Cellular ATP Demand and the Creation of Metabolically Distinct Mitochondrial Subpopulations

Source: Nature (November 6th, 2024) – Most authors work at  Memorial Sloan Kettering Cancer Center, New York City. Link: https://www.nature.com/articles/s41586-024-08146-w

Author list: Keun Woo Ryu, Tak Shun Fung, Daphne C. Baker, Michelle Saoi, Jinsung Park, Christopher A. Febres-Aldana, Rania G. Aly, Ruobing Cui, Anurag Sharma, Yi Fu, Olivia L. Jones, Xin Cai, H. Amalia Pasolli, Justin R. Cross, Charles M. Rudin & Craig B. Thompson.

Introduction: The Multifunctional Role of Mitochondria

Mitochondria play a majior role in cellular bioenergetic, but their capabilities extend beyond that. These organelles can use surplus substrates to generate macromolecular precursors, such as amino acids, which are essential for supporting cell growth and maintaining physiological functions.

In addition to oxidative phosphorylation (OXPHOS)—the process of ATP production through oxidative mechanisms—mitochondria can also participate in reductive biosynthesis. Notably, this includes the production of:

• Proline: A building block for proteins like collagen, which contributes to skin healing, joint and tendon function, and immunity.
• Ornithine: A non-proteinogenic amino acid involved in the urea cycle, which converts ammonia into urea in the liver, mitochondria, and cytoplasm.

While both reductive  (biosynthetic) and oxidative (bioenergetic) functions of mitochondria are well understood individually, how these processes are coordinated under bioenergetic and nutrient stress remains unclear. The study addresses this gap by exploring how distinct mitochondrial subpopulations arise and function under varying conditions.

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Experimental Setup and Key Findings

To investigate mitochondrial behavior, the researchers cultured cells under different conditions, including nutrient-rich (serum, galactose) and nutrient-starved environments. Their experiments focused on the enzyme Pyrroline-5-carboxylate synthase (P5CS), which plays a crucial role in the synthesis of proline and ornithine.

Formation of Mitochondrial Subpopulations

As bioenergetic demand increased, the researchers observed that P5CS progressively formed filamentous clusters. These clusters contributed to the emergence of two distinct mitochondrial subpopulations through repeated cycles of mitochondrial fusion and fission:

1. ATP synthase-enriched mitochondria
2. Mitochondria containing filamentous P5CS

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Functional Specialization of the Subpopulations

Each subpopulation exhibited distinct structural and metabolic characteristics:

P5CS-Containing Mitochondria

• These mitochondria support reductive biosynthesis.
• They maintain electron transport chain (ETC) activity and show increased membrane potential, despite lacking cristae structures.
• They are largely devoid of ATP synthase, and the membrane potential is used up by biosynthetic reactions.

ATP Synthase-Enriched Mitochondria

• These mitochondria are optimized for oxidative phosphorylation (OXPHOS).
• They feature highly ordered cristae and are freed from competition for reducing equivalents.
• This specialization increases their capacity for efficient ATP production.

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Reversibility and Implications

An interesting  aspect is the reversibility of mitochondrial subpopulation specialization. When bioenergetic stress decreases, the distinct roles of the subpopulations can revert to a more uniform state. It is intriguing how mitochondrial fusion and fission enable metabolic adaptability within cells.

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Conclusion: A Step Toward Understanding Mitochondrial Coordination

The findings highlight the capability of mitochondria to form metabolically distinct subpopulations tailored to specific cellular demands. The division of labor—between reductive biosynthesis and oxidative phosphorylation—provides insights into how cells manage metabolic challenges. Additionally, the study highlights another important function of mitochondrial dynamics (fusion and fission).

Further research into this phenomenon could enhance our understanding of mitochondrial function under stress and its implications for cellular health and disease.

Updating the Free Radical Theory of Aging

https://www.frontiersin.org/articles/10.3389/fcell.2020.575645/full 

Adam S. Ziada, Marie-Soleil R. Smith and Hélène C. F. Côté.

INTRODUCTION - TRANSITION AND TRASNVERSION MUTATIONS

Transversions are point mutations in which a purine (A or G) is changed for a pyrimidine (T or C) or vice-versa. Transitions are point mutations that change a purine for another purine or a pyrimidine for another pyrimidine.

Although there are twice as many possible transversions as transitions, the latter are more common (approximately 2/3 of point mutations are transitions).

A POLYMERASE γ - CENTRIC THEORY OF MITOCHONDRIAL AGEING

The free radical theory of aging hypothesizes that oxidative damage to the mtDNA induces random de novo mtDNA mutations which gradually accumulate over time, potentially reaching pathological levels. The authors summarise recent studies have showing that transition mtDNA mutations  rather than transversion mutations  gradually build up overtime and are amplified, via clonal expansion, to pathological levels. 

Given that transition mutations are generally associated with replication errors made by the mitochondrial polymerase γ, the age associated accumulation of mtDNA mutations could result from free radicals interacting with polymerase γ, potentially reducing its fidelity and/or inhibiting mtDNA replication. This would in turn lead to random de novo transition mutations and their subsequent clonal amplification. Conditions hypothesized to induce accelerated aging via oxidative damage/stress could include chronic infections such as HIV, chronic inflammatory conditions, or tobacco smoking

The authors conclude suggesting the possibility that free radicals, rather than directly contributing to mtDNA mutations via oxidative lesions, affect the mitochondrial polymerase and decrease its fidelity, indirectly increasing somatic transition mutations. 

Metformin Improves Mitochondrial Respiratory Activity through Activation of AMPK

https://www.sciencedirect.com/science/article/pii/S2211124719312677?via%3Dihub

Wang Y., An H., Liu T., Qin C., Sesaki H., Guo S., Radovick S., Hussain M., Maheshwari A., Wondisford F. E. , O'Rourke B., He L.

• Metformin is the first-line medication for the treatment of type 2 diabetes (T2D), particularly in people who are overweight. It is estimated that around 150 million people around the world. 
• Metformin works mainly by improving patients' hyperglycemia (suppressing liver's glucose production) and alleviating insulin resistance. However, its mechanisms of actions are currently not understood.
• It is known that mitochondrial dysfunctions are involved in the development of T2D and that patients with T2D have decreased mitochondrial copy number and respiration.
• The author show that therapeutic doses of metformin increase mito respiration, ATP level and membrane potential and promote mitochondrial fission in liver cells. Through knock-out studies, they determine that the enzyme AMPK is required for metformin to be effective.
• The author also report that very high concentration of the drug can lead to a stop of respiration, by depleting cellular ADP levels. Respiration was restored through the addition of exogenous ADP.

Mitochondria as multifaceted regulators of cell death

https://www.nature.com/articles/s41580-019-0173-8

Florian J. Bock, Stephen W. G. Tait

INTRODUCTION

It might look paradoxical that mitochondria are central to life as well as to cell death. However, programmed cell death is essential for health. The authors discuss the roles of mitochondria in cell death and their implications for health and disease. Here, I summarise the information about the involvement of mitochondria in apoptosis and other, recently described, forms of cell death.

1. The role of mitochondria is well established in apoptosis, where mitochondrial outer membrane permeabilization (MOMP) initiates a signalling cascade that leads to cell death. Recently, it has been appreciated that there are non-lethal functions of MOMP, triggering inflammation and immune response. See this blog post for more detail and a reference.
2. Necroptosis is a programmed form of cell death that shares morphological and inflammatory characteristics with necrosis, an unregulated and passive form of cell death due to disease, injury, or failure of the blood supply. Mitochondria are involved at least in some cell types: levels of ROS may be an important determinant as to whether a cell initiates necroptosis. Therefore, progressive mitochondrial dysfunction, like that observed during ageing, may increase the propensity of cells to undergo necroptosis. It has been observed, however, that necroptosis can proceed independently of mitochondria.
3. Pyroptosis is a highly inflammatory form of programmed cell death. It occurs most frequently upon infection with intracellular pathogens and is probably part of the antimicrobial response. There is little evidence that mitochondria play an important role in pyroptosis, but there is extensive crosstalk exists between pyroptosis and mitochondrial apoptosis.
4. Ferroptosis is a type of regulated cell death  triggered by lipid peroxides that kill the cell by attacking lipid membranes. It is dependent on iron (hence the name) and ROS (hence the mitochondrial involvement) in that lipid peroxides are produced through the Fenton reaction, requiring iron and peroxides.  Ferroptosis is characterized morphologically by morphological aberration of mitochondria.

Even though the role of mitochondria in 2-4 appears less crucial, or at least context dependent, these different cell death modalities crosstalk with one another and this crosstalk involves mitochondria.

