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Fig. 5 | Journal of Neuroinflammation

Fig. 5

From: Microbial BMAA elicits mitochondrial dysfunction, innate immunity activation, and Alzheimer’s disease features in cortical neurons

Fig. 5

Bacterial effectors disrupt mitochondrial network and expose cardiolipin. In primary mouse cortical neurons pre-treated with 1 μM CCCP, 3 mM BMAA, and 1 μg/μl LPS for 48 h, we evaluated a representative images of mitochondrial network immunostained with TOM20. Respective histograms of mitochondrial footprint, mitochondrial network branches, and mitochondrial branch length were calculated using Mitochondrial Network Analysis (MiNA) toolset, a relatively simple pair of macros making use of existing ImageJ plug-ins, allowing for semi-automated analysis of mitochondrial networks in cultured mammalian cells. MiNA is freely available at https://github.com/ScienceToolkit/MiNA. This MiNA toolset allows the analysis and quantification of various parameters of mitochondrial network namely mitochondrial footprint (distribution), network branching, and branch length. The panel corresponding to the calculated mitochondrial morphology shows mitochondrial network without the noise that characterizes fluorescent images. This filter is applied to a specified radius of two pixels and the pixel value is changed to the median of the surrounding pixels of that radius, removing noise from the image, allowing a clearer view of mitochondrial footprint. Values are mean ± SEM of four independent experiments and are expressed relative to untreated neurons. The statistical significance was determined using one-way ANOVA followed by Dunnett’s test *p < 0.05, **p < 0.01 versus untreated neurons. b Mitochondrial fission by detecting pDrp1 s616 fission protein by western blotting. Representative immunoblot and quantification analysis of pDrp1 s616 protein levels was performed. Equal protein amounts were loaded and confirmed with succinate dehydrogenase complex, subunit A (SDHA) staining. Data represent mean ± SEM values derived from four independent determinations. The statistical significance was determined using one-way ANOVA followed by Dunnett’s test *p < 0.05, when compared to untreated neurons and c cardiolipin exposure using the fluorescent dye 10-N-nonyl acridine orange (NAO), which is extensively used for location and quantitative assays of cardiolipin in living cells. Cardiolipin fluorescence intensity was calculated with ImageJ. Values are mean ± SEM of four independent experiments and are expressed relative to untreated neurons. The statistical significance was determined using one-way ANOVA followed by Dunnett’s test *p < 0.05, **p < 0.01 versus untreated neurons

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