, we characterized novel roles for miR-124 in control of mitochondria localization and function. We further demonstrated that the intermediate filament Vimentin is a key target of miR-124 in this system. Our data establishes a new pathway for control of mitochondria function in motor neurons, revealing the value of a neuron-specific miRNA gene as a mechanism for the re-shaping of otherwise ubiquitously-expressed intermediate filament network, upstream of mitochondria activity and cellular metabolism.

Figure 1

High content image analysis reveals the impact of miR-124 on primary motor neuron morphology. (a) Values for nine individual miRNAs, all transfected at 0.5 ng/µl, to mouse primary motor neurons. miRNA expression levels displayed on the Y-axis as 40 minus qPCR cycle threshold (40-Ct), on a Log₂ scale. All miRNAs were significantly overexpressed. (b) A diagram describing the method: Spinal motor neurons were isolated from E13.5 mouse embryos and seeded on a 384 multiwell plate. Culture was transfected with different miRNA mimics using Bravo automated liquid handling robot. 72 hrs later, cells were fixed, stained with anti Tuj1 antibody and DAPI. Two fluorescent micrographs were captured per well (ImageXpress Micro and MetaXpress2 software, Molecular Devices). (c) Cell numbers (Cell), neurite outgrowth per cell (outgrowth) and number of branches per cell (branches), were quantified with serial doses of the stress-inducing agent, Sodium Arsenite (15, 30 and 60 µM, for 60 minutes). See Methods and Sup. Figure 2. (d) None of the nine miRNAs tested influenced cell numbers. miR-124 was the only miRNA to reduce mean axonal outgrowth per cell and mean number of branches per cell. 500 Tuj1+ neurons quantified per field, 2 fields/well and 6 wells per treatment in five independent experimental repeats. Data collected from >30,000 Tuj1+ neurons per treatment. Averages $\pm$ SEM, Student’s t-test. *P-value < 0.05.

Figure 2

Unbiased bioinformatics analysis of miR-124 targets. Next generation sequencing was performed on RNA extracted from primary motor neurons transfected with either miR-124 mimics (N = 3) or control mimics (N = 6). (a) A dendrogram depicting the hierarchical clustering of Pearson correlation coefficients for global gene expression from miR-124 overexpressing motor neurons (blue) and controls (black). (b) Enrichment landscape plot for all 876 7mer motifs complementary to canonical mouse miRNA seed regions, gained by Sylamer analysis. Sorted 6500 mRNAs expressed in primary motor neurons, ranked from down- to up-regulated after overexpressing of miR-124, or control mimic. Assessment of over- and under-represented miRNA recognition sequences (seed-matches) for all known miRNAs, identified two enriched motifs, both matching the miR-124 ‘seed’ sequence (blue, 7mer–2; light blue, 7mer-1A). (c) Top seven terms (by gene count) from gene ontology (GO) analysis on all mRNAs significantly up- or down- regulated following miR-124 overexpression (corrected P-value < 0.05), using database for annotation, visualization and integrated discovery (DAVID) software. This analysis revealed an enrichment for mitochondrial-related genes and is further described in Table 1.

