Journal: Experimental & Molecular Medicine

Article Title: Astrocyte–neuron crosstalk through extracellular vesicle-shuttled miRNA-382-5p promotes traumatic brain injury

doi: 10.1038/s12276-024-01355-3

Figure Lengend Snippet: a Representative Western blot (WB) and image of Coomassie brilliant blue staining (loading control) of EVs isolated from the plasma of TBI patients and normal controls. b Representative electron micrograph of isolated EVs. Scale bar, 50 nm. c The size distribution and number of particles per milliliter in samples isolated from plasma-derived EVs are shown. d , e The concentration and size of plasma-derived EVs isolated under the experimental conditions are shown ( n = 6 per group; Student’s t test). f Volcano plots showing the differential expression of miRNAs in plasma EVs from TBI patients and healthy controls (TBI = 12, CON = 12; |log2FC| ≥ 1 and p < 0.05). g Related fold changes in the mRNA levels of miRNA-382-5p in plasma EVs between TBI patients and healthy controls ( n = 60 per group; Student’s t test). h Volcano plots showing the differential expression of miRNAs in the brain-derived EVs of mice in the TBI group compared with those in the sham group (TBI group=6, sham group=6; |log2FC | ≥1 and p < 0.05). i Venn diagram showing that miRNA-382-5p was the only significantly upregulated miRNA between the human and mouse groups. j , k Related fold changes in the expression of miRNA-382-5p and premiR-382 in each organ from mice in the sham and TBI groups ( n = 6 per group; Student’s t test). The data are presented as the means ± SDs; * p < 0.05, ** p < 0.01, *** p < 0.001, and NS not significant.

Article Snippet: Once the primary neurons reached 70% confluence, they were transfected with 100 nM miRNA-382-5p mimic (sense, 5’-GAAGUUGUUCGUGGUGGAUUCG-3’ and antisense, 5’-AAUCCACCACGAACAACUUCUU-3’) or miRNA mimic NC (sense, 5’-UUCUCCGAACGUGUCACGUTT-3’ and antisense, 5’-ACGUGACACGUUCGGAGAATT-3’) (GenePharma Inc., Shanghai, China) using Lipofectamine RNAiMAX (Thermo Fisher, #13778150) for 24 h, according to the manufacturer’s guidelines.

Techniques: Western Blot, Staining, Control, Isolation, Clinical Proteomics, Derivative Assay, Concentration Assay, Quantitative Proteomics, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Astrocyte–neuron crosstalk through extracellular vesicle-shuttled miRNA-382-5p promotes traumatic brain injury

doi: 10.1038/s12276-024-01355-3

Figure Lengend Snippet: a , b Relative expression levels of miRNA-382-5p and premiR-382 in neurons, microglia and astrocytes at 24 h after scratch injury ( n = 6 per group; Student’s t test). c Relative miRNA-382-5p expression levels among NDEVs, MDEVs, and ADEVs at 24 h after scratch injury ( n = 6 per group; Student’s t test). d Representative images of immunofluorescence staining of mouse brain sections obtained 2 weeks after the stereotactic injection of AAV-SMPD3-shRNA containing a GFAP-specific promoter. Scale bars, 50 µm. e qRT‒PCR analysis of the level of EV-derived miRNA-382-5p in the perilesional cortex and hippocampus of TBI mice with or without astrocyte-specific knockdown of SMPD3 ( n = 6 per group; one-way ANOVA). f qRT‒PCR analysis of the level of miRNA-382-5p in neurons isolated from TBI mice with or without astrocyte-specific knockdown of SMPD3 ( n = 6 per group; one-way ANOVA). g Translocation of the extracellular vesicle Cy3-miR-382-5p from astrocytes to primary neurons. Confocal imaging of primary neurons. h – k ADEVs promoted apoptosis ( h , i ), decreased cell viability ( j ), and increased lactate dehydrogenase (LDH) ( k ) release from scratched primary neurons ( n = 6 per group; one-way ANOVA). l Triton X-100/RNase treatment prevented the transfer of miRNA-382-5p from astrocytes into primary neurons ( n = 6 per group; one-way ANOVA). The data are presented as the means ± SDs; * p < 0.05, ** p < 0.01, *** p < 0.001, and NS not significant.

Article Snippet: Once the primary neurons reached 70% confluence, they were transfected with 100 nM miRNA-382-5p mimic (sense, 5’-GAAGUUGUUCGUGGUGGAUUCG-3’ and antisense, 5’-AAUCCACCACGAACAACUUCUU-3’) or miRNA mimic NC (sense, 5’-UUCUCCGAACGUGUCACGUTT-3’ and antisense, 5’-ACGUGACACGUUCGGAGAATT-3’) (GenePharma Inc., Shanghai, China) using Lipofectamine RNAiMAX (Thermo Fisher, #13778150) for 24 h, according to the manufacturer’s guidelines.

Techniques: Expressing, Immunofluorescence, Staining, Injection, shRNA, Derivative Assay, Knockdown, Isolation, Translocation Assay, Imaging

Journal: Experimental & Molecular Medicine

Article Title: Astrocyte–neuron crosstalk through extracellular vesicle-shuttled miRNA-382-5p promotes traumatic brain injury

doi: 10.1038/s12276-024-01355-3

Figure Lengend Snippet: a Overlap of potential targets of miRNA-382-5p predicted by the TargetScan, miRWalk, miRDB, RNAhybrid, and miRanda databases. b Venn diagram of putative target genes of miRNA-382-5p overlapping with mitochondria-related genes. c Wild-type and mutant OPA1 3′-UTR reporter constructs. d Luciferase reporter assay results for HEK293T cells cotransfected with the indicated wild-type or mutant 3’-UTR constructs and the miRNA-382-5p mimic ( n = 6 per group; one-way ANOVA). e , f WB analysis and densitometric quantification of OPA1 levels in primary neurons transfected with or without the miRNA-382-5p mimic ( n = 6 per group; Student’s t test). g MitoTracker was used to label mitochondria in primary neurons, mitochondrial morphology was examined via fluorescence microscopy, and mitochondrial morphological features were quantified (aspect ratios) using ImageJ software. h The boxed area next to each micrograph shows an magnified version of the white square ( n = 6 per group, one-way ANOVA). i , j Representative images of MitoSOX fluorescence and quantitative comparison of mitochondria-derived ROS levels ( n = 6 per group; one-way ANOVA). k , l Representative images of fluorescence staining and quantitative comparison of JC-1 aggregates (red fluorescence) and JC-1 monomers (green fluorescence) in cultured primary neurons from different experimental groups ( n = 6 per group; one-way ANOVA). The data are presented as the means ± SDs; ** p < 0.01, *** p < 0.001, and NS not significant.

Article Snippet: Once the primary neurons reached 70% confluence, they were transfected with 100 nM miRNA-382-5p mimic (sense, 5’-GAAGUUGUUCGUGGUGGAUUCG-3’ and antisense, 5’-AAUCCACCACGAACAACUUCUU-3’) or miRNA mimic NC (sense, 5’-UUCUCCGAACGUGUCACGUTT-3’ and antisense, 5’-ACGUGACACGUUCGGAGAATT-3’) (GenePharma Inc., Shanghai, China) using Lipofectamine RNAiMAX (Thermo Fisher, #13778150) for 24 h, according to the manufacturer’s guidelines.

Techniques: Mutagenesis, Construct, Luciferase, Reporter Assay, Transfection, Fluorescence, Microscopy, Software, Comparison, Derivative Assay, Staining, Cell Culture