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. 2021 Sep 22;10:e69795. doi: 10.7554/eLife.69795

Figure 1. The zebrafish NTR-MTZ chemo-genetic DA neuron ablation model suffers from mitochondrial dysfunction that can be counteracted by PD-associated mitochondrial quality control gene activity.

(A) Confocal images of ventral forebrain DA neurons in 0.2% DMSO control and 9 mM MTZ-treated 6 days post fertilization (dpf) transgenic larval zebrafish brains show significant difference in normalized fluorescent intensity (n = 10; p < 0.05, unpaired t test). The red fluorescence in the eyes is due to pigment-derived autofluorescence. (B) Long-range PCR of mitochondrial DNA versus nuclear DNA products using ventral forebrain DA neurons from control and MTZ-treated 6 dpf larval zebrafish brains anterior to the mid-hindbrain boundary (4.5 mM, 8 hrs) (n = 4 pools of 25 larval brains per pool; p < 0.01, unpaired t test). (C) Live confocal imaging of mitochondrial dynamics with mitochondria-targeted DsRed and mitochondria-targeted photoactivatable GFP in 5dpf larvae treated with 0.2% DMSO (control) or 4.5 mM MTZ for 16 hr. Arrowheads point to the elongated appearance of mitochondria in DA axons of MTZ-treated animals. (D–H) Analysis of mitochondrial dynamics including total mitochondrial count, length, % moving, velocity, and direction of movement between control and MTZ-treated samples (n = 8–10; **p < 0.01, ****p < 0.0001, unpaired t test). (I–M) Overexpression of PD-associated human genes including PARK2, PARK6, PARK7, PARK1, and associated mutant forms. mRNAs were microinjected into one-cell stage transgenic embryos and treated with 4.5 or 9 mM MTZ at 30hpf for 24 hrs to determine the neuroprotective effect of experimental conditions compared to control GFP-encoding mRNA injection (n = 10–12; *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test).

Figure 1—source data 1. Dopamine neuron intensity quantification and statistical tests.
DMSO Control vs 9 mM MTZ. Human Parkin overexpression. Human PINK1 overexpression. Human DJ1 overexpression. Human alpha synuclein overexpression. Human alpha synuclein A53T overexpression.
Figure 1—source data 2. Labeled mitochondrial DNA damage gel.
Figure 1—source data 3. Labeled nuclear DNA damage gel.
Figure 1—source data 4. Raw mitochondria DNA damage gel.
Figure 1—source data 5. Raw nuclear DNA damage gel.

Figure 1.

Figure 1—video 1. Movies showing mitochondrial dynamics in DA neuronal axons.
Download video file (1.9MB, mp4)
DA neuronal axons with mitochondria-targeted DsRed and mitochondria-targeted photoactivatable GFP were visualized and recorded under a confocal microscope in 5 dpf larvae treated with 0.2% DMSO (control, Figure 1—video 1) or 4.5 mM MTZ (Figure 1—video 2) for 16 hr.
Figure 1—video 2. Movies showing mitochondrial dynamics in DA neuronal axons.
Download video file (719KB, mp4)
DA neuronal axons with mitochondria-targeted DsRed and mitochondria-targeted photoactivatable GFP were visualized and recorded under a confocal microscope in 5 dpf larvae treated with 0.2% DMSO (control, Figure 1—video 1) or 4.5 mM MTZ (Figure 1—video 2) for 16 hr.