Fig. 6.
Mitochondrial transfer underlies the beneficial effects of astrocytes on calcium dynamics in neurons damaged by cisplatin. a. Neurons were treated with and without cisplatin for 24 h and co-cultured with astrocytes transfected with mito-mCherry (labeled mCh) for 17 h. The calcium response to 20 mM KCl was compared in cisplatin-treated neurons cultured without astrocytes (black bar), or with astrocytes separated into mCherry negative neurons (orange bar; do not contain astrocytic mitochondria) and mCherry positive neurons (blue bar; contain astrocyte-derived mitochondria). The mean increase in the F340:F380 ratio of Fura-2 fluorescence in response to 20 mM KCl was calculated as in Fig. 5. Data represents the mean ± SEM of 3 independent experiments with n = 24 neurons in total. One-way ANOVA, followed by Tukey’s post-hoc test: (**p < 0.01, ***p < 0.0001). b. Resting F340:F380 ratio of Fura-2 of neurons treated with cisplatin followed by co-culture with astrocytes transfected with mito-GFP and scr siRNA or Miro-1 siRNA. c. Mean F340:F380 ratio was calculated for each neuron for first 50 s and data represents the mean ± SEM of 3 independent experiments with n > 80 neurons for each group. Two-way ANOVA: cisplatin x transfected astrocyte interaction: ***p < 0.001 followed by Tukey’s post-hoc test: (***p < 0.001, ****p < 0.0001). d. Mean traces and SEM of the F340:F380 ratio in response to 20 mM KCl stimulus in neurons treated with and without cisplatin and co-cultured with astrocytes transfected with scrambled (Scr) or Miro-1 siRNA. The mean F340:F380 ratio KCl responses were normalized to baseline. e. The change in F340:F380 ratio between baseline and maximum peak value from the 20 mM KCl stimulus was calculated which correlated with [Ca2+]i increase in neurons. Mean increase in F340:F380 ratio ratio was calculated for each neuron for 3 s before 20 mM KCl and subtracted from mean F340:F380 ratio at the maximum response to 20 mM KCl. Data represents the mean ± SEM of 3 independent experiments with n > 80 neurons for each group. Two-way ANOVA: cisplatin x transfected astrocyte interaction: (****p < 0.0001), followed by Tukey’s post-hoc test (****p < 0.0001)