Variant load of mitochondrial DNA in single human mesenchymal stem cells.

Heteroplasmic mitochondrial DNA (mtDNA) variants accumulate as humans age, particularly in the stem-cell compartments, and are an important contributor to age-related disease. Mitochondrial dysfunction has been observed in osteoporosis and somatic mtDNA pathogenic variants have been observed in animal models of osteoporosis. However, this has never been assessed in the relevant human tissue. Mesenchymal stem cells (MSCs) are the progenitors to many cells of the musculoskeletal system and are critical to skeletal tissues and bone vitality. Investigating mtDNA in MSCs could provide novel insights into the role of mitochondrial dysfunction in osteoporosis. To determine if this is possible, we investigated the landscape of somatic mtDNA variation in MSCs through a combination of fluorescence-activated cell sorting and single-cell next-generation sequencing. Our data show that somatic heteroplasmic variants are present in individual patient-derived MSCs, can reach high heteroplasmic fractions and have the potential to be pathogenic. The identification of somatic heteroplasmic variants in MSCs of patients highlights the potential for mitochondrial dysfunction to contribute to the pathogenesis of osteoporosis.

The rise and rise of mitochondrial DNA mutations.

How mitochondrial DNA mutations clonally expand in an individual cell is a question that has perplexed mitochondrial biologists for decades. A growing body of literature indicates that mitochondrial DNA mutations play a major role in ageing, metabolic diseases, neurodegenerative diseases, neuromuscular disorders and cancers. Importantly, this process of clonal expansion occurs for both inherited and somatic mitochondrial DNA mutations. To complicate matters further there are fundamental differences between mitochondrial DNA point mutations and deletions, and between mitotic and post-mitotic cells, that impact this pathogenic process. These differences, along with the challenges of investigating a longitudinal process occurring over decades in humans, have so far hindered progress towards understanding clonal expansion. Here we summarize our current understanding of the clonal expansion of mitochondrial DNA mutations in different tissues and highlight key unanswered questions. We then discuss the various existing biological models, along with their advantages and disadvantages. Finally, we explore what has been achieved with mathematical modelling so far and suggest future work to advance this important area of research.

The contribution of non-essential Schizosaccharomyces pombe genes to fitness in response to altered nutrient supply and target of rapamycin activity.

Nutrient fluctuations in the cellular environment promote changes in cell metabolism and growth to adapt cell proliferation accordingly. The target of rapamycin (TOR) signalling network plays a key role in the coordination of growth and cell proliferation with the nutrient environment and, importantly, nutrient limitation reduces TOR complex 1 (TORC1) signalling. We have performed global quantitative fitness profiling of the collection of Schizosaccharomyces pombe strains from which non-essential genes have been deleted. We identified genes that regulate fitness when cells are grown in a nutrient-rich environment compared with minimal environments, with varying nitrogen sources including ammonium, glutamate and proline. In addition, we have performed the first global screen for genes that regulate fitness when both TORC1 and TORC2 signalling is reduced by Torin1. Analysis of genes whose deletions altered fitness when nutrients were limited, or when TOR signalling was compromised, identified a large number of genes that regulate transmembrane transport, transcription and chromatin organization/regulation and vesicle-mediated transport. The ability to tolerate reduced TOR signalling placed demands upon a large number of biological processes including autophagy, mRNA metabolic processing and nucleocytoplasmic transport. Importantly, novel biological processes and all processes known to be regulated by TOR were identified in our screens. In addition, deletion of 62 genes conserved in humans gave rise to strong sensitivity or resistance to Torin1, and 29 of these 62 genes have novel links to TOR signalling. The identification of chromatin and transcriptional regulation, nutritional uptake and transport pathways in this powerful genetic model now paves the way for a molecular understanding of how cells adapt to the chronic and acute fluctuations in nutrient supply that all eukaryotes experience at some stage, and which is a key feature of cancer cells within solid tumours.

Pentose phosphate pathway function affects tolerance to the G-quadruplex binder TMPyP4.