Individual cristae within the same mitochondrion display different membrane potentials and are functionally independent

https://www.embopress.org/doi/10.15252/embj.2018101056

Dane M Wolf, Mayuko Segawa, Arun Kumar Kondadi, Ruchika Anand, Sean T Bailey, Andreas S Reichert, Alexander M van der Bliek, David B Shackelford, Marc Liesa, Orian S Shirihai

• It is often supposed that the inner mitochondrial membrane is at a uniform membrane potential (ΔΨm). 
• The authors develop an approach to evaluate ΔΨm at the level of individual cristae.
• The authors find the existence of heterogeneity in ΔΨm throughout the inner mitochondrial membrane, with individual cristae possessing different membrane potentials.
• Interventions causing acute depolarization to a particular crista may leave other cristae unchanged in their membrane potential.
• In other words, individual cristae seen to act as independent bioenergetic units, so that the failure of a specific one does not spread to the entire mitochondrion. Therefore, mitochondria should be thought of not as  electric wires, but as sets of batteries.
• The loss of this cristae compartmentalization, causing the spread of damage among regions of a mitochondrion, may be implied in pathological states. Several diseases are associated with structural perturbations in cristae. Restoring the heterogeneity of ΔΨm could represent a therapeutic avenue. 
• A fascinating area of future investigation would be to link cristae membrane heterogeneity to mitochondrial genetics.

Chemoptogenetic damage to mitochondria causes rapid telomere dysfunction

https://www.pnas.org/content/early/2019/08/22/1910574116.long

Wei Qian, Namrata Kumar, Vera Roginskaya, Elise Fouquerel, Patricia L. Opresko, Sruti Shiva, Simon C. Watkins, Dmytro Kolodieznyi, Marcel P. Bruchez, and Bennett Van Houten

• The authors develop a chemoptogenetic technology to specifically induce mitochondrial reactive oxygen species with precise spatio-temporal control by using light stimulation.
• The authors show that induction of mitochondrial reactive oxygen species can result in increased hydrogen peroxide levels inside the nucleus, resulting in telomere loss.

Mitochondrially-targeted APOBEC1 is a potent mtDNA mutator affecting mitochondrial function and organismal fitness in Drosophila

https://www.nature.com/articles/s41467-019-10857-y

Simonetta Andreazza, Colby L. Samstag, Alvaro Sanchez-Martinez, Erika Fernandez-Vizarra, Aurora Gomez-Duran, Juliette J. Lee, Roberta Tufi, Michael J. Hipp, Elizabeth K. Schmidt, Thomas J. Nicholls, Payam A. Gammage, Patrick F. Chinnery, Michal Minczuk, Leo J. Pallanck, Scott R. Kennedy & Alexander J. Whitworth

• The authors describe a new mtDNA mutator model, whereby a cytidine deaminase is targetted to mitochondria to induce mutations (mito-APOBEC1), in fruit flies.
• The most established system for understanding the physiological consequences of mtDNA mutation is to knock-in a proofreading deficient version of the mtDNA polymerase (POLG). Doing so introduces high levels of point mutations, and also small indels, but has surprisingly limited impact on organismal longevity or fitness in flies, given the level of mutation which this mutation induces (see here). In contrast, mito-APOBEC1 exclusively introduces C:G>T:A transitions (which is the most predominant mutation profile in human ageing), with no indels or mtDNA depletion. The authors argue that mutations of this type (rather than those induced by the POLG mutation) cause dramatic reduction in organismal fitness, even at modest heteroplasmy.

A nanoscale, multi-parametric flow cytometry based platform to study mitochondrial heterogeneity and mitochondrial DNA dynamics

https://www.nature.com/articles/s42003-019-0513-4

Julie A. MacDonald, Alisha M. Bothun, Sofia N. Annis, Hannah Sheehan, Somak Ray, Yuanwei Gao, Alexander R. Ivanov, Konstantin Khrapko, Jonathan L. Tilly, and Dori C. Woods

• The authors describe a new technology for isolation and analysis of single mitochondria using flow cytometry, called "fluorescence-activated mitochondria sorting" (FAMS).
• Mitochondria isolated from liver tissue exhibited intact outer and inner membranes, and cristae structure, when evaluated by electron microscopy.
• Staining samples with the DNA stain DAPI, the authors found correlation between side-scatter of organelles and DNA content, suggesting that larger organelles, containing larger amounts of DNA, have larger side-scatter. 
• The authors used the membrane potential sensor dye JC-1 to categorise mitochondria into high/low membrane potential populations. They found that whilst both low and high-membrane potential populations generated ATP when provided with ADP, high-membrane potential mitochondria produced approximately x6 more ATP, and approximately x3 more Mt-ND1 and Mt-Nd4, than low-membrane potential mitochondria. The low-membrane potential mitochondria had ~2.5x lower FSC-PMT, potentially indicating their smaller size [Question: do differences in mitochondrial size confound the inference of differential membrane potential using the JC-1 dye, due to the surface area to volume ratio affecting the aggregation rate? If smaller mitochondria have a higher surface area to volume ratio then perhaps the true difference in mitochondrial membrane potential is even larger.]
• The authors generated mixed samples for two mouse strains, with two different mtDNA haplotypes, and performed single-molecule PCR. Of 54 organelles measured, 2 showed mixtures of mtDNA sequences, suggesting a relatively low rate of artificial fusion of mitochondrial in mixed samples.
• The authors measured the median number of mtDNAs per mitochondrion was 3, ranging from 1 to 22 molecules per sorted organelle.
• The authors used beads to calibrate FSC-PMT and SSC to define two gates: ~0.22-0.5 um, and 0.5-1um, and found that the small gate had approximately 1-2 mtDNAs per organelle, whereas the large gate had 6.5-7.5 mtDNAs per organelle. 

Energetic costs of cellular and therapeutic control of stochastic mitochondrial DNA populations

https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1007023

Hanne Hoitzing, Payam A. Gammage, Lindsey Van Haute, Michal Minczuk, Iain G. Johnston, and Nick S. Jones


Background on mitochondrial DNA dynamics and control

Mitochondria have their own genomes (mtDNAs). These genomes can mutate upon division and at any one given time, mixture of normal (wildtype, w) and mutated (m) mtDNA can exist within a cell. Heteroplasmy is defined as the fraction of mutant mtDNA molecules.

The birth and death of mtDNAs is a stochastic process, their numbers fluctuating over time. Some kind of feedback control must be present, as mtDNA numbers in normal healthy cells tend to remain within certain bounds.

Treatments exist to reduce the load of mutant mtDNAs inside cells. For example, nucleases which are targeted to the specific sequence of a mutant mtDNA can be introduced in cells. They will bind to these mutant sequences and cut the (though off-target cutting of the wildtype genomes is a problem).

Thinking about controlling levels of mtDNA gives rise to various questions:

•  What exactly is this feedback control? What is the quantity that is being controlled (e.g. is it total mtDNA copy number, or is it the overall energy level)? 
• How does the type of control influence heteroplasmy levels? Does one type of control lead to faster mutant accumulation than another?
• How does the cell choose a particular feedback control? Does it do this randomly or does it minimize some 'cost function'?
• Can we somehow interfere with the cellular feedback control to reduce mutant loads?

 
Paper results

This paper investigates these questions a bit more closely.  Some of the main findings are:

• Many different forms of feedback control (e.g. linear, quadratic, etc..) can give rise to similar mtDNA dynamics and heteroplasmy dynamics.
• What makes all the difference, however, is which quantity is being controlled (rather than how it is controlled). Is it total copy number (w + m)? Is it only the number of wildtypes (w)? Is it some more general linear combination (w + 𝛿 m)?
• The more strongly one species is controlled, the more control is lost over the other
• A mitochondrial cost function is introduced, and it is shown that it can actually be more expensive for a cell to contain a mixture of mutant and wildtype molecules, rather than only mutants!
• A control based on energy levels seems to make more sense than blindly controlling total mtDNA copy number. This means that if mutants produce less energy, the quantity being controlled is (w + 𝛿 m) with 𝛿 < 1.
• Variance of mtDNA dynamics is important! An increase in variance in mutant and/or wildtype copy numbers (which will always occur over time) can lead to an increase in cost of maintaining a tissue
• Gene therapies specifically targeting mutant mtDNAs can successfully lower heteroplasmy levels, but this becomes hard when high tissue heteroplasmy levels are caused by only a small fraction of cells (i.e. a few cells have very high heteroplasmy levels and most cells are ok). Again, it's the mtDNA variance that's important!
• Long and weak gene therapies seem to reach lower overall heteroplasmy levels compared to short and strong therapies.

Mitochondrial Network State Scales mtDNA Genetic Dynamics

https://doi.org/10.1534/genetics.119.302423

Juvid Aryaman, Charlotte Bowles, Nick S. Jones and Iain G. Johnston

(Mirrored from Evolution, Energetics & Noise)

Mitochondrial DNA (mtDNA) populations within our cells encode vital energetic machinery. MtDNA is housed within mitochondria, cellular compartments lined by two membranes, that lead a very dynamic life. Individual mitochondria can fuse when they meet, and fused mitochondria can fragment to become individual smaller mitochondria, all the while moving throughout the cell. The reasons for this dynamic activity remain unclear (we’ve compared hypotheses about them before here and here, with blog articles here). But what influence do these physical mitochondrial dynamics have on the genetic composition of mtDNA populations?

MtDNA populations can, naturally or as a result of gene therapies, consist of a mixture of different mtDNA types. Typically, different cells will have different proportions of, say, type A and type B. For example, one cell may be 20% type A, another cell may be 40% type A, and a third may be 70% type A. This variability matters because when a certain threshold (often around 60%) is crossed for some mtDNA types, we get devastating diseases.

