OxPhos Dysfunction Causes Hypermetabolism and Reduces Lifespan in Cells and in Patients with Mitochondrial Diseases

Patients with primary mitochondrial diseases present with fatigue and multi-system disease, are often lean, and die prematurely, but the mechanistic basis for this clinical picture remains unclear. Integrating data from 17 cohorts of patients with mitochondrial diseases (n=690), we find that clinical mitochondrial disorders increase resting energy expenditure, a state termed hypermetabolism. In a longitudinal cellular model of primary patient-derived fibroblasts from multiple donors, we show that genetic and pharmacological disruptions of oxidative phosphorylation (OxPhos) similarly trigger increased energy consumption in a cell-autonomous manner, despite near-normal OxPhos coupling efficiency. Hypermetabolism was associated with mtDNA instability, activation of the integrated stress response, increased extracellular secretion of age-related cytokines and metabokines including GDF15, as well as an accelerated rate of telomere erosion and epigenetic aging, and a reduced Hayflick limit. Finally, we generate a longitudinal RNASeq and DNA methylation resource dataset, which reveals conserved, energetically demanding, genome-wide recalibrations to OxPhos dysfunction. Hypermetabolism, or the increased energetic cost of living in mitochondrial diseases, has important biological and clinical implications.

The Role of Outer-Membrane Vesicles in Intercellular Communication in Pseudomonas Aeruginosa

<p>Gram-negative bacteria produce outer-membrane vesicles (OMVs) that have biological roles ranging from biofilm formation, modulation of host-cell interactions & delivery of virulence factors. Several studies have shown a role for OMVs to act as intracellular signals to co-ordinate the behaviour of bacteria. This study showed OMVs generated at sub-lethal ciprofloxacin concentrations were capable of programming naïve P. aeruginosa cultures resulting in premature entry into stationary-phase and a significantly lower final culture density reached after 14 hrs. Pyoverdine production was also initiated after 6 hrs in cultures treated with OMVs. Heat-inactivation of OMVs failed to impede OMV-mediated growth inhibition & pyoverdine production. Chloroform-disruption of OMVs prevented OMV-mediated growth inhibition but did not inhibit OMV-induced pyoverdine production. It is likely that these effects are mediated by multiple signals as opposed to a single mechanism. This suggests that a protein is not responsible for OMV-mediated growth inhibition and an intact OMV lipid bilayer is required. Induction of pyoverdine production is likely due to a lipid (such as a homo-serine lactone) or small molecule present within OMVs. Preincubation with OMVs for 2-4 hrs resulted in a substantial decrease in the final culture density from cultures that were exposed to OMVs during the course of growth. This suggests that OMV fusion is capable of programming naïve bacteria to set a predetermined division limit on subsequent daughter cells. We coin this as the ‘Dayflick’ limit due to the similarities of the Hayflick limit in eukaryotic cells. This shows that OMVs act as intercellular messaging vehicles between bacteria that communicate and program naïve bacteria to adapt to the environment under which they were generated in, aiding survival in harsh environments. Further study is needed to determine what OMV components are responsible for initiating these responses and to determine how long the programming is stable.</p>

The Role of Outer-Membrane Vesicles in Intercellular Communication in Pseudomonas Aeruginosa

<p>Gram-negative bacteria produce outer-membrane vesicles (OMVs) that have biological roles ranging from biofilm formation, modulation of host-cell interactions & delivery of virulence factors. Several studies have shown a role for OMVs to act as intracellular signals to co-ordinate the behaviour of bacteria. This study showed OMVs generated at sub-lethal ciprofloxacin concentrations were capable of programming naïve P. aeruginosa cultures resulting in premature entry into stationary-phase and a significantly lower final culture density reached after 14 hrs. Pyoverdine production was also initiated after 6 hrs in cultures treated with OMVs. Heat-inactivation of OMVs failed to impede OMV-mediated growth inhibition & pyoverdine production. Chloroform-disruption of OMVs prevented OMV-mediated growth inhibition but did not inhibit OMV-induced pyoverdine production. It is likely that these effects are mediated by multiple signals as opposed to a single mechanism. This suggests that a protein is not responsible for OMV-mediated growth inhibition and an intact OMV lipid bilayer is required. Induction of pyoverdine production is likely due to a lipid (such as a homo-serine lactone) or small molecule present within OMVs. Preincubation with OMVs for 2-4 hrs resulted in a substantial decrease in the final culture density from cultures that were exposed to OMVs during the course of growth. This suggests that OMV fusion is capable of programming naïve bacteria to set a predetermined division limit on subsequent daughter cells. We coin this as the ‘Dayflick’ limit due to the similarities of the Hayflick limit in eukaryotic cells. This shows that OMVs act as intercellular messaging vehicles between bacteria that communicate and program naïve bacteria to adapt to the environment under which they were generated in, aiding survival in harsh environments. Further study is needed to determine what OMV components are responsible for initiating these responses and to determine how long the programming is stable.</p>