Figure 3

miR-124 overexpression impacts mitochondrial localization and function. (a) Confocal fluorescent micrographs of primary motor neurons, 72 hrs post transfection with scrambled control or miR-124 mimics, stained with MitoTracker Deep Red FM (green) and TMRE (red) to depict functional mitochondria. Merged signal (yellow) relative to nuclei (blue, Hoechst), scale bars, 20 μm. Oligomycin A (1 µM) or miR-124 overexpression diminished mitochondrial activity. Bar graphs quantification of fluorescent signal intensity in (b) axons and (c) soma. Quantification of 3 random positions per compartment; 9 neurons per condition; Data collected from three replicates and the study performed in > four independent experimental repeats. Averages ± SEM, one way ANOVA with post hock Neuman-Keuls. *P-value < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Vimentin regulates neuron morphology and mitochondria function (a) Venn diagram of predicted miR-124 targets (TargetScan) and transcripts that were experimentally repressed >2 fold by miR-124 overexpression in primary motor neurons, relative to control conditions. (b) qPCR analysis of the three mRNAs that obeyed both criterions: Vim, Ptbp1 and Mdk, in primary motor neurons transfected with miR-124 oligos (N = 3), relative to controls (N = 3). (c) miR-124 recognition element at Vim 3′UTR is conserved in vertebrate species. Knockdown of Vim by shRNA lentiviruses downregulated (d) Vim mRNA and (e) VIM protein expression, relative to non-targeting shRNA controls. A full view of the Western blot is in Sup. Figure 6. (f) High content image analysis of cell numbers, mean axonal outgrowth per cell and mean number of branches per cell. 100 Tuj1+ neurons quantified per field, 8 fields/well, 6 replicates per treatment. Data collected from >24,000 Tuj1+ neurons per treatment. (g) Confocal fluorescent micrographs of primary motor neurons, 72 hrs. post transduction with scrambled-shRNA or Vim-shRNA, stained with TMRE (red) and nuclei (blue, Hoechst). Transduction efficacy >80%. Scale bars, 20 μm. Bar graphs quantification of fluorescent signal intensity in (h) axons and (i) soma. Quantification of 3 random positions per compartment; 9 neurons per condition. Data from three replicates and the study performed in three independent experimental repeats. All graphs present averages $\pm$ SEM, Student’s t-test. *P-value < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

miR-124 regulates mitochondria activity via Vimentin. Primary motor neurons of control, miR-124 overexpression alone or transduced in addition with lentiviral vector for Doxycyclin-dependent expression of Vim (>80% transduction efficacy, transduction 24 hrs. before transfection of miRNA mimics), without or with the chemical inducer (Dox). (a) Vim mRNA and (b) VIM protein expression without or with Dox. Three experimental repeats, Averages $\pm$ SEM, Student’s t-test. A full view of the Western blot is in Sup. Figure 6. (c) Confocal fluorescent micrographs of primary motor neurons, stained with MitoTracker (green) and TMRE (red), merged signal (yellow) and nuclei (blue, Hoechst). Scale bars, 20 μm. Bar graphs quantification of fluorescent signal intensity in (d) axons and (e) soma. Quantification of three random positions per compartment; 9 neurons per condition; Data from three replicates and the study performed in three independent experimental repeats. Averages ± SEM, ANOVA + Duncan’s new multiple range test (MRT) *P-value < 0.05, **p < 0.01, ***p < 0.001. (f) Snapshot from mitochondria live imaging along axons of primary motor neurons. Bar graphs quantification of (g) axonal mitochondria density/μm and (h) mean TMRE intensity in control axons (n = 48), miR-124 overexpression alone (n = 52), miR-124 and Vim without Dox (n = 34) or with Dox (n = 34). Averages $\pm$ SEM, Student’s t-test, P-value *p < 0.05, **p < 0.01.

Figure 6

miR-124 and Vimentin co-regulate mitochondria motility in primary motor neurons. Primary motor neurons of control, miR-124 overexpression alone or transduced in addition with lentiviral vector for Doxycycline-dependent expression of Vim (>80% transduction efficacy, performed 24 hrs. before transfection of miRNA mimics), without or with the chemical inducer (Dox). (a) Representative captures from mitochondria live imaging and motor axons kymographs, depicting anterograde movement. Green arrows indicate the movement of one representative mitochondrion. Running mitochondria were defined by moving a distance of >10 µm at average speed >0.2 µm/sec. Paused mitochondria are a subpopulation of the running mitochondria, which paused in the same location for ≥3 frames in succession. Proportion of pausing and continuously running mitochondria in (b) anterograde or (c) retrograde directions. Horizontal scale bar − 10 µm. Vertical scale bar − 60 seconds. Test of proportion statistics, *p < 0.05, **p < 0.01. (d) Anterograde or (e) retrograde measurement of pause duration (sec). Averages $\pm$ SEM, Student’s t-test, P-value *p < 0.05.