We previously showed mathematically (blog) and experimentally (blog) that this cell-to-cell variability in mtDNA proportions (often called “heteroplasmy variance” and sometimes referred to via the “mtDNA bottleneck”) is expected to increase linearly over time. However, this analysis pictured mtDNAs as individual molecules, outside of their mitochondrial compartments. When mitochondria fuse to form larger compartments, their mtDNA is more protected: smaller mitochondria (and their internal mtDNA) are subject to greater degradation. More degradation means more replication, and more opportunities for the fraction of a particular type of mtDNA to change per unit time. In a new paper here in Genetics, we show that this protection can dramatically influence cell-to-cell mtDNA variability. Specifically, the rate of heteroplasmy variance increase is scaled by the proportion of mitochondria that exist in a fragmented state. (It turns out that it's the proportion of mitochondria that are fragmented that's important -- not whether the rate of fission-fusion is fast or slow).

This has knock-on effects for how the cell can best get rid of low-quality mutant mtDNA. In particular, if mitochondria are allowed to fuse based on their quality (“selective fusion”), we show that intermediate rates of fusion are best for removing mutants. Too much fusion, and all mtDNA is protected; too little, and good mtDNA cannot be sorted from bad mtDNA using the mitochondrial network. This mechanism could help explain why we see different levels of mitochondrial fusion in different conditions. More broadly, this link between mitochondrial physics and genetics (which we’ve also speculated about here (blog) and here) suggests one way that selective pressures and tradeoffs could influence mitochondrial dynamics, giving rise to the wide variety of behaviours that remain unexplained. Juvid, Nick, and Iain

Respiratory Syncytial Virus co-opts host mitochondrial function to favour infectious virus production

https://elifesciences.org/articles/42448

MengJie Hu, Keith E Schulze, Reena Ghildyal, Darren C Henstridge, Jacek L Kolanowski, Elizabeth J New, Yuning Hong, Alan C Hsu, Philip M Hansbro, Peter AB Wark, Marie A Bogoyevitch, David A Jans

• Respiratory syncytial virus (RSV) is responsible for more deaths each year than influenza. Here, the authors investigate how RSV hijacks mitochondria for viral production.
• The authors suggest that RSV induces perinuclear clustering of mitochondria, reduction in mitochondrial respiration, impaired mitochondrial membrane potential, and increased reactive oxygen species production. 
• The authors find that inhibiting the dynein motor protein, or inhibiting mitochondrial ROS production, suppresses RSV production in vivo.

RNA sequence analysis reveals macroscopic somatic clonal expansion across normal tissues

https://science.sciencemag.org/content/364/6444/eaaw0726?ijkey=747d2d8299edcfd1fdfe566522ccbcf3ba841b1f&keytype2=tf_ipsecsha

Keren Yizhak, François Aguet, Jaegil Kim, Paz Polak, Kristin G. Ardlie, Gad Getz and others

• The authors study the RNA sequence of >6000 samples across 29 normal tissues (using a method they call RNA-MuTect), and find multiple macroscopic somatic mutations in normal tissues.
• Genes which are highly expressed may be investigated for evidence of somatic mosaicism
• Sun-exposed skin, esophagus, and lung have a higher mutation load than other tested tissues, suggesting an evironmental role
• Mutation burden was associated with age and tissue-specific proliferation rate
• Normal tissues were found to harbour mutations in known cancer genes
• See also Cristian Tomasetti's summary here

Mitochondrial Stress Response in Neural Stem Cells Exposed to Electronic Cigarettes

https://www.sciencedirect.com/science/article/pii/S2589004219301713

Atena Zahedi, Rattapol Phandthong, Angela Chaili, Sara Leung, Esther Omaiye, Prue Talbot

A WORD ON MITOCHONDRIAL DYNAMICS (from this publication)

• Mitochondria of healthy cells continually divide and fuse with each other, forming an ever-changing mitochondrial network. This is referred to as mitochondrial dynamics.
• Fusion promotes exchange of mtDNA and other vital components, thus reinvigorating the mitochondrial network.
• Fission allows for disposal of faulty mitochondrial fragments through mitophagy. Moreover, when cells become committed to apoptosis, they shatter their mitochondrial networks.
• Modest levels of stress (well below the threshold to induce apoptosis) lead mitochondria to fuse extensively. This response was called stress‐induced mitochondrial hyperfusion (SIMH),  and might counter stress by optimizing mitochondrial ATP production.

FINDINGS OF THE PAPER

• Stem cells are critical to our wellbeing (controlling organ development and tissue renewal/repair) and the damage they accumulate over life can lead to disease.
• During development, neural stem cells are highly sensitive to toxicants and more vulnerable to stress than differentiated cells. Mitochondria are good indicators of stress in stem cells.
• Electronic cigarettes are marketed as a healthy substitute to cigarettes, and are targeted at youth and pregnant women.
• The authors exposed stem cells to EC fluid in a set of in vitro experiments. They argue that the nicotine present in EC fluid causes SIMH of stem cells. SIMH is a survival response in stem cells and is accompanied by increased oxidative stress and alterations in mitochondrial morphology and dynamics.
• Further, an interruption of autophagy was observed when stem cells were exposed to nicotine. Since autophagy is a defense mechanism of the cell, clearing damaged mitochondria, its inhibition is deleterious the the stem cell population.
• The main message of the study is that EC are not as harmless as they are claimed to be, and that similar findings could apply to any product containing nicotine.

DNA Microscopy: Optics-free Spatio-genetic Imaging by a Stand-Alone Chemical Reaction

https://www.sciencedirect.com/science/article/pii/S0092867419305471

Joshua A. Weinstein, Aviv Regev, and Feng Zhang

• The authors develop a novel method of determining spatial localisation of transcripts within the cell through "DNA Microscopy". 
• The method consists, firstly, of randomly tagging individual transcripts or DNA molecules with DNA unique molecular identifiers (UMIs), which are random nucleotide sequences of a particular length. 
• The UMI-concatenated molecules are then amplified through PCR, and diffuse in the cell. UMI tags are designed to contain overhanging complementary regions, such that tagged molecules are subsequently able to bind to another complementary molecule which is in close spatial proximity (called "beacon" and "target" amplicons). Through this process, "unique event identifiers" (UEIs) are generated. The cell can then be lysed, and sequenced through next-generation sequencing.
• The rate at which UMIs bound to a particular molecule concatenate indicates the distance between their points of origin.
• A computational algorithm then decodes molecular proximities from these UEIs to infer the spatial distribution of transcripts at cellular resolution. 

Atlas of Subcellular RNA Localization Revealed by APEX-Seq

https://www.cell.com/cell/fulltext/S0092-8674(19)30555-0

Fazal FM, Han S, Parker KR, Kaewsapsak P, Xu J, Boettiger AN, Chang HY, Ting AY

• The authors introduce the method APEX-seq, which is a method for whole-transcriptome spatial profiling in living cells. It is based on direct proximity labelling of RNA using the peroxidase enzyme APEX2. 
• The APEX protein may be localised to different cellular subcomponents, such as the nucleolus, nuclear pore, endoplasmic reticulum, nuclear lamina, outer mitochondrial membrane, and mitochondrial matrix. Once there, APEX biotinylates mRNAs and proteins, allowing mRNAs from the targeted region to be purified and sequenced through RNA-seq.

Cell population heterogeneity driven by stochastic partition and growth optimality

https://arxiv.org/pdf/1805.07768.pdf

Jorge Fernandez-de-Cossio-Diaz, Roberto Mulet, Alexei Vazquez

• The authors suggest that a cellular quantity which i) has an optimal value for growth rate; ii) is stochastically partitioned at cell division; may display a bimodal distribution in the population. 
• Whether the distribution is unimodal or bimodal depends on the sharpness of (i) and the extent of noise in (ii). The authors suggest mitochondria as a potential cellular component for which their theory is applicable.

Quasi-Mendelian Paternal Inheritance of mitochondrial DNA: A notorious artifact, or anticipated mtDNA behavior?

https://www.biorxiv.org/content/10.1101/660670v1?ct=

Sofia Annis, Zoe Fleischmann, Mark Khrapko, Melissa Franco, Kevin Wasko, Dori Woods, Wolfram S. Kunz, Peter Ellis, Konstantin Khrapko

• A recent publication suggested that biparental inheritance of mtDNA may sometimes occur in humans
• It has since been suggested that these observations may be explained by the presence of mtDNA nuclear pseudogenes (NUMTs) in the father's nuclear genome, rather than biparental inheritance
• The authors of this article suggest another interpretation: that the original authors did in fact observe biparental inheritance of mtDNA, and that the paternal mtDNA was inherited by nascent cells with low copy number, and that the paternal mtDNA had a selective advantage. 
• Using computational modelling (based on this publication), the authors predict a somatic mosaic distribution of paternal mtDNA in the resulting progeny, including in the germline.