TINF2 is a haploinsufficient tumor suppressor that limits telomere length

Telomere shortening is a presumed tumor suppressor pathway that imposes a proliferative barrier (the Hayflick limit) during tumorigenesis. This model predicts that excessively long somatic telomeres predispose to cancer. Here, we describe cancer-prone families with two unique TINF2 mutations that truncate TIN2, a shelterin subunit that controls telomere length. Patient lymphocyte telomeres were unusually long. We show that the truncated TIN2 proteins do not localize to telomeres, suggesting that the mutations create loss-of-function alleles. Heterozygous knock-in of the mutations or deletion of one copy of TINF2 resulted in excessive telomere elongation in clonal lines, indicating that TINF2 is haploinsufficient for telomere length control. In contrast, telomere protection and genome stability were maintained in all heterozygous clones. The data establish that the TINF2 truncations predispose to a tumor syndrome. We conclude that TINF2 acts as a haploinsufficient tumor suppressor that limits telomere length to ensure a timely Hayflick limit.

Telomerase Inhibition for Killing Cancer Cells

There is an upper limit to the number of times a cell can divide before it stops growing and enters a phase which is called senescence. For human embryonic diploid fibroblasts, the Hayflick limit is about 60 doublings. This limit is related to the end-replication problem faced by DNA polymerase as it moves along a template strand synthesizing a new strand. DNA polymerase must initiate synthesis from an RNA primer and it synthesizes a new strand beginning with the primers 3 end. The need for the RNA primer means that the DNA polymerase cannot replicate the very 5 end of the lagging strand because the primer occupies the last few bases of the template strand. As a consequence, the DNA of a chromosome shortens by 50-100 nucleotides each time the chromosome replicates. Eukaryotic chromosomes have telomeres at their ends, which are long sections made up of a repeat sequences, TTAGGG in human cells. These sequences protect the ends of the chromosomes, thus preventing the chromosomes from interacting with each other. Telomeres are eroded with each division until they no longer function properly, with catastrophic results for the cell.

Roles of Telomere Biology in Cell Senescence, Replicative and Chronological Ageing

Telomeres with G-rich repetitive DNA and particular proteins as special heterochromatin structures at the termini of eukaryotic chromosomes are tightly maintained to safeguard genetic integrity and functionality. Telomerase as a specialized reverse transcriptase uses its intrinsic RNA template to lengthen telomeric G-rich strand in yeast and human cells. Cells sense telomere length shortening and respond with cell cycle arrest at a certain size of telomeres referring to the “Hayflick limit.” In addition to regulating the cell replicative senescence, telomere biology plays a fundamental role in regulating the chronological post-mitotic cell ageing. In this review, we summarize the current understandings of telomere regulation of cell replicative and chronological ageing in the pioneer model system Saccharomyces cerevisiae and provide an overview on telomere regulation of animal lifespans. We focus on the mechanisms of survivals by telomere elongation, DNA damage response and environmental factors in the absence of telomerase maintenance of telomeres in the yeast and mammals.

Human Lung Epithelial Cells Divide >200 Population Doublings without Engaging a Telomere Maintenance Mechanism

The “Hayflick limit” is a “mitotic clock” and primary cells have a finite lifespan that correlates with telomere length. However, introduction of the telomerase catalytic protein component (TERT) is insufficient to immortalize most, but not all, human cell types under typical cell culture conditions. Originally, telomerase activity was only detected in cancer cells but is now recognized as being detectable in transit amplifying cells in tissues undergoing regeneration or in extreme conditions of wound repair. Here we report thatin vitrolow stress culture conditions allow normal human lung basal epithelial cells to grow for over 200 population doublings without engaging any telomere maintenance mechanism. This suggests that most reported instances of telomere-based replicative senescence are due to cell culture stress-induced premature senescence.One Sentence SummaryHuman lung cells growing in reduced stress conditions can divide well beyond the Hayflick limit.

The Hayflick Limit and Age-Related Adaptive Immune Deficiency

The adaptive immune system (AIS) acquires significant deficiency during chronological ageing, making older individuals more susceptible to infections and less responsive to vaccines compared to younger individuals. At the cellular level, one of the most striking features of this ageing-related immune deficiency is the dramatic loss of T-cell diversity that occurs in elderly humans. After the age of 70 years, there is a sharp decline in the diversity of naïve T cells, including a >10-fold decrease in the CD4+ compartment and a >100-fold decrease in the CD8+ compartment. Such changes are detrimental because the AIS relies on a diverse naïve T-cell pool to respond to novel pathogens. Recent work suggests that this collapse of naïve T-cell diversity results from T cells reaching the Hayflick limit and being eliminated through both antigen-dependent and -independent pathways. The progressive attrition of telomeres is the molecular mechanism that underlies this Hayflick limit. Therefore, we propose that by measuring the telomere lengths of T cells with high resolution, it is possible to develop a unique biomarker of immune deficiency, potentially much better correlated with individual susceptibility to diseases compared to chronological age alone.