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. 2022 Feb 14;9:820105. doi: 10.3389/fcell.2021.820105

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Copyright © 2022 Sainio, Rasila, Molchanova, Järvilehto, Torregrosa-Muñumer, Harjuhaahto, Pennonen, Huber, Herukka, Haapasalo, Zetterberg, Taira, Palmio, Ylikallio and Tyynismaa.

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Movement of mitochondria and lysosomes is increased in iPSC-MN axons lacking NFL. Mitochondria and lysosome movement along the axons of motor neurons reveals increased movement in PT and KO cells. (A) Mask of CTR mitochondria tracking video frame 1. Total axon mitochondrial movements by time-lapse imaging of entire micrographic fields (n = 15 fields from three independent differentiations). Scale bar 50 µm. (B) Organelle movement by kymographs of axonal segments (n = 9 micrograph frames from three independent differentiations, total 45 axon segments/cell line). Shown is an example kymograph with retrograde and anterograde movement, stationary mitochondria, and mitochondria with less than 0.2 µm movement during 60 s (Dyn. Pausing = Dynamic pausing). (C) Mitochondria total displacement/number of mitochondria is increased in axons lacking NFL (PT/KO1/KO2 vs. CTR p > .0173). (D) Average run length of a mitochondria normalized to the count of organelles per kymograph is increased in PT and KO1 (CTR vs. KO1 p = .0007, CTR vs. PT p = .0138). (E) Number of mitochondria per video (first frame) (CTR vs. KO1 p = .0013). (F) Lysosome total displacement/number of lysosomes is increased in PT and KO1 motor neuron axons (CTR vs. KO1 p < .0001 and CTR vs. PT p = .0003). (G) Average run length of a lysosome normalized to the count of lysosomes. (H) Number of lysosomes per video (first frame) (CTR vs. KO1 p = .0099). (I) Average velocity of a moving mitochondria. (J). Average velocity of a moving lysosomes. (K) Proportions of anterograde moving (Ant.), retrograde moving (Ret.), anterograde and retrograde combined (Tot.), dynamic pausing (DP.) and stationary (Stat.) organelles in kymographs. KO1 iPSC-MN have proportionally more anterograde moving mitochondria (CTR vs. KO1 p = .0029). Data shown as mean with SD. *p < .05, **p <.01, ***p < 0.001, ****p < .0001. One-way ANOVA followed with Dunnett’s multiple comparison post hoc test vs. CTR. ^# indicates abnormal karyotype.