Mitochondrial behaviors prime the selective inheritance against harmful mitochondrial DNA mutations

https://www.biorxiv.org/content/biorxiv/early/2019/05/24/646638.full.pdf

Zhe Chen, Zong-Heng Wang, Guofeng Zhang and Hong Xu

• The authors investigate the mechanism of selective inheritance of a deleterious temperature-sensitive mitochondrial DNA mutation in the germline of Drosophila. At 29C, this allele is selected against.
• They show that mitochondria become fragmented such that >90% of organelles contain a single mitochondrial nucleoid in the germarium 2A region of developing Drosophila ovaries. Nucleoids were found to contain 1.3 mtDNAs on average, suggesting that intra-nucleoid complementation is limited.
• Inhibition of fission caused the inter-generational selection against the mutation to essentially be eliminated. 
• They show that in region 2B, mitochondrial transcripts are expressed (shown via fluoresence in-situ hybridization), and the TMRM:MitoTracker ratio is increased by ~x3 fold.
• Knock-down of cox5A resulted in diminished selection, suggesting that activation of mitochondrial respiration is necessary for selection. Similarly, expression of AOX, which by-passes the electron transport chain, resulted in diminished selection. Also, inhibition of mtDNA replication diminished selection (although mean heteroplasmy was also lower in the control setting, at the permissive temperature of 18C, in this case).
• To summarise, the authors demonstrate that mitochondrial fission, combined with a suppression of mtDNA replication, in proliferating germ cells segregates mtDNA into individual organelles. The expression of mtDNA induces a genotype-phenotype correspondence for individual organelles, whereby defective organelles are removed and consequently an elimination of mutated molecules of mtDNA.

Mitochondrial fusion supports increased oxidative phosphorylation during cell proliferation

https://elifesciences.org/articles/41351

Cong-Hui Yao, Rencheng Wang, Yahui Wang, Che-Pei Kung, Jason D Weber, Gary J Patti

• The authors show that mouse fibroblasts increase oxidative phosphorylation by nearly x2, and mitochondrial coupling efficiency by ~30%, during proliferation. Both of these changes are supported by mitochondrial fusion.
• Modulating mitochondrial fusion through Mfn2 levels caused modulation in proliferation rate. Decreases in fusion decreased OXPHOS but not ATP levels.
• The authors suggest that cell proliferation requires increased OXPHOS supported by mitochondrial fusion.

Mammalian cell growth dynamics in mitosis

https://elifesciences.org/articles/44700

Teemu P Miettinen, Joon Ho Kang, Lucy F Yang, Scott R Manalis

• The authors use a suspended microchannel resonator and protein synthesis assays to measure the accumulation of cell mass through the cell cycle, for single mammalian cells.
• For various animal cell types, the growth rate in prophase (the first stage of the cell cycle) is comparable to or larger than interphase (the phase where DNA is copied) growth rates. Growth is only stopped in the metaphase-to-anaphase transition. 
• The authors find that a range of mitotic arrest mechanisms inhibit cell growth. Their results counter the traditional idea that cell growth is negligible during mitosis.

Germline selection shapes human mitochondrial DNA diversity

https://science.sciencemag.org/content/364/6442/eaau6520.abstract

Wei Wei, Salih Tuna, Michael J. Keogh, Katherine R. Smith, Timothy J. Aitman, F. Lucy Raymond, Mark Caulfield, Ernest Turro, Patrick F. Chinnery and others

• The authors analyse 1526 mother-offspring pairs from rare-disease patients in the 100,000 genomes project, to show that 45% of individuals display heteroplasmy at >1% variant allele frequency (VAF).
• The authors define 3 kinds of variant: transmitted/inherited (present in both mother and offspring and heteroplasmic in at least one; transmitted = mother, inherited = offspring), lost (present in mother, absent in offspring) and de novo (present in offspring, absent in mother). Absence is defined as VAF < 1%.
• Transmitted variants had a much larger heteroplasmic fraction than lost and de novo variants. 
• Transmitted VAF correlates with inherited VAF (in logit-transformed space).
• Heteroplasmy transmission/inheritance did not display a significantly skewed distribution in the inter-generational VAF shift, which is compatible with this set of mutations undergoing neutral drift.
• The D-loop had an approximately 4 times higher inter-generational mutation rate per base pair than the rest of the mitochondrial genome, suggesting the existence of stronger selective pressures against mutation on the reset of the genome, or potentially an intrinsically lower de novo mutation rate.
• tRNA, rRNA, and non-synonymous mutations tended to have a lower VAF than D-loop and synonymous mutations, suggesting the existence of selection.
• The authors identified haplogroup-matched (92%) and haplogroup-mismatched (2.3%) groups within their dataset (6% could not be identified). Haplogroup mismatching arises from mixed-race ancestry. The heteroplasmic variants in the mismatched group were significantly more likely to match the ancestry of the nuclear genetic background than the mtDNA background on which the heteroplasmy occurred.

Epigenetic Control of Mitochondrial Fission Enables Self-Renewal of Stem-likeTumor Cells in Human Prostate Cancer

.https://www.ncbi.nlm.nih.gov/pubmed/31130467

Gianluca Civenni, Roberto Bosotti, Andrea Timpanaro, Ramiro Vàzquez, Jessica Merulla, Shusil Pandit, Simona Rossi, Domenico Albino, Sara Allegrini, Abhishek Mitra, Sarah N. Mapelli, Luca Vierling, Martina Giurdanella, Martina Marchetti, Alyssa Paganoni, Andrea Rinaldi, Marco Losa, Enrica Mira-Catò, Rocco D’Antuono, Diego Morone, Keyvan Rezai, Gioacchino D’Ambrosio, L’Houcine Ouafik, Sarah Mackenzie, Maria E. Riveiro, Esteban Cvitkovic, Giuseppina M. Carbone and Carlo V. Catapano

INTRODUCTION

• Prostate cancer (PC) is the most common neoplasy in men and one of the main causes of cancer death in developed countries.

• Cancer stem cells (CSCs) are a small subset of cancer cells with stem-cell like properties. They contribute to treatment failure and relapse. Understanding the mechanisms which regulate their self-renewal, differentiation and senescence could lead to new therapeutic strategies.

• Mitochondrial reprogramming has important functions in CSCs. Mitochondrial dynamics control  asymmetric cell division, self-renewal, and the fate of stem cells. Fission and clearance of dysfunctional mitochondria avoid senescence and prevent stem cell exhaustion.

MAIN FINDINGS OF THE PAPER

• The authors uncover a novel link between the protein BRD4, mitochondrial dynamics and self-renewal of CSCs.

• Genetic knockdown of BRD4 or chemical inhibitors blocked mitochondrial fission and caused CSC exhaustion and loss of tumorigenic properties. This is mediated through the  inhibition of  mitochondrial fission factor (Mff) caused by BRD4 knockdown.

• Evidence for this is that suppression of Mff transcription reproduced the effects of BRD4 knockdown, whereas ectopic expression of Mff rescued CSCs from exhaustion. Therefore the authors conclude that targeting mitochondrial plasticity in CSCs is a promising avenue for new and more effective therapies. 

Mutational signatures of redox stress in yeast single-strand DNA and of aging in human mitochondrial DNA share a common feature

https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000263

Natalya P. Degtyareva, Natalie Saini, Joan F. Sterling, Victoria C. Placentra, Leszek J. Klimczak, Dmitry A. Gordenin, Paul W. Doetsch

• The authors report on the mutational spectra of redox stress in single-stranded DNA of budding yeast and in human mitochondrial DNA in the context of healthy aging, finding that the predominance of C>T transitions is a predominant feature in both.
• The authors find that the frequencies of hydrogen peroxide-induced mutations in proof-reading deficient yeast mutants supports the conclusion that this form of mutagenesis is the result of direct damage to DNA, rather than misincorporation errors.
• They propose that mutations may occur to the heavy strand of mtDNA when DNA replication starts at the light chain, temporarily making the displaced, heavy strand more vulnerable to damage.

An aerobic eukaryotic parasite with functional mitochondria that likely lacks a mitochondrial genome

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482013/pdf/aav1110.pdf

John U, Lu Y, Wohlrab S, Groth M, Janouškovec J, Kohli GS, Mark FC, Bickmeyer U, Farhat S, Felder M, Frickenhaus S, Guillou L, Keeling PJ, Moustafa A, Porcel BM, Valentin K, Glöckner G

• A long-standing debate in the field of mitochondrial physiology is the purpose of mitochondrial DNA. The "co-location for redox regulation" (CoRR) hypothesis states that mitochondrial genomes are necessary to provide local control of the electron transport chain.
• The authors describe an aerobic eukaryotic parasite (Amoebophyra ceratii) with functional mitochondria, but have completely lost their mitochondrial genome, finding that all mitochondrial proteins appear to be lost or encoded in the nucleus. 
• This finding challenges the CoRR hypothesis, and potentially suggests the possibility of complete transfer of the mitochondrial genome into the nuclear genome for more complex organisms.

