Functional characterization of two variants in the mitochondrial topoisomerase gene TOP1MT that impact regulation of the mitochondrial genome

TOP1MT encodes a mitochondrial topoisomerase that is important for mtDNA regulation, and is involved in mitochondrial replication, transcription and translation. Two variants predicted to affect TOP1MT function (R199C and V338L) were identified by exome sequencing of a newborn with hypertrophic cardiomyopathy. As no pathogenic TOP1MT variants have been confirmed previously, we characterized these variants for their ability to rescue several TOP1MT functions in knockout cells. Consistent with a role for these TOP1MT variants contributing to the patient phenotype, comprehensive characterization suggests that both variants had impaired topoisomerase activity and demonstrates that neither variant was able to restore steady state levels of mitochondrial encoded proteins, nor reduced oxidative phosphorylation. However, the two variants behaved differently in some respects. While the R199C variant was better at restoring transcript levels, the V338L variant was able to restore mtDNA copy number and replication. These findings suggest that the different TOP1MT variants affect distinct TOP1MT functions. Altogether, these findings begin to provide insight into the many roles that TOP1MT plays in the maintenance and expression of the mitochondrial genome, and how impairments in this important protein may lead to human pathology.

IL-6 Reduces Mitochondrial Replication, and IL-6 Receptors Reduce Chronic Inflammation in NAFLD and Type 2 Diabetes

Interleukin (IL)-6 family cytokines act through a receptor complex with gp130 subunits. IL-6 is a pleiotropic cytokine that regulates inflammation and liver regeneration. Mitochondria are the first to respond to stress and adapt their dynamics in conditions of damage. In this regard, the study aimed to investigate the role of the IL-6 cytokine family (sIL-6Ra, gp130/sIL-6Rb, and IL-11) in the regulation of mitochondrial dynamics in the liver in obese patients and to assess the contribution of these cytokines to the pathogenesis of type 2 diabetes mellitus (T2DM). We studied 134 obese patients with and without T2DM and 41 healthy donors. We found that increasing the concentration of sIL-6Ra and gp130/sIL-6Rb protected against carbohydrate disorders in obese patients and prevented non-alcoholic fatty liver disease (NAFLD) progression in obese patients. An increase in plasma IL-6 levels is associated with decreased, mitochondrial transcription factor A (TFAM) protein production in liver biopsies in obese patients with and without T2DM. Replication, transcription, and division processes in liver biopsy were reduced in patients with T2DM. Inflammatory processes stimulate liver cell apoptosis in obese patients with T2DM. The increase in IL-11 levels is associated with decreased pro-apoptotic Bcl-2-associated X protein (BAX) protein production in obese patients with and without T2DM.

Disentangling the intertwined roles of mutation, selection and drift in the mitochondrial genome

Understanding and quantifying the rates of change in the mitochondrial genome is a major component of many areas of biological inquiry, from phylogenetics to human health. A critical parameter in understanding rates of change is estimating the mitochondrial mutation rate (mtDNA MR). Although the first direct estimates of mtDNA MRs were reported almost 20 years ago, the number of estimates has not grown markedly since that time. This is largely owing to the challenges associated with time- and labour-intensive mutation accumulation (MA) experiments. But even MA experiments do not solve a major problem with estimating mtDNA MRs—the challenge of disentangling the role of mutation from other evolutionary forces acting within the cell. Now that it is widely understood that any newly generated mutant allele in the mitochondria will initially be at very low frequency (1/ N , where N is the number of mtDNA molecules in the cell), the importance of understanding the effective population size ( N e ) of the mtDNA and the size of genetic bottlenecks during gametogenesis and development has come into the spotlight. In addition to these factors regulating the role of genetic drift, advances in our understanding of mitochondrial replication and turnover allow us to more easily envision how natural selection within the cell might favour or purge mutations in multi-copy organellar genomes. Here, we review the unique features of the mitochondrial genome that pose a challenge for accurate MR estimation and discuss ways to overcome those challenges. Estimates of mtDNA MRs remain one of the most widely used parameters in biology, thus accurate quantification and a deeper understanding of how and why they may vary within and between individuals, populations and species is an important goal. This article is part of the theme issue ‘Linking the mitochondrial genotype to phenotype: a complex endeavour’.

An Ecologist’s Guide to Mitochondrial DNA Mutations and Senescence

Longevity plays a key role in the fitness of organisms, so understanding the processes that underlie variance in senescence has long been a focus of ecologists and evolutionary biologists. For decades, the performance and ultimate decline of mitochondria have been implicated in the demise of somatic tissue, but exactly why mitochondrial function declines as individual’s age has remained elusive. A possible source of decline that has been of intense debate is mutations to the mitochondrial DNA. There are two primary sources of such mutations: oxidative damage, which is widely discussed by ecologists interested in aging, and mitochondrial replication error, which is less familiar to most ecologists. The goal of this review is to introduce ecologists and evolutionary biologists to the concept of mitochondrial replication error and to review the current status of research on the relative importance of replication error in senescence. We conclude by detailing some of the gaps in our knowledge that currently make it difficult to deduce the relative importance of replication error in wild populations and encourage organismal biologists to consider this variable both when interpreting their results and as viable measure to include in their studies.

PrimPol is required for the maintenance of efficient nuclear and mitochondrial DNA replication in human cells

Eukaryotic Primase-Polymerase (PrimPol) is an enzyme that maintains efficient DNA duplication by repriming replication restart downstream of replicase stalling lesions and structures. To elucidate the cellular requirements for PrimPol in human cells, we generated PrimPol-deleted cell lines and show that it plays key roles in maintaining active replication in both the nucleus and mitochondrion, even in the absence of exogenous damage. Human cells lacking PrimPol exhibit delayed recovery after UV-C damage and increased mutation rates, micronuclei and sister chromatin exchanges but are not sensitive to genotoxins. PrimPol is also required during mitochondrial replication, with PrimPol-deficient cells having increased mtDNA copy number but displaying a significant decrease in replication rates. Deletion of PrimPol in XPV cells, lacking functional Pol Eta, causes an increase in DNA damage sensitivity and pronounced fork stalling after UV-C treatment. We show that, unlike canonical TLS polymerases, PrimPol is important for allowing active replication to proceed, even in the absence of exogenous damage, thus preventing the accumulation of excessive fork stalling and genetic mutations. Together, these findings highlight the importance of PrimPol for maintaining efficient DNA replication in unperturbed cells and its complementary roles, with Pol Eta, in damage tolerance in human cells.

Mitochondrial DNA replication in mammalian cells: overview of the pathway

Mammalian mitochondria contain multiple copies of a circular, double-stranded DNA genome and a dedicated DNA replication machinery is required for its maintenance. Many disease-causing mutations affect mitochondrial replication factors and a detailed understanding of the replication process may help to explain the pathogenic mechanisms underlying a number of mitochondrial diseases. We here give a brief overview of DNA replication in mammalian mitochondria, describing our current understanding of this process and some unanswered questions remaining.