Fig. 4

BMAA treatment increases mitROS, decreases ΔΨmit, and impairs neuronal glycolysis rates. 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 mitochondrial ROS production using the fluorescent probe MitoPY1. MitoPY1 is selective for hydrogen peroxide of mitochondria in living cells. After baseline reading, neurons were exposed to 5 μM rotenone (mitochondrial complex I inhibitor). Bars depict maximum RFU after rotenone minus basal RFU reading. Data represent the mean ± SEM derived from six independent experiments and are expressed in relation to untreated neurons. The statistical significance was determined using one-way ANOVA followed by Dunnett’s test *p < 0.05 versus untreated neurons. b Mitochondrial membrane potential using the fluorescent cationic dye TMRM⁺. After baseline reading, neurons were exposed to 1 μM of CCCP plus 2.5 μM oligomycin to prevent reversal of ATP synthase. Bars depict maximum RFU after CCCP + oligomycin minus basal RFU reading. Data represent the mean ± SEM derived from six independent experiments and are expressed in relation to untreated neurons. The statistical significance was determined using one-way ANOVA followed by Dunnett’s test *p < 0.05 and ***p < 0.001 versus untreated neurons. c Extracellular acidification rate (ECAR) representative curve and respective histograms of glycolysis and glycolytic capacity were evaluated were evaluated in primary mouse cortical neurons pre-treated with 1 μM CCCP, 3 mM BMAA, and 1 μg/μl LPS for 48 h, using a Seahorse XF24 extracellular flux analyzer. Incubations were performed in quadruplicate and data are the mean values of such experiments. Data represent the mean ± SEM derived from four independent experiments and are expressed relative to untreated cells. The statistical significance was determined using one-way ANOVA followed by Dunnett’s test **p < 0.01 and ***p < 0.001 versus untreated neurons