Mitochondrial fragmentation drives selective removal of deleterious mtDNA in the germline

https://www.nature.com/articles/s41586-019-1213-4

Toby Lieber, Swathi P. Jeedigunta, Jonathan M. Palozzi, Ruth Lehmann & Thomas R. Hurd

• The authors generate mutated fruit flies by transfering mitochondria from a wild-type strain of Drosophila yakuba into a strain of Drosophila melanogaster in which the mtDNA contain a temperature-sensitive mutation in Complex IV of the electron transport chain.
• The authors designed fluorescent probes to specifically bind to the D-loop of either D. yakuba or D. melanogaster, allowing them to visualise heteroplasmy.
• At 18C, the point mutation does not affect Complex IV activity, whereas at the inhibitory temperature of 29C, Complex IV activity is greatly reduced and is selected against 
• Selection first manifests during oogenesis, where a reduction in mitofusins causes fragmentation of the mitochondrial network
• The authors identify the proteins Atg1 and BNIP3  as necessary for the selective removal of mitochondria with mutated mtDNAs
• A reduction in Atg1 or BNIP3 decreases the amount of wild-type mtDNA, suggesting a link between mitochondrial degradation and replication
• At the restrictive temperature, selection occured in the germline but not in the somatic cells which surround the germline in the ovariole, and was largely absent in the male germ line (possibly because only female mtDNA is inherited)
• Inhibiting cell death through over-expression of the cell-death inhibitor p35 did not block selection
• Expression of the alternative oxidase protein (AOX) bypasses the function of complex IV and partially blocked selection, suggesting that the selection process senses defects in oxidative phosphorylation
• The authors observed greater fragmentation in the germline mitochondria relative to the soma. Using photoactivatable GFP, the authors show that mitochondrial contents rarely pass from one mitochondrion to another, suggesting that the purpose of fragmentation is to reduce complementation so that the genotype of individual mitochondria may be sensed through their phenotype.
• Reducing Mitofusin expression in somatic cells also induced selection
• The authors inhibited the protein IF1, to allow ATP synthase to run in reverse and maintain mitochondrial membrane potential by burning ATP. In doing so, the authors did not observe statistically significant selection, which may suggest that membrane potential sensing is the mechanism by which mitochondria are selected.
• Expression of a dominant-negative form of ATP synthase caused a reduction in mtDNA copy number of both mutants and wild-types

Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single cell resolution: a focus on aging and cancer

https://www.biorxiv.org/content/biorxiv/early/2019/03/16/579789.full.pdf

Ryan O’Hara, Enzo Tedone,, Andrew Ludlow, Ejun Huang, Beatrice Arosio, Daniela Mari, Jerry W. Shay

• The authors develop a protocol to measure single-cell mtDNA copy number using digital droplet PCR
• The authors find ~10-fold inter-cellular variability in mtDNA copy number (in an immortalised human cell line, H1299), which could not be fully explained by cell cycle variations.
• The authors investigated how mtDNA copy number changes after stimulation of peripheral blood mononuclear cells (PBMCs, a heterogeneous cellular population largely consisting of T cells). Previous studies have shown a decline in mtDNA copy number with ageing in this cell population. The authors studied stimulated PBMCs in young, old, and healthy/frail centenarians. Healthy centenarians tended to have higher mtDNA copy number than frail, or ~70 year old, individuals.  

Intramitochondrial transfer and engineering of mammalian mitochondrial genomes in yeast

https://www.sciencedirect.com/science/article/pii/S1567724918300370?via%3Dihub

Yoon YG, Koob MD

• The authors demonstrate that entire mouse mtDNA can be stably transferred to the mitochondrial network in yeast which have been depleted of their own native mtDNA.
• The yeast cells which contained the full mouse mtDNA genome, replicated the mouse mtDNA molecules without detectable sequence alterations or rearrangements.

Mitochondria-specific drug release and reactive oxygen species burst induced by polyprodrug nanoreactors can enhance chemotherapy

https://www.nature.com/articles/s41467-019-09566-3

Zhang W, Hu X, Shen Q, Xing D

• Many cancer cells over-produce reactive oxygen species by ~10-fold relative to normal cells, providing a biomarker for cancer cells.
• The authors sought to design a chemical system which targets ROS-overproducing cells, and then further stimulate long-term ROS overproduction inside mitochondria, to induce apoptosis.

Mitochondrial Protein Synthesis and mtDNA Levels Coordinated through an Aminoacyl-tRNA Synthetase Subunit

https://www.cell.com/cell-reports/fulltext/S2211-1247(19)30329-8?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2211124719303298%3Fshowall%3Dtrue

Picchioni D, Antolin-Fontes A, Camacho N, Schmitz C, Pons-Pons A, Rodríguez-Escribà M, Machallekidou A, Güler MN, Siatra P, Carretero-Junquera M, Serrano A, Hovde SL, Knobel PA, Novoa EM, Solà-Vilarrubias M, Kaguni LS, Stracker TH, Ribas de Pouplana L

• The authors investigate a signalling pathway which couples mtDNA translation to mtDNA copy number 
• They identify a protein (SLIMP) which is essential for mitochondrial respiration, and is involved in tRNA-serine aminoacylation
• SLIMP interacts with the protein LON, which degrades TFAM. TFAM is a protein involved in forming mtDNA-protein complexes known as nucleoids. Reduction in TFAM levels is associated with mtDNA depletion.
• Hence mitochondrial translation is directly coupled to mtDNA copy number.

Mitochondrial volume fraction controls translation of nuclear-encoded mitochondrial proteins

https://www.biorxiv.org/content/biorxiv/early/2019/01/25/529289.full.pdf

Tatsuhisa Tsuboi, Matheus P. Viana, Fan Xu, Jingwen Yu, Raghav Chanchani, Ximena G. Arceo, Evelina Tutucci, Joonhyuk Choi, Yang S. Chen, Robert H. Singer, Susanne M. Rafelski, Brian M. Zid

• The authors investigate the physiological impact of nuclear-encoded mitochondrial mRNA localization to mitochondria in yeast
• They observe that as yeast switches to oxidative metabolism, the cytoplasmic density of mitochondria increases (i.e. the ratio of mitochondrial volume to cytoplamic volume)
• Increases in mitochondrial density drives the localisation of nuclear-encoded mitochondrial mRNAs to the mitochondrial surface, increasing mitochondrial protein production
• Sequestering mRNAs away from the mitochondrial surface is sufficient to reduce mitochondrial protein production
• This suggests that mitochondrial density is a physiologically important parameter, which is sensed to regulate mitochondrial gene expression via mRNA localisation.

A non‐death function of the mitochondrial apoptosis apparatus in immunity

http://emboj.embopress.org/content/early/2019/04/12/embj.2018100907

Dominik Brokatzky, Benedikt Dörflinger, Aladin Haimovici, Arnim Weber, Susanne Kirschnek, Juliane Vier, Arlena Metz, Julia Henschel, Tobias Steinfeldt, Ian E. Gentle and Georg Häcke

INTRODUCTION
Apoptosis mostly proceeds through mitochondria: the outer mitochondrial membrane is permeabilized, in a process called mitochondrial outer membrane permeabilization (MOMP). This releases cytochrome c to activate cytosolic caspases, which execute apoptosis through proteolysis of numerous substrates.

Recently, it has been observed that apoptosis signaling may be initiated at a low level, in the absence of cell death. Only few mitochondria are permeabilized, and small amounts of cytochrome c are released, causing only limited caspase activation. Apoptosis appears to be triggered but then aborted before the point of no return. The process has been termed minority MOMP and the cell stays alive and can presumably repair any damage caused

MAIN FACTS OF THE PAPER
The author hypothesise that low-level (sub-lethal) “apoptosis” signaling (minority MOMP) can trigger cytokine secretion, causing inflammation and immune alert. Full apoptosis activates caspases that counteract this immune function. The small amounts of caspase activated during sub-lethal signaling following minority MOMP may however be too low to turn the signal off, resulting in cytokine secretion and immune activation.

Infecting HeLa cells by several agents, they tested this possibility and reported that human cells can react to low-level apoptosis induction with cytokine secretion. Minority MOMP was detected with all infectious agents tested. This suggests that minority MOMP is a very common occurrence during infection. In addition, the authors show that the ability of epithelial cells to fight the growth of parasites is decreased when proapoptotic signaling is deleted.


CONCLUSIONS
Their results suggest that mitochondria have a function in the detection of microbial infection and cell-autonomous immunity, through a sub-lethal (low-intensity) activation of the mitochondrial apoptosis apparatus.

They also report damage to the genomic DNA caused by the mitochondrial apoptosis signaling. Since MOMP seems like a frequent occurrence, this suggests that infection-associated damage to the genomic DNA is widespread. Therefore, this study also identifies DNA damage as a common occurrence during infection and indicate the possibility that infection-associated mutations, potentially leading to cancer, may be a side effect of this system of microbial detection.

