Generation, screening and validation of mcu^−/− zebrafish. (A) CRISPR/Cas9-based gene editing was used to generate mcu^−/− zebrafish. Schematics showing the targeted genomic sequence for the introduction of the indel mutation in the exon 3 containing the mcu coding gene. The identified 18 base pair deletion (shown in faded font) and 70 bp insertion (shown in highlighted font) is denoted in the cDNA sequence. (B) The indel mutation leads to a frameshift mutation with a premature stop codon. The predicted protein product for mcu mutant allele is shown in the lower panel. (C) RT-PCR data showing wt mcu allele in the first column; the second column shows the wt and mutant allele in heterozygous mutants and the third column shows the mcu mutant allele in mcu homozygous zebrafish. (D) q-PCR data show significant downregulation (***P<0.0001) of mcu gene expression in mcu^−/− zebrafish when compared to wt, indicating mutation-induced RNA decay. Statistical analysis with t-test of three different experiments with n=3. (E) Representative images showing in situ hybridization using mcu riboprobe. mcu expression is abolished in mcu^−/− 3 dpf zebrafish. (F) Impaired mitochondrial calcium ions influx/efflux in isolated mitochondria from 24 hpf larvae in mcu^−/− zebrafish. Control mitochondria (green line) had an uptake in calcium added to solution, which created a drop in the fluorescence. After ∼5 min, we observed calcium leakage (rise in fluorescence). Blank control (black line) was mitochondria without added calcium. Ruthenium Red (pink line) prevented calcium uptake. CCCP (yellow line) caused leakage of calcium, observed as rising fluorescence. Uptake in mcu^−/− (blue line) was completely blocked in given circumstances, being like the dynamics of Ruthenium Red treatment.

Mitochondrial calcium homeostasisis altered in pink1^−/− zebrafish. (A–C) Tg(HuC:GCaMP5G) line with AP localization (A,B) and calcium efflux (C). (A) Lateral view of zebrafish head. Confocal image from GCaMP5G is merged with bright field view image. Scale bar: 100 μm. (B) Dorsal view of AP localization. In A and B, AP is highlighted with a magenta box. Scale bar: 200 μm. (C) The process of calcium efflux in AP neurons before (C1) and after (C2) CCCP treatment. Brighter color represents higher calcium concentration. Scale bar: 10 μm. (D) Fluorescence ratio of calcium released from mitochondria to the cytoplasm to the basal calcium in the cytoplasm, presented as individual single-measured neurons. (E) The same ratios, but presented as individual fish (average from nine neurons). The statistical significance (P-value) of two groups of values was calculated using a two-tailed, two-sample unequal variance t-test calculated in GraphPad Prism 5. *P<0.05, ****P<0.001. Horizontal bars are means with s.e.m. (wt: n=13 fish, n=117 neurons; pink1^−/−: n=15 fish, n=134 neurons). (F) Calcium ions influx/efflux in isolated mitochondria from 24 hpf larvae. On the timeline we can compare the dynamics of calcium influx/efflux in wt, pink1^−/− and (pink1; mcu)^−/− mutants. Uptake in pink1^−/− was lower than in wt, whereas in (pink1; mcu)^−/−was completely blocked in given circumstances. Results are mean with s.e.m. [wt had 11, pink1^−/− had 13, mcu^−/− had three and (pink1; mcu)^−/− had four independent replications] gathered during six independent experiments. Every replication was done on 200 larvae per variant.

Dopaminergic neurons are rescuedafter deleting mcu in pink1^−/− zebrafish. (A–D) Representative images of wt (A), pink1^−/− (B), mcu^−/− (C) and (pink1; mcu)^−/− (D) 3 dpf larvae after WISH using TH-specific riboprobe. (E–F) Representative images of wt (E), pink1^−/− (F), mcu^−/− (G) and (pink1; mcu)^−/− (H) 3 dpf larvae after whole-mount FISH using TH-specific riboprobe and TSA/Cy3-based signal amplification. There was a significant decrease (P<0.001) in number of dopaminergic neurons in pink1^−/− zebrafish when compared to wt. In (pink1; mcu)^−/− zebrafish there was a significant increase (P<0.05) in number of dopaminergic neurons when compared to pink1^−/− zebrafish. (I–L) Representative images of wt (I), pink1^−/− (J), mcu^−/− (K) and (pink1; mcu)^−/− (L) 3 dpf larvae after immunohistochemistry using TH-specific antibody. Arrowheads show absence of dopaminergic neurons. (M) Graphical representation of chromogenic WISH. There was a significant decrease (**P<0.01) in number of dopaminergic neurons in pink1^−/− zebrafish when compared to wt. In (pink1; mcu)^−/− zebrafish, there was a significant increase (***P<0.001) in number of dopaminergic neurons when compared to pink1^−/− zebrafish. (N) Graphical representation of immunohistochemistry. There was a significant decrease (***P<0.001) in number of dopaminergic neurons in pink1^−/− zebrafish when compared to wt. In (pink1; mcu)^−/− zebrafish there was a significant increase (***P<0.001) in number dopaminergic neurons when compared to pink1^−/− zebrafish. The mean number of diencephalic dopaminergic neurons for wt, pink1^−/−, mcu^−/− and (pink1; mcu)^−/− was calculated over three independent experiments (n=10 embryos per genotype and experiment). Scale bars: 100 μm.

