Multiple Molecular Mechanisms Rescue mtDNA Disease in C. elegans

Abstract

Genetic instability of the mitochondrial genome (mtDNA) plays an important role in human aging and disease. Thus far, it has proven difficult to develop successful treatment strategies for diseases that are caused by mtDNA instability. To address this issue, we developed a model of mtDNA disease in the nematode C. elegans, an animal model that can rapidly be screened for genes and biological pathways that reduce mitochondrial pathology. These worms recapitulate all the major hallmarks of mtDNA disease in humans, including increased mtDNA instability, loss of respiration, reduced neuromuscular function, and a shortened lifespan. We found that these phenotypes could be rescued by intervening in numerous biological pathways, including IGF-1/insulin signaling, mitophagy, and the mitochondrial unfolded protein response, suggesting that it may be possible to ameliorate mtDNA disease through multiple molecular mechanisms.

Keywords: IGF-1/insulin signaling; RNAi; mitochondrial DNA depletion; mitochondrial disease; mitochondrial genome; mitochondrial unfolded protein response; mitophagy; mutation; neuromuscular dysfunction; polymerase gamma.

Figure 1. Characterization of the polg-1(srh1) Worms

(A) The exonuclease (green) domain of DNA polymerase γ contains three highly conserved regions (red) that control the fidelity of DNA synthesis, including an aspartic acid in exonuclease domain II that is essential for the proofreading activity of polymerase γ across the tree of life. We used CRISPR/Cas9 technology to mutate this residue to alanine in the error-prone allele polg-1(srh1). (B) The mtDNA mutation frequency of the polg-1(srh1) worms is >70-fold higher compared to WT worms. (C) The mutation spectrum of WT and polg-1(srh1) depicted as peaks above and below the WT nucleotide according to standard electrophoretogram color coding (red, T; blue, C; black, G; green, A). The percentage of each type of mutation is listed by the peaks. (D) The mtDNA copy number is reduced by 56% in the polg-1(srh1) worms. (E) The basal respiration of polg-1(srh1) worms worsens with age compared to WT worms. (F) The mitochondria of polg-1(srh1) worms display reduced reserve capacity upon FCCP treatment at 5 days of age. (G and H) Confocal images of day 10 polg-1(srh1) and WT worms (G) do not reveal a significant difference in oxidation stress (H). (I–K) Neuromuscular function assessed by chemotaxis (I), thrashing (J), and a gentle touch assay (K) reveals increased dysfunction in polg-1(srh1) worms compared to WT animals. (L) The polg-1(srh1) worms have a median lifespan of 13 days compared to 16 days for WT worms (log-rank test p < 0.01). Data for WT and polg-1(srh1) worms are in blue and pink, respectively (in B, D–F, and H–L). Bar graphs represent the mean ± SEM of at least three biological replicates, and the lifespan assay was performed using at least 100 worms per genotype. Unpaired t tests were performed to determine significance (*p < 0.05, **p < 0.01; ns, no significant difference). Also see Figure S1 and Movies S1 and S2.

Figure 2. The Phenotype of the polg-1(srh1) Worms Worsens from Generation to Generation

(A) The outer circle represents the worm mitochondrial genome highlighting the protein-coding genes (green), rDNA (blue), AT-rich region (yellow), and all the mutations (red) that arose over 35 generations of maintaining the polg-1(srh1) mutator allele. The inner circle represents the regions that were sequenced (blue). (B) mtDNA mutations were tracked over 35 generations in WT and polg-1(srh1) worms. At generations 5, 15, 25, and 35, ~11 kb of mtDNA of 3 progenies of a single WT and polg-1(srh1) worm were sequenced. The mutations depicted here were present in all 3 progenies, indicating that they were successfully transmitted through the germline. Three mutations, here denoted in purple, brown, and orange polg-1(srh1) were present across multiple generations and showed substantial genetic drift. No new mutations arose in the WT strain. (C–E) Neuromuscular dysfunction was assessed by chemotaxis (C), thrashing (D), and gentle touch (E) revealed progressive dysfunction with increasing generation in the polg-1(srh1) worms. (F) Representative electrophoretograms tracking the T2085 deletion that results in an early stop codon in the ND1 gene in both the parent with polg-1(srh1) allele and its resultant progeny where the mutator allele has been mated out. Three individuals without the polg-1(srh1) allele in the F1 generation, 10 individuals in the F2 generations, and 21 individuals in the F3 generation were isolated and tested for the transmission of T2085 heteroplasmy in a background WT for polg-1. Bar graphs represent the mean ± SEM of at least three biological replicates. Unpaired t tests were performed to determine significance (*p < 0.05, **p < 0.01; ns, no significant difference). Also see Figure S2.

Figure 3. An RNAi Screen Identified Multiple Genes That Control mtDNA Disease in the polg-1(srh1) Worms

(A and B) 20–35 WT (A) and polg-1(srh1) (B) worms were aged for 5 days and spotted inside the red circle. A chemo-attractant was spotted 5 cm away from the worms (green circle), and the worms were allowed to crawl toward the chemo-attractant for 1 hr. Over this time span, most WT worms reached the chemo-attractant, while the polg-1(srh1) worms performed significantly worse. (C) Twenty 5-day-old polg-1(srh1) adults on RNAi against age-1 perform similar to WT worms. (D) A list of genes that rescued the polg-1(srh1) neuromuscular defect by >30% when suppressed by RNAi. See also Figure S3 and Table S1.

Figure 4. Verification of the RNAi Screen with Genetic Mutants and Small Molecules

(A) The daf-2(e1370) allele rescues the neuromuscular defect of 5-day-old polg-1(srh1) worms. (B) The daf-16(mu86) allele worsens the neuromuscular defect of 5-day-old polg-1(srh1) worms. This set of experiments was performed at generation 7 instead of generation 30, when the phenotype of the mutator worms is not significantly different from the WT worms until 7 days of age. Accordingly, the detrimental effect of the daf-16(mu86) allele is better illustrated. (C) The daf-2(e1370) allele rescues the basal respiration rate of 10-day-old polg-1(srh1) worms. (D and E) The daf-2(e1370) allele (D) increases mtDNA copy number by 118%, but (E) has no effect on the mutation frequency of polg-1(srh1) worms. (F) The pdr-1(gk448) allele rescues the chemotaxis defect of 5-day-old polg-1(srh1) worms. (G) Induction of mitopagy with 50 µM urolithin A (UA) worsens the chemotaxis phenotype of the 5-day-old polg-1(srh1) worms. (H) The pdr-1(gk448) allele rescues the basal respiration rate of 10-day-old polg-1(srh1) worms. (I and J) The pdr-1(gk448) allele (I) increases mtDNA copy number by 25% and (J) results in a 5.5-fold increase in the mutation frequency of polg-1(srh1) worms. (K and L) The atfs-1(et15) allele (K) rescues the chemotaxis defect of 5-day-old polg-1(srh1) worms and (L) the reserve capacity of 5-day-old polg-1(srh1) worms. (M and N) The atfs-1(et15) allele (M) increases mtDNA copy number by 63%, but (N) has no effect on the mutation frequency of polg-1(srh1) worms. Bar graphs represent the mean ± SEM of at least three biological replicates. Unpaired t tests were performed to determine significance (*p < 0.05, **p < 0.01; ns, no significant difference).