Mitochondrial origins of fractional control in regulated cell death

https://www.nature.com/articles/s41467-019-09275-x.pdf

Luís C. Santos, Robert Vogel, Jerry E. Chipuk, Marc R. Birtwistle, Gustavo Stolovitzky & Pablo Meyer

• The authors investigate cell-to-cell variability in cell death in response to TNF-related apoptosis inducing ligand (TRAIL), an apoptosis-inducing drug.
• The authors find that with successively increasing doses of TRAIL, the probability distribution of mitochondrial density (which the authors define as MitoTracker Deep Red fluoresence intensity divided by forward scatter) becomes increasingly enriched for cells with high mitochondrial density -- suggesting that high cytoplasmic mitochondrial density is required for TRAIL resistance (in contrast to this)
• The authors point out that the steepness of a dose-response curve is a measure of cell-to-cell variability in cellular sensitivity to a stimulus, and derive a formalism to convert a Hill function into a probability distribution over cellular stimulus thresholds for a binary response variable (such as cell death). They derive an approximate expansion of the sensitivity threshold in terms of the half maximal inhibitory concentration (IC50), and the log ratio of mitochondrial density to mean mitochondrial density. This results in an expression for the conditional probability of a cell being alive given a biological quantity of interest, e.g. mitochondrial density (see Eq 4)
• The authors attribute this correlation to variable effective concentrations of pro-apoptotic proteins on the mitochondrial outer membrane
• The authors suggest that anti-apoptotic Bcl-2 family proteins may increase the variance in cell death response, potentially enhancing resistance to treatment

NAD+ metabolism governs the proinflammatory senescence-associated secretome

https://www.nature.com/articles/s41556-019-0287-4

Nacarelli T, Lau L, Fukumoto T, Zundell J, Fatkhutdinov N, Wu S, Aird KM, Iwasaki O, Kossenkov AV, Schultz D, Noma KI, Baur JA, Schug Z, Tang HY, Speicher DW, David G, Zhang R

• THe authors show that the enzyme nicotinamide phosphoribosyltransferase (NAMPT), which is a rate-limiting enzyme of the NAD+ salvage pathway, is involved in the senesence-associated secretory phenotype (SASP), independent of the senesence-associated growth arrest.
• The signalling pathway the authors identify is promotes the SASP by enhancing glycolysis and mitochondrial respiration. 
• The tumour-promoting effects of SASP suggests that anti-ageing dietary NAD+ augmentation should be administered with care.

The NAD-Booster Nicotinamide Riboside Potently Stimulates Hematopoiesis through Increased Mitochondrial Clearance

https://www.sciencedirect.com/science/article/pii/S1934590919300621

Vannini N, Campos V, Girotra M, Trachsel V, Rojas-Sutterlin S, Tratwal J, Ragusa S, Stefanidis E, Ryu D, Rainer PY, Nikitin G, Giger S, Li TY, Semilietof A, Oggier A, Yersin Y, Tauzin L, Pirinen E, Cheng WC, Ratajczak J, Canto C, Ehrbar M, Sizzano F, Petrova TV, Vanhecke D, Zhang L, Romero P, Nahimana A, Cherix S, Duchosal MA, Ho PC, Deplancke B, Coukos G, Auwerx J, Lutolf MP, Naveiras O

• Boosting NAD+ via dietary supplementation of nicotinamide riboside (NR) in mice
• Reduces mitochondrial activity in hematopoetic stem cells 
• Increases mitophagy in HSCs (determined in the mitoQC mouse)
• Reduces mitochondrial membrane potential (perhaps related to the increased mitophagy)
• Leading to increased asymmetric division of mitochondria (quantified by TMRM/mitochondrial mass) in HSCs, resulting in expansion of the hematopoetic progenitor compartment.
• NR supplementation enhanced survival and blood cell production following HSC transplantation.

Nuclear genetic regulation of the human mitochondrial transcriptome

https://elifesciences.org/articles/41927

Aminah T Ali, Lena Boehme, Guillermo Carbajosa, Vlad C Seitan, Kerrin S Small, Alan Hodgkinson

• The authors analyse >11k RNA sequencing libraries across 36 tissue types and investigate the variability in transcription of the mitochondrial genome
• The authors identify 64 nuclear genetic loci associated with expression of mitochondrially-encoded genes. 
• The authors replicate ~21% of associations with independent tissue-matched datasets.

Systems biology identifies preserved integrity but impaired metabolism of mitochondria due to a glycolytic defect in Alzheimer’s disease neurons

https://onlinelibrary.wiley.com/doi/full/10.1111/acel.12924

Pierre Theurey, Niamh M. C. Connolly, Ilaria Fortunati, Emy Basso, Susette Lauwen, Camilla Ferrante, Catarina Moreira Pinho, Alvin Joselin,  Anna Gioran, Daniele Bano, David S. Park,  Maria Ankarcrona, Paola Pizzo,  Jochen H. M. Prehn

• Many studies have focussed on the role of mitochondrial dysfunctions in Alzheimer's disease (AD), but most of them cannot tell whether mitochondrial defects are a cause or a consequence of AD.
• The authors use a combined experimental and computational approach to study mitochondrial function in the neurons of a transgenic mouse model.
• Experiments show that AD neurons have limited respiratory capacity. The computational model predicted that this could not be explained by any defect in the respiratory chain (RC),  but could be observed by simulating an impairment in the NADH flux to the RC.
• The authors used NAD(P)H autofluorescence measurements to validate the computationally predicted mitochondrial NADH defect in transgenic AD neurons.

Additionally, the authors investigated the cause of these reduced NADH flux and the resulting  mitochondrial NAD(P)H dyshomeostasis.

• Extracellular acidification experiments  measure the rate of excretion of lactic acid after its conversion from pyruvate, a product of glycolysis. These experiments  showed an impaired glycolytic flux in the transgenic AD neurons of the study.
• The authors supplemented neurons with pyruvate (therefore bypassing glycolysis), and this suppressed the NAD(P)H  impairment and the mitochondrial defects.
• This supports the hypothesis that a glycolytic defect is responsible for the unbalance of NAD(P)H observed in AD neurons.

The study shows that defects in glucose metabolism in vitro are detectable in neurons before the onset of any sign of pathology in transgenic AD mice.

Cardiolipin remodeling by ALCAT1 links mitochondrial dysfunction to Parkinson’s diseases

https://onlinelibrary.wiley.com/doi/full/10.1111/acel.12941

Chengjie Song  Jun Zhang  Shasha Qi  Zhen Liu  Xiaoyang Zhang  Yue Zheng  John‐Paul Andersen  Weiping Zhang  Randy Strong  Paul Anthony Martinez  Nicolas Musi  Jia Nie Yuguang Shi

• Parkinson's disease's (PD) causes remain elusive, but oxidative stress, mitochondrial dysfunction, and defective mitophagy are all considered as the primary pathogenic mechanisms.
• Cardiolipin (CL) is a phospholipid which is almost exclusively located in the inner mitochondrial membrane, where it is biosynthesized.
• ROS-induced damage of CL  is implicated in the pathogenesis of PD, but the mechanism remains unclear.
• The authors induced PD in a mouse model, induced by MPTP (a chemical that caused PD when injected, and has been used to study disease models in various animal studies). They oxidative stress, mtDNA mutations, and mitochondrial dysfunction in the midbrain.
• Then, they ablated  the ALCAT1 gene and treated mice with MPTP. This prevented MPTP‐induced neurotoxicity, apoptosis, and motor deficits and mitigated mitochondrial dysfunction.
• Mitophagy, which removes dysfunctional mitochondria, is also compromised in PD. The pharmacological inhibition of ALCAT1 significantly improved mitophagy, by stimulating the recruitment of Parkin to dysfunctional mitochondria and their association.
• These results show that ALCAT1 may be a promising drug target in the treatment of PD.

Alternative assembly of respiratory complex II connects energy stress to metabolic checkpoints

https://www.nature.com/articles/s41467-018-04603-z

Ayenachew Bezawork-Geleta, He Wen, LanFeng Dong, Bing Yan, Jelena Vider, Stepana Boukalova, Linda Krobova, Katerina Vanova, Renata Zobalova, Margarita Sobol, Pavel Hozak, Silvia Magalhaes Novais, Veronika Caisova, Pavel Abaffy, Ravindra Naraine, Ying Pang, Thiri Zaw, Ping Zhang, Radek Sindelka, Mikael Kubista, Steven Zuryn, Mark P. Molloy, Michael V. Berridge, Karel Pacak, Jakub Rohlena, Sunghyouk Park & Jiri Neuzil

• The authors show that depletion of mtDNA causes a shift in CII assembly from its full tetrameric form to an alternative 100 kDa form
• The authors suggest that cells may modulate their energy consumption by altering DNA synthesis and cell cycle progression. This modulation is mediated by the alternative form of CII

Lineage Tracing in Humans Enabled by Mitochondrial Mutations and Single-Cell Genomics

https://www.cell.com/cell/pdf/S0092-8674(19)30055-8.pdf

Leif S. Ludwig, Caleb A. Lareau, Jacob C. Ulirsch, Elena Christian, Christoph Muus, Lauren H. Li, Karin Pelka, Will Ge, Yaara Oren, Alison Brack, Travis Law, Christopher Rodman, Jonathan H. Chen, Genevieve M. Boland, Nir Hacohen, Orit Rozenblatt-Rosen, Martin J. Aryee, Jason D. Buenrostro, Aviv Regev, and Vijay G. Sankaran

INTRODUCTION

• Lineage tracing involves inferring the developmental history of an organism, with respect to its ancestors. Since single cells divide and proliferate, an emerging field is the inference of lineages of single-cells.
• In model organisms, this can be achieved through engineered genetic labels and single-cell RNA sequencing. These two approaches cannot be used together in humans, because of the genetic manipulations required to tag cells with heritable marks.
• Therefore, to date lineage tracing studies in humans have relied on the detection of naturally occurring somatic mutations in the nuclear genome. However, these mutations have high error rates and their detection is costly and difficult to perform at scale.
• The mitochondrial genome provides an attractive target for inferring cellular lineages for several reasons: 
• MtDNA is large enough to show substantial levels of variation
• It is short enough to be cost-effective for targetted sequencing: 18,000 mitochondrial genomes (17k bases) can be sequenced at 100-fold coverage for the same cost as a single nuclear genome (3.2bn bases) at 10-fold coverage. 
• Its mutation rate is reported to be 10-100 times larger than the nuclear genome.
• MtDNA is held in high copy number per cell (100-1000s), therefore less amplification is necessary.
• Mutations in mtDNA often reach a variant allele fraction of ~100% due to partitioning noise, random genetic drift, and faster replication relative to nuclear DNA.
• Existing methods (ATAC-seq and single-cell RNA-seq) can be used to detect mtDNA sequences and genetic variation.

