s opa1  (Addgene inc)

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: Neuron-specific OPA1 knockout mice were generated by crossing Opa1 f/f mice (Cyagen, USA, Inc., CA, USA) with Map2-CreERT2 mice (Shanghai Model Center) and then treating the offspring with 75 mg/kg tamoxifen (#S1238, Selleck) for 5 days.

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

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 Cartoon illustrating the process used to construct OPA1 conditional knockout mice. b Results showing the identification of the neuron-specific OPA1 conditional knockout mice. c TEM images showing cortical mitochondrial crista remodeling in the Opa1 f/f group or Opa1 CKO group subjected to sham surgery or TBI. d Fifty randomly selected mitochondria per sample were scored in three categories ( n = 6 per group, one-way ANOVA): more than four cristae (Class I), between two and three cristae (Class II), and one or no cristae (Class III) per mitochondrion. e Fifty mitochondria per sample were scored according to matrix density and swelling ( n = 6 per group, one-way ANOVA): dense matrix (Class A) and swollen mitochondria with a hypodense matrix (Class B). f The length of the mitochondria in each group, which is represented by the mean length of 50 randomly selected mitochondria ( n = 6 per group; one-way ANOVA). g , h Representative MR images and statistical analysis of histological impairments at 24 h after TBI ( n = 6 per group; Student’s t test). i Statistical analysis of brain edema. n = 6 per group; Student’ s t test) j – l The neurological function deficit scores of the Opa1 f/f group or Opa1 CKO group ( n = 12 per group, two-way ANOVA). The data are presented as the means ± SDs; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Neuron-specific OPA1 knockout mice were generated by crossing Opa1 f/f mice (Cyagen, USA, Inc., CA, USA) with Map2-CreERT2 mice (Shanghai Model Center) and then treating the offspring with 75 mg/kg tamoxifen (#S1238, Selleck) for 5 days.

Techniques: Construct, Knock-Out

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 WB analysis and densitometric quantification of the levels of the OPA1 protein in the perilesional cortex of mice injected with or without RVG-miR-382-5pi-EVs ( n = 6 per group; one-way ANOVA). c TEM images showing that the loss of mitochondrial cristae in the perilesional cortex was inhibited in the RVG-NC-EVs group and the RVG-miR-382-5pi-EVs group. d Fifty mitochondria per sample were assigned to three categories: more than four cristae (Class I), between two and three cristae (Class II), and one or no cristae (Class III) per mitochondrion ( n = 6 per group, one-way ANOVA). e Fifty mitochondria per sample were scored according to matrix density and swelling: dense matrix (Class A) and swollen mitochondria with a hypodense matrix (Class B) ( n = 6 per group, one-way ANOVA). f Representative quantitative results of the length of 50 mitochondria per experiment ( n = 6 per group; one-way ANOVA). g , h Representative MR images and statistical analysis of histological impairments at 24 h after TBI ( n = 6 per group; one-way ANOVA). i Statistical analysis of brain edema ( n = 6 per group, one-way ANOVA). j‒l Neurological function deficit scores of the RVG-NC-EVs group and RVG-miR-382-5pi-EVs group ( n = 12 per group, two-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: Neuron-specific OPA1 knockout mice were generated by crossing Opa1 f/f mice (Cyagen, USA, Inc., CA, USA) with Map2-CreERT2 mice (Shanghai Model Center) and then treating the offspring with 75 mg/kg tamoxifen (#S1238, Selleck) for 5 days.

Techniques: Injection