Fig. 6: NMJ function is dependent on mitochondria and local synthesis, and their absence leads to decreased NMJ activity and axon degeneration. | Nature Communications

Fig. 6: NMJ function is dependent on mitochondria and local synthesis, and their absence leads to decreased NMJ activity and axon degeneration.

From: Axonal TDP-43 condensates drive neuromuscular junction disruption through inhibition of local synthesis of nuclear encoded mitochondrial proteins

Fig. 6

a Schematic illustration of Mito-Killer-Red (MKR) experimental setup, used for specifically targeting oxidative stress to NMJ mitochondria. b Images of MKR in NMJ pre-synapse before and after bleach (white line=bleached region). Lower panel indicates post-synaptic muscle labeled with calcium indicator Oregon-Green-BAPTA (OGB). Scale bar=10 µm. n = 13,12 NMJs from 3 independent experiments. c, d Representative OGB time-trace of OGB indicating muscle contraction (c) and quantification (d) of contraction ratio before and after MKR bleaching. As control, ChAT::tdTomato-expressing axons were bleached instead of MKR-expressing axons. n = 13,12 NMJs from 3 independent experiments. Data are presented as mean values ± SD. Unpaired-t-test, two-sided. ****p < 0.0001. e Schematic illustration of experimental procedure for puromycin local protein synthesis inhibition in NMJ pre-synapse using puromycin resistant muscles. f Left panel: Time-series images of OGB-labeled co-cultures treated, or not with puromycin in NMJ compartment. Right panel: Demonstration of paired axon-muscle calcium activity only in control NMJs and its absence upon puromycin application. Scale bar=20 µm. n = 7,9 MFCs from 3 independent experiments. g Time traces of OGB in pre-synaptic neurons and post-synaptic muscles in control (upper plot), and in puromycin-treated (lower plot) cultures. h Quantification of the percent of innervated and contracting muscles after puromycin application. n = 7,9 MFCs from 3 independent experiments. Data are presented as mean values ± SD. Unpaired-t-test, two-sided. ****p < 0.0001. i Images and j quantification of the percent of degenerating axons in TDP∆NLS and control MN cultures following 16 and 24 h of puromycin treatment. Scale bar=50 µm. n = 4,3 MFCs from 3 independent experiments. Data are presented as mean values ± SD. Two-way-ANOVA with Holm-Sidak correction. *p = 0.0407. a.u stands for arbitrary units. Source data are provided as a Source Data file.

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