Mutant C. elegans mitofusin leads to selective removal of mtDNA heteroplasmic deletions across generations to maintain fitness

Abstract

Background Mitochondrial DNA (mtDNA) is present at high copy numbers in animal cells, and though characterized by a single haplotype in each individual due to maternal germline inheritance, deleterious mutations and intact mtDNA molecules frequently co-exist (heteroplasmy). A number of factors, such as replicative segregation, mitochondrial bottlenecks, and selection, may modulate the exitance of heteroplasmic mutations. Since such mutations may have pathological consequences, they likely survive and are inherited due to functional complementation via the intracellular mitochondrial network. Here, we hypothesized that compromised mitochondrial fusion would hamper such complementation, thereby affecting heteroplasmy inheritance. Results We assessed heteroplasmy levels in three Caenorhabditis elegans strains carrying different heteroplasmic mtDNA deletions (ΔmtDNA) in the background of mutant mitofusin (fzo-1). Animals displayed severe embryonic lethality and developmental delay. Strikingly, observed phenotypes were relieved during subsequent generations in association with complete loss of ΔmtDNA molecules. Moreover, deletion loss rates were negatively correlated with the size of mtDNA deletions, suggesting that mitochondrial fusion is essential and sensitive to the nature of the heteroplasmic mtDNA mutations. Introducing the ΔmtDNA into a fzo-1;pdr-1;+/ΔmtDNA (PARKIN ortholog) double mutant resulted in a skewed Mendelian progeny distribution, in contrast to the normal distribution in the fzo-1;+/ΔmtDNA mutant, and severely reduced brood size. Notably, the ΔmtDNA was lost across generations in association with improved phenotypes. Conclusions Taken together, our findings show that when mitochondrial fusion is compromised, deleterious heteroplasmic mutations cannot evade natural selection while inherited through generations. Moreover, our findings underline the importance of cross-talk between mitochondrial fusion and mitophagy in modulating the inheritance of mtDNA heteroplasmy.

Multiple distinct evolutionary mechanisms govern the dynamics of selfish mitochondrial genomes

“…30,32,36 Conversely, genes involved in mitochondrial fission and autophagy reportedly contribute to intra-organismal selection against mutant mtDNA. 34,36,37,39,[58][59][60] Given these findings, we propose that host genotypes and environmental factors, especially those linking physiological stress with mtDNA replication and turnover, will be important determinants of the cooperator-cheater dynamics described here and should be considered in future research.…”

“…One possible molecular basis is that some mutant genomes are more vulnerable to cheater-suppression mechanisms, namely mitochondrial autophagy, consistent with prior empirical work. 34,36,37,39,[58][59][60] Alternatively, the dynamics of mitochondrial fission and fusion may permit the diffusion of gene products throughout the mitochondrial network, resulting in the genetic complementation of a mutation by nearby wildtype genomes. In either case, such scenarios may enable a mutation to persist in a heteroplasmic state by remaining neutral or nearly neutral at the organismal level.…”

Gonadotropin-releasing hormone-like receptor 2 inversely regulates somatic proteostasis and reproduction in Caenorhabditis elegans

“…Mutant strains were outcrossed into our N2 strain ( n ≥ 3). Standard genetic crossing techniques were used to construct mutant strains and mutation were verify using single worm PCR (Phire Animal Tissue Direct PCR Kit, Thermo Scientific) as previously described ( Meshnik et al, 2022 ). Nematodes were cultured using standard techniques.…”

HLH-1 Modulates Muscle Proteostasis During Caenorhabditis elegans Larval Development

“…Cross-strains were generated using standard C. elegans procedures. Animals’ genotype was confirmed by single worm PCR using Phire Animal Tissue Direct PCR Kit (F-170L, Thermo Scientific) with primers that amplified the area of the mutation as in ( Meshnik et al, 2022 ). Primer sequences used in this study are listed in Supplementary Table S2 .…”

“…For cDNA synthesis, RNA was reverse-transcribed using the iScript TM cDNA Synthesis Kit (1708891, Bio-Rad). mRNA levels were measured using quantitative PCR, performed on a C1000 Thermal Cycler (Bio-Rad) with KAPA SYBRFAST qPCR Master Mix (KK4602, KAPA Biosystems), as in ( Meshnik et al, 2022 ). Relative transcript levels were determined by averaging the C T of triplicate values for the genes examined and normalizing those to C T values obtained for 18S rRNA of the same sample using the 2 −ΔΔCT T method ( Livak and Schmittgen, 2001 ).…”