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. 2023 Apr 29;12(9):1287. doi: 10.3390/cells12091287

Figure 7.

Figure 7

CRMP2 ablation using siRNA-augmented mitochondrial fragmentation and reduced mitochondrial motility in cultured cortical neurons (12–14 DIV) from WT mice. The results of representative experiments with cultured cortical neurons are shown. Neurons were isolated from P1 wild-type C57BL/6;C3H mice. In (A), phase-contrast bright-field image. In (B–D), typical immunochemistry staining is shown. (B), CRMP2 immunostaining. (C), visualization of mitochondria with mito-eYFP. (D), overlapped mitochondria and CRMP2 images. Arrows, neuron with successful transfection. Delivery of anti-CRMP2 siRNA (ACTCCTTCCTCGTG TACATTT) led to ablation of CRMP2 (B). In (E–G), mitochondrial morphology in cultured cortical neurons is shown. In (E), scramble siRNA did not change mitochondrial morphology. In (F), anti-CRMP2 siRNA resulted in an increased mitochondrial fission. In (G), a neuron with deleted CRMP2 and treated with 10 μM (S)-LCM for 7 days; mitochondria remained fragmented. Mito-eYFP was used to visualize mitochondria in live neurons. Mitochondrial morphology was assessed employing z-stacks of serial images and 3D reconstruction. In (H–J), representative kymographs illustrating mitochondrial motility are shown. Mitochondrial traffic was documented for 5 min at 37 °C. In (H), mitochondrial motility in a neuron treated with scramble siRNA. In (I), CRMP2 ablation with siRNA was accompanied by reduced mitochondrial traffic. In (J), a neuron with deleted CRMP2 and treated with 10 μM (S)-LCM for 7 days; mitochondrial traffic remained suppressed. In (K), the length of neuronal mitochondria in µm. In (L), fractions of moving organelles and mitochondria trafficking in retrograde and anterograde directions shown as percentage from total number of analyzed mitochondria. In (K,L), data are mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, N = 5 separate experiments with neurons from different platings. Green circles, neurons transfected with scramble siRNA; red circles, neurons transfected with anti-CRMP2 siRNA; blue circles, neurons transfected with anti-CRMP2 siRNA and treated with (S)-LCM. Data were analyzed by nonparametric Kruskal–Wallis ANOVA on ranks followed by Tukey test.