G-quadruplexes form in guanine-rich regions of DNA and the presence of these structures at telomeres prevents the activity of telomerase in vitro. Ligands such as the cationic porphyrin TMPyP4 stabilise G-quadruplexes and are therefore under investigation for their potential use as anti-cancer drugs. In order to investigate the mechanism of action of TMPyP4 in vivo, we carried out a genome-wide screen in the budding yeast Saccharomyces cerevisiae. We found that deletion of key pentose phosphate pathway (PPP) genes increased the sensitivity of yeast to the presence of TMPyP4. The PPP plays an important role in the oxidative stress response and sensitivity to TMPyP4 also increased when genes involved in the oxidative stress response, CCS1 and YAP1, were deleted. For comparison we also report genome wide-screens using hydrogen peroxide, which causes oxidative stress, RHPS4, another G-quadruplex binder and hydroxyurea, an S phase poison. We found that a number of TMPyP4-sensitive strains are also sensitive to hydrogen peroxide in a genome-wide screen. Overall our results suggest that treatment with TMPyP4 results in light-dependent oxidative stress response in budding yeast, and that this, rather than G-quadruplex binding, is the major route to cytotoxicity. Our results have implications for the usefulness and mechanism of action of TMPyP4.

A senescent cell bystander effect: senescence-induced senescence.

Senescent cells produce and secrete various bioactive molecules including interleukins, growth factors, matrix-degrading enzymes and reactive oxygen species (ROS). Thus, it has been proposed that senescent cells can damage their local environment, and a stimulatory effect on tumour cell growth and invasiveness has been documented. However, it was unknown what effect, if any, senescent cells have on their normal, proliferation-competent counterparts. We show here that senescent cells induce a DNA damage response, characteristic for senescence, in neighbouring cells via gap junction-mediated cell-cell contact and processes involving ROS. Continuous exposure to senescent cells induced cell senescence in intact bystander fibroblasts. Hepatocytes bearing senescence markers clustered together in mice livers. Thus, senescent cells can induce a bystander effect, spreading senescence towards their neighbours in vitro and, possibly, in vivo.

Genome-wide analysis to identify pathways affecting telomere-initiated senescence in budding yeast.

In telomerase-deficient yeast cells, like equivalent mammalian cells, telomeres shorten over many generations until a period of senescence/crisis is reached. After this, a small fraction of cells can escape senescence, principally using recombination-dependent mechanisms. To investigate the pathways that affect entry into and recovery from telomere-driven senescence, we combined a gene deletion disrupting telomerase (est1Δ) with the systematic yeast deletion collection and measured senescence characteristics in high-throughput assays. As expected, the vast majority of gene deletions showed no strong effects on entry into/exit from senescence. However, around 200 gene deletions behaving similarly to a rad52Δest1Δ archetype (rad52Δ affects homologous recombination) accelerated entry into senescence, and such cells often could not recover growth. A smaller number of strains similar to a rif1Δest1Δ archetype (rif1Δ affects proteins that bind telomeres) accelerated entry into senescence but also accelerated recovery from senescence. Our genome-wide analysis identifies genes that affect entry into and/or exit from telomere-initiated senescence and will be of interest to those studying telomere biology, replicative senescence, cancer, and ageing. Our dataset is complementary to other high-throughput studies relevant to telomere biology, genetic stability, and DNA damage responses.

Quantitative assessment of markers for cell senescence.

Cellular senescence, the irreversible loss of replicative capacity, might be a tumour suppressor and a contributor to age-related loss of tissue function. The absence of quantitative tests for reliability of candidate markers for senescent cells is a major drawback in cell population studies. Fibroblasts in culture constitute mixed populations of proliferation-competent and senescent cells, with transition between these with increasing population doublings (PD). We estimated senescent fraction in human and mouse fibroblasts with high precision from easily observed growth curves using a dynamic simulation model. We also determined senescent fractions, at various PD (over a wide range of senescent cell frequencies) using candidate senescence markers: Ki67, p21 (CDKN1A), γH2AX, SAHF and Sen-ß-Gal either alone or in combination, and compared with those derived from growth curves. This comparison allowed ranking of candidate markers. High rankings were obtained for Sen-ß-Gal, SAHFs and the combination of Ki67 negativity with high (>5 per nucleus) γH2A.X foci density in MRC5 fibroblasts. We demonstrate that this latter marker combination, which can easily be performed in paraffin-embedded tissue, gives quantitative senescent cell frequency estimates in mouse embryonic fibroblast cultures and in mouse intestinal sections. The technique presented is a framework for quantitative assessment of markers for senescence.