Figure 1.

Figure 1.

Disruption of retrograde mitochondrial movement impacts mitochondrial distribution in neurons. A, Image of a 4 dpf TgBAC(neurod:egfp)^nl1 transgenic zebrafish larva expressing mitochondrially localized TagRFP (shown in magenta) mosaically in neurons. Regions imaged in B, D shown in white boxes. B, C, pLLg neurons have cytosolic GFP and a subset express TagRFP in mitochondria (arrows). In WT animals, mitochondria (magenta in B; white in B') fill the soma. C, In actr10^nl15 mutants, cell body mitochondrial area is reduced. D, E, Mitochondria accumulate in axon terminals of actr10^nl15 mutants. F, Quantification of cell body mitochondrial load (mitochondrial area/cell body area) at 4 dpf shows a reduction in actr10^nl15 and p150a/b mutants (ANOVA); 4 dpf, WT: 0.58 ± 0.04; actr10^nl15: 0.24 ± 0.04; p150a/b: 0.21 ± 0.05; 6 dpf, WT: 0.47 ± 0.04; actr10^nl15: 0.26 ± 0.05. G, Axon terminal mitochondrial load (mitochondrial area/axon terminal area) at 4 dpf is increased in actr10^nl15 and p150a/b mutants (ANOVA). Changes in mitochondrial load are also observed at 6 dpf in actr10^nl15 animals; 4 dpf – WT: 0.16 ± 0.03; actr10^nl15: 0.26 ± 0.03; p150a/b: 0.31 ± 0.03; 6 dpf, WT: 0.16 ± 0.03; actr10^nl15: 0.26 ± 0.04. H–N, shRNA-mediated knock-down of Actr10 in rat hippocampal neurons results in loss of cell body mitochondrial load. Images of hippocampal neuron cell bodies (H, I) stained for TOM20 with a cytoplasmic EGFP fill and either vector only (H) or actr10 shRNA#2 co-transfection (I). Arrows points to mitochondrial in axon terminals. J, K, Western blotting and quantification showing knock-down of Actr10 by the shRNAs tested in HEK cells (top, Actr10; bottom, GAPDH; **p < 0.01, ***p < 0.005; ANOVA; n = 3). L, Quantification of mitochondrial load in Actr10 knock-down hippocampal neurons (ANOVA). WT: 0.40 ± 0.01; actr10 shRNA2: 0.32 ± 0.01. Sample sizes (F, G, number of larvae; L, number of neurons) indicated on graph. Scale bars: 10 µm. All data are mean ± SEM.