MAIN FACTS OF THE PAPER

• The authors established 65 individual sub-clonal populations, over 8 generation, in an immortalised cell line. They derived subclones (populations of cells derived from a single cell) from the parental colony at each generation, and performed bulk mitochondria individual cells’ l genome sequencing  through ATAC-seq. The authors used high-confidence mtDNA mutations to reconstruct clonal relations between the subpopulations, allowing them to predict the most recent common ancestor with >80% accuracy (See Fig 1C, 1E and 1F).
• Since mtDNA is almost entirely transcribed, the authors hypothesized that single-cell RNA-seq would also be able to detect heteroplasmic mutations in mtDNA. The authors tested 6 protocols and found that full-length scRNA-seq methods showed better coverage of the mitochondrial genome than 3'-end-directed methods, with Smart-Seq2 having the best performance. 
• The authors performed whole-genome sequencing and single-cell RNA-seq simultaneously for single cells using SIDR, finding that several mutations were highly heteroplasmic in RNA, but not present in the genome, suggesting: RNA editing, transcription errors or technical errors in sc-RNA seq (Fig 2B). 
• To investigate inter- and intra-individual heterogeneity in mtDNA mutations, the authors analysed bulk RNA-seq data from 8.8k samples, spanning 49 tissues from at least 25 donors, as well as 426 donors with at least 10 tissues (GTEx project). 
• The authors found 2.7k mutations that were tissue-specific within an individual donor at a minimum of 3% heteroplasmy 
• Typically, ~25% of total mRNA originates from the mitochondrial genome across tissues, although this can be much larger in tissues such as the brain and heart. Tissues with a large proportion of mitochondrial mRNA tend to show very large variability -- see Fig 4B.
• Mitochondrial mutations around 10% are not uncommon across the whole mitochondrial genome (Fig 4D) and somatic mtDNA mutations with levels as low as 5% heteroplasmy can be stably propagated and serve as clonal markers in primary human cells.
• Every tissue had at least one tissue-specific mtDNA mutation across all individual donors, which likely arose via somatic mutation in a tissue-specific manner
• The authors used primary hematopoietic stem cells from two individual donors, and found that the mtDNA mutation profile separates single cells according to their donor of origin, as well as their single-cell-derived colony of origin via highly heteroplasmic mtDNA mutations.
• The authors performed bulk ATAC-seq and scRNA-seq on cells from colorectal adenocarcinoma primary tumor resection. Upon sequencing 238 cells, the authors found 12 distinct clusters of mtDNA mutations, suggesting clonal heterogeneity. 
• The authors provide an improved mutation detection framework, where mutation are first identified through bulk sequencing, and then called in scRNA-seq data. 

CONCLUSION AND OBSERVATIONS

• A potential limitation of inferring cell lineage from mtDNA sequence data comes from horizontal transfer of mtDNA between cells. However, the authors show that horizontal transfer would have to be relatively large to confound their analysis.
• Mapping the phenotypic impact of such genotypic diversity remains an open challenge.
• The authors use techniques for which reads mapping to the  mitochondrial genome are usually considered an unwanted by-product. Using assays focussed on the mitochondrial genomescan reduce costs and increase coverage.

The exceptional longevity of the naked mole‐rat may be explained by mitochondrial antioxidant defenses

https://onlinelibrary.wiley.com/doi/full/10.1111/acel.12916

Daniel Munro, Cécile Baldy, Matthew E. Pamenter, Jason R. Treberg

TWO THEORIES OF AGEING

The oxidative damage theory of ageing postulates that  a slow and steady accumulation of oxidative damage to macromolecules, which increases with age, causes the decline of physiologic functions. The oxidative damage is caused by reactive oxygen species (ROS) of mitochondrial origin.

The mitochondrial oxidative stress hypothesis states that ageing is primarily driven by loss of mitochondrial function with time, caused by oxidative stress. This theory stems from the fact that ROS are mostly released inside mitochondria, therefore directly exposing them to damage.

THE PRESENT STUDY

Naked mole-rat (NMR) can live >30 years in lab conditions, with a very long healthy lifespan, in comparison to <4 years for mice. Studies have shown that NMRs are subject to extensive oxidative damage (evidence found in liver cells) and high ROS productions, as much as mice. Therefore, their longevity has been widely used to contradict the oxidative damage theory of ageing. The mitochondrial oxidative stress hypothesis cannot explain these observations.

The authors, writing in Aging Cell, showed that NMRs mitochondria are much more efficient than mice's in consuming ROS. They also find evidence that skeletal muscle and heart mitochondria of mice and NMRs produce similar quantities of ROS.  Therefore, the authors conclude that the marked difference in longevity between the two species is to be attributed to the much greater capacity of NMRs mitochondria to clean ROS.

This finding supports the mitochondrial oxidative stress hypothesis, without positing that NMRs mitochondria produce less ROS. Further research could tell whether other long-lived species share this greater mitochondrial detoxifying capacity.

Mechanisms of organelle biogenesis govern stochastic fluctuations in organelle abundance

Shankar Mukherji, Erin K O'Shea

https://elifesciences.org/articles/02678

• Modelled the dynamics of organelle biogenesis, finding that fluctuations in organelle abundance depend strongly on the specific mechanisms that influence organelle number
• Model predicts the experimentally measured size of the Golgi apparatus and vacuole abundance fluctuations
• Work provides a general framework for exploring stochastic organelle biogenesis

Excessive Cell Growth Causes Cytoplasm Dilution And Contributes to Senescence

https://www.sciencedirect.com/science/article/pii/S0092867419300510

Gabriel E. Neurohr, Rachel L. Terry, Jette Lengefeld, Megan Bonney, Gregory P. Brittingham, Fabien Moretto, Teemu P. Miettinen, Laura Pontano Vaites, Luis M. Soares, Joao A. Paulo, J. Wade Harper, Stephen Buratowski, Scott Manalis, Folkert J. van Werven, Liam J. Holt, Angelika Amon

• Cells of a particular type tend to display a relatively narrow range of cell sizes (relative to the orders-of-magnitude difference in cell size between cells of different types). 
• When cell cycle is blocked in budding yeast, cells continue to grow. The authors were able to reversibly arrest the cell cycle by perturbing a particular gene (CDC28), providing them with a x12-fold variation in cell volume. Note that denying these cells with glucose, or applying cyclohexamide, prevented the mutants from growing large.
• When allowed to re-enter the cell cycle, larger cells proliferated more slowly, and delays cell cycle progression.
• The authors observed that cell cycle regulators are produced at a lower rate in oversized cells (although the pool-size was comparable to normal cells).
• When cells exceeded ~200 fL, cellular growth shifted from exponential to linear
• In the linear growth regime, cell volume increased faster than total RNA and protein, suggesting dilution of cellular macro-molecules. Direct measurement of cellular density showed a 36% reduction in total cell density, largely explained by reductions in protein and RNA mass.
•  Transcriptome and proteome analysis suggested that general transcription and translation machineries becomes limiting in large cells.
• Using nocodazole to generate diploid cells, large diploid cells grew faster than large haploid cells, and also progressed faster through the cell cycle. The authors therefore suggest that the nDNA:(cytoplasmic volume) ratio is what limits cell growth in oversized cells.
• The authors found that the majority of old yeast cells (>16 cell divisions) were >200 fL and display many of the phenotypes of oversized cells. 
• Excessive increase in cell size was sufficient to reduce lifespan
• The authors tested many of these observations in human fibroblasts.

Thoughts
----------------------

• Some of the findings in here, especially relating to cellular growth rates, remind me of this
• Quantifying single-cell mtDNA copy number in this system would be extremely interesting! 

Dimers of mitochondrial ATP synthase induce membrane curvature and self-assemble into rows

Thorsten B. Blum, Alexander Hahn, Thomas Meier, Karen M. Davies, and Werner Kühlbrandt

 

• ATP synthase is known to form dimers which form rows along curved ridges of mitochondrial cristae
• It has been suggested previously through computer simulation that these rows of ATP synthase cause local curvature
• This study shows experimentally, for the first time, that ATP synthase dimers spontaneously assemble into rows, and that these rows bend the membrane.
• The authors suggest that assembly of ATP synthase dimers into rows is likely the first step in the formation of cristae

Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells

https://doi.org/10.1016/j.cmet.2018.10.014

Bajzikova M, Kovarova J, Coelho AR, Boukalova S, Oh S, Rohlenova K, Svec D, Hubackova S, Endaya B, Judasova K, Bezawork-Geleta A, Kluckova K, Chatre L, Zobalova R, Novakova A, Vanova K, Ezrova Z, Maghzal GJ, Magalhaes Novais S, Olsinova M, Krobova L, An YJ, Davidova E, Nahacka Z, Sobol M, Cunha-Oliveira T, Sandoval-Acuña C, Strnad H, Zhang T, Huynh T, Serafim TL, Hozak P, Sardao VA, Koopman WJH, Ricchetti M, Oliveira PJ, Kolar F, Kubista M, Truksa J, Dvorakova-Hortova K, Pacak K, Gurlich R, Stocker R, Zhou Y, Berridge MV, Park S, Dong L, Rohlena J, Neuzil J.