Mitochondrial membrane potential partly restored after mcu deletion. The graph represents ratio of JC-1 dye emission spectrum (590 nm to 530 nm). wt mitochondria showed a slight uptake of JC-1, which turned into leakage after ∼18 min after the start of the experiment, when mitochondria began to lose membrane integrity in an in vitro environment. pink1^−/− mitochondria did not show uptake, instead we observed JC-1 leakage starting at ∼6 min after experiment initialization. Both variants started and ended at similar levels. Results are mean with s.e.m. [wt had two, pink1^−/− had two and pink1; mcu)^−/− had one independent replication(s)] gathered during two independent experiments. Every replication was done on 200 larvae per variant.

Mitochondrial dynamics is alteredin pink1^−/− zebrafish. (A–L) Representative images of immunohistochemistry performed on wt, pink1^−/−, mcu^−/− and (pink1; mcu)^−/− dissected 4 dpf zebrafish larval brain. DA neurons (red) are marked with anti-TH antibody, mitochondrial structures (green) are marked with anti-Tom20 antibody and DAPI (blue) is shown as nuclear stain. Arrowheads show mitochondrial morphology in muscle fibers. (M–O) Representative images of mitochondrial volumetric analysis in muscle fibers of transgenic wt, pink1^−/− and (pink1; mcu)^−/− 3 dpf zebrafish expressing mitochondria-localized GFP. Arrowheads point out mitochondrial structures in somites. (P) Graphical representation and statistical analysis of mitochondrial area with one-way ANOVA and post-hoc analysis using Tukey’s multiple comparison test of two different experiments with n=15. There was a significant (*P>0.05) decrease in DA neuronal mitochondrial area in pink1^−/− zebrafish when compared to wt; mitochondria area was restored (**P>0.01) in (pink1; mcu)^−/− zebrafish. (Q,R) Graphical representation and statistical analyses of muscle mitochondrial volume and sphericity with one-way ANOVA and post-hoc analysis using Tukey’s multiple comparison test of three different experiments with n=30. There was a slight decrease in mitochondrial volume in pink1^−/− zebrafish when compared to wt. The cumulative sphericity index is significantly (***P>0.001) reduced in pink1^−/− zebrafish, unlike wt and (pink1; mcu)^−/− zebrafish.

mcu inactivation renders neuroprotection against MPTP. (A) Representative images of MPTP-untreated wt (a), pink1^−/− (c), (pink1; mcu)^−/− (e), mcu^−/− (g) and MPTP-treated wt (b), pink1^−/− (d), (pink1; mcu)^−/− (f) and mcu^−/− (h) 3 dpf larvae after WISH using TH-specific riboprobe. MPTP-treated wt (b) and pink1^−/− (d) zebrafish larvae were most susceptible to MPTP toxicity while MPTP-untreated (pink1; mcu)^−/− (e) and mcu^−/− (g) were most resistant to MPTP toxicity. (B) Graphical representation of chromogenic WISH. There was a significant decrease (***P<0.001) in number of dopaminergic neurons in pink1^−/− zebrafish treated with MPTP when compared to untreated pink1^−/− zebrafish. MPTP-treated (pink1; mcu)^−/− zebrafish showed a significant increase (***P<0.001) in number of dopaminergic neurons when compared to MPTP-treated pink1^−/− zebrafish. Statistical analysis with one-way anova and post hoc analysis using Tukey's multiple comparison test of two different experiments with n=20. (C) Kaplan–Meier survival curves depicting survival rate for 5 days of wt, pink1^−/−, mcu^−/− and (pink1; mcu)^−/− zebrafish treated with 25 µg ml-1 MPTP (n=100). Scale bars:100 µm.