Fig. 4: Local deregulation of EV-loaded miR-126a-5p in NMJs may directly regulate TDP-43 expression.
From: Muscle-derived miR-126 regulates TDP-43 axonal local synthesis and NMJ integrity in ALS models
a, Volcano plot of small RNA-seq from muscle-derived EVs and whole-cell lysates of muscle cultures. Data are shown as log2FC of EV miRNAs over whole-cell lysates. Blue dots indicate miRNA with log2FC > 0.5 and −log10Padj > 2. n = 3 cultures/isolated EVs. Wald test with correction for multiple comparisons. b, Quantitative TaqMan RT–PCR for miR-126a-5p in whole-cell muscle lysates versus muscle-derived EVs. n = 3 cultures/isolated EVs. **P = 0.0073. c, Representative images of miR-126a-5p FISH in NMJs of EDL muscles. Scale bar, 10 µm. d, TaqMan RT–qPCR analysis of miR-126a-5p levels in GFP-infected and miR-126-infected primary MNs. U6 was used as loading control. n = 3 neuronal cultures. ****P = 0.000014. e, Top, scheme of TDP-43 transcript variant 3 mRNA with marking of the approximate location of two miR-126a-5p binding sites. Bottom, RT–qPCR for mmu-Tardbp transcript variant 3 in primary MNs infected with either empty GFP backbone or miR126–GFP vector. n = 3 neuronal cultures. **P = 0.0046. f,g, Scheme and quantitative analysis (g) of dual-luciferase assay for miR-126a-5p and mmu-Tardbp mRNA interaction. One hundred base pairs (bp) of the 3’ UTR of mmu-Tardbp mRNA, including one miR-126a-5p (wt) or a mutant miR-126a-5p (mut), were inserted into dual-luciferase reporter plasmid. n = 9 replicates from three repeats. ****P = 2.46 × 10−7. h, Representative images and quantification of western blots for TDP-43 (43 kD) in primary MNs infected with GFP or with miR126–GFP vector. Tubulin (55 kD) was used as loading control. n = 4 neuronal cultures. *P = 0.0258. i, TaqMan RT–qPCR analysis for miR-126-5p in serum-derived EVs of patients with sALS (n = 7) and healthy controls (n = 5). ****P = 0.000019. j,k, TaqMan RT–qPCR for miR-126-5p in P60 SOD1G93A GC muscles and P60 SOD1G93A SC (j) and P357 SOD1G37R GC muscles and P357 SOD1G37R SC (k). U6 was used as loading control. n = 3 (SC), n = 3 (j; GC) and n = 6–8 (k; GC) mice in each group. One-tailed (j; GC) or two-tailed (j,k) unpaired t-test, *P (j) < 0.0491, NS (j) = 0.6973, ***P (k) < 0.0004, NS (k) = 0.9591. For b,d,e,h–k, data are shown as the mean ± s.e.m. For g, data are shown as the mean ± s.d. For b, two-tailed paired Student’s t-test. For d,e,g,h,i,j (SC),k, two-tailed unpaired Student’s t-test. For j (GC), one-tailed unpaired Student’s t-test. For c, images represent eight repeats in mouse muscles. FC, fold change; CDS, coding sequence; GC, gastrocnemius; mut, mutant; NS, not significant; Padj, adjusted P value; SC, spinal cord; wt, wild-type.