• The authors graft cancer cells lacking mtDNA (ρ⁰) onto mice, and show horizontal transfer of mtDNA into the cancer cells after a lag period. After the transfer of mtDNA, a tumour subsequently develops. 
• The authors show that OXPHOS-derived ATP is not essential for tumorigenesis 
• Pyrimidine biosynthesis is dependent on respiration, and is required for cell-cycle progression

Mitochondrial complex III is essential for suppressive function of regulatory T cells

https://doi.org/10.1038/s41586-018-0846-z

Samuel E. Weinberg, Benjamin D. Singer, Elizabeth M. Steinert, Carlos A. Martinez, Manan M. Mehta, Inmaculada Martínez-Reyes, Peng Gao, Kathryn A. Helmin, Hiam Abdala-Valencia, Laura A. Sena, Paul T. Schumacker, Laurence A. Turka & Navdeep S. Chandel

• Regulatory T-cells (T-regs) are a sub-population of T cells (T cells being a kind of immune cell) which have immunosuppressive activities. They tend to down-regulate the induction and proliferation of effector T cells.
• In this study, the authors ablate complex III in T-regs of mice, and show that this induces fatal inflammatory disease early in life. 
• Mice lacking complex III in T-regs displayed a loss of ability to downregulate T-cell activity, without affecting T-reg proliferation and survival. 
• Loss of complex III in T-regs was associated with increased DNA methylation

How mitochondria can vary, and consequences for human health

Mitochondria are components of the cell which are involved in generating “energy currency” molecules called ATP across much of complex life. Since many mitochondria exist within single cells (often hundreds or thousands), it is possible for the characteristics of individual mitochondria to vary within cells, and within tissues. This variation of mitochondrial characteristics can affect biological function and human health.

Since mitochondria possess their own, small, circular, DNA molecules (mtDNA), we can split mitochondrial characteristics into two categories: genetic and non-genetic. In our review, we discuss a number of aspects in which mitochondria vary, from both genetic and non-genetic perspectives. 

In terms of mitochondrial genetics, the amount of mtDNA per cell is variable. When a cell divides, its daughters receive a share of its parents mtDNA, but the split isn’t precisely 50/50, so cell division can cause variability in the number of mtDNAs per cell. As mtDNAs are replicated and degraded over time, errors in the copying process may give rise to mtDNA mutations, which may spread throughout a cell. Factors such as: the total amount, the rate of degradation/replication, the mean fraction of mutants, and the extent of fragmentation in the mitochondrial network, can all influence how variable the fraction of mutated mtDNAs becomes through time (see here for a preview of some upcoming work on this topic). The total amount, and mutated fraction of mtDNAs, are implicated in diseases such as neurodegeneration, as well as the ageing process.

Apart from genetic variations, there are many non-genetic features of mitochondria which also vary within and between cells. Changes in mtDNA sequence can change the amino-acid sequence of the proteins encoded by mtDNA, causing structural changes in the molecular machines which generate ATP. The shape of the membranes of mitochondria are also highly variable, and respond to mitochondrial activity through quantities such as pH, where mitochondrial activity itself may depend on mtDNA sequence. The previous two examples (mitochondrial protein and membrane structure) demonstrate how the genetic state of mitochondria may influence their non-genetic characteristics. Mitochondrial non-genetic characteristics may also influence the genetic state: for instance, mitochondrial membrane potential can influence the probability of a mitochondria being degraded, along with its mtDNA.

The inter-dependence of genetic and non-genetic characteristics demonstrate the complex feedback loops linking these two aspects of mitochondrial physiology. We suggest here that, since changes in mitochondrial genetics occur more slowly than most physical aspects of mitochondrial physiology, understanding mitochondrial genetics may be especially important in explaining phenomena such as ageing, which appears to be closely related to mitochondrial heterogeneity. You can freely access our work, which has recently been published in Frontiers in Genetics, as “Mitochondrial Heterogeneity” https://www.frontiersin.org/articles/10.3389/fgene.2018.00718/full Juvid, Iain and Nick.

 

Investigating mitonuclear interactions in human admixed populations

https://www.nature.com/articles/s41559-018-0766-1

Arslan A. Zaidi & Kateryna D. Makova

• The authors explore signatures of mitonuclear incompatibility and coevolution in six admixed human populations from the Americas
• They hypothesize that incompatibility might arise between e.g. mtDNA origins of replication and nuclear-encoded mtDNA replication machinery and therefore, might lead to a decrease in mtDNA replication efficiency. The authors therefore predict that if mito/nuclear discordance is increased in admixed individuals, mtDNA copy number may consequently decrease.
• Given two admixed human populations with different mitochondrial haplotypes, if all females are from population 1, and all males from population 2, inherited autosomal loci will be a mixture of the two populations whereas the mtDNA will be purely from population 1. This may place selection in favour of nuclear-encoded mitochondrial genes from population 1, and such progeny may suffer mito-nuclear mismatch (see Fig 1b). 
• The authors found statistically significant negative correlation between mtDNA copy number and mitonuclear DNA discordance in admixed individuals, although the relationship was rather noisy (Fig 3a, R^2=0.04).
• They find significant enrichment of ancestry at nuclear-encoded mitochondrial genes towards the source populations contributing the most prevalent mtDNA haplogroups, indicating compensatory selective effects.

Mitochondrial Populations Exhibit Differential Dynamic Responses to Increased Energy Demand during Exocytosis In Vivo

https://www.sciencedirect.com/science/article/pii/S2589004218302669

Natalie Porat-Shliom, Olivia J. Harding, Lenka Malec, Kedar Narayan, and Roberto Weigert

• This study leverages in vivo visualisation of mitochondrial physiology in mouse salivary epithelium, in live animals through intravital microscopy (see here for further fascinating work in this system).
• The authors generate videos of mitochondrial dynamics, at single cell resolution, in all 3 spatial dimensions.
• The authors find evidence for two distinct mitochondrial populations existing within secretory cells: one juxtaposed to the plasma membrane, and another dispersed throughout the cytosol. These populations differ in their motility and propensity to undergo mitochondrial fusion/fission.
• The authors found that increasing energy demand in these cells enhanced fusion and motility in central mitochondria

Memory of ancestral mitochondrial stress

https://www.nature.com/articles/s41556-018-0255-4

Sarah-Lena Offenburg, Marcos Francisco Perez and Ben Lehner

A WORD ON EPIGENETIC MODIFICATIONS
There are two main types of epigenetic modifications, DNA methylation and histone modifications.

In DNA methylations, a methyl group is added to DNA. These reactions are catalysed by enzymes known as DNA methyltransferases. This modification result in the creation binding sites for other proteins, which bind and recruit or are associated with other proteins which can act on histones (determining histone modifications, see below).In eukaryotes, the most prevalent DNA methylation concerns cytosine nucleotides and gives origin to 5-methylcytosine.

Histone modifications affect the DNA-protein interactions, modifying the structure of chromatin (mixture of DNA and proteins which form chromosomes). This, in turn, alters the ability for a gene to be transcribed and expressed. 

THE RECENT FINDING

Dna methylation was thought to be absent in the roundworm C. elegans, since its genome does not contain 5-methylcytosine. Another methylation, N6-methyldeoxyadenine (6mA) was recently detected in C. elegans (and other species), but its functions remain elusive.

In a recent work, published in Nature Cell Biology, Ma et al. show that C. elegans can inherit resistance to stress and give evidence for the involvement of 6mA into this process.

The authors used antimycin, an antibiotic, to stress the mitochondria of the roundworm. The effect of antimycin is to inhibit the mitochondrial respiratory chain and that, in turn, slows down the development of worms. 

It was observed the progeny of animals exposed to the antibiotic developed faster when exposed to the same stressor. Unexposed offspring was protected up to four generations.

Interestingly, the resistance is not inherited through mitochondria themselves, since it can also be transmitted through male parents.

The authors found that the worms defective in a specific histone modification (H3K4me3) were unable to inherit resistance. A previous study in C. elegans showed a crosstalk between H3K4me3 and the methylation 6mA. Moreover, animals deficient in a known 6mA me methyltransferase were unable to transmit the resistance.

Open questions remain about the precise roles of 6mA and H3K4me3 in the observed phenomenon.

The involvement of mitochondria is important because C. elegans may be exposed to bacteria-induced mitochondrial stress in its natural habitat, which makes the finding more relevant. The inheritance of this stress resistance is one of the few documented cases of a trans-generational memory of a kind of stimulus which can occur in nature.