Wednesday, 14 February 2018

Myosin VI-Dependent Actin Cages Encapsulate Parkin-Positive Damaged Mitochondria

Antonina J. Kruppa,Chieko Kishi-Itakura, Thomas A. Masters, Joanna E. Rorbach, Guinevere L. Grice, John Kendrick-Jones, James A. Nathan, Michal Minczuk, Folma Buss

  • The authors identify a protein MYO6 which triggers the formation of F-actin cages to form around damaged mitochondria (mitochondria were damaged using a variety of pharmacological means)
  • These cages form a physical barrier, preventing damaged mitochondria from refusing with the network
  • MYO6 interacts with other proteins known to recruit autophagosomes to damaged mitochondria

Friday, 9 February 2018

Affinity purification of cell-specific mitochondria from whole animals resolves patterns of genetic mosaicism

Arnaud Ahier, Chuan-Yang Dai, Andrea Tweedie, Ayenachew Bezawork-Geleta, Ina Kirmes & Steven Zuryn

  • The authors demonstrate a technique called Cell-specific mitochondrial affinity purification (CS-MAP) to yield intact, functional, mitochondrial with >96% enrichment, >96% purity at single-cell and single-animal resolution in C. elegans.
  • CS-MAP consists of tagging the outer mitochondrial membrane protein TOMM-20 with a fluorophore and a particular epitope (which is something that an antibody can bind to) called HA. They placed the fusion protein under the control of a tissue-specific promoter so that e.g. muscle-specific mitochondria could be tagged. Mitochondria can then be purified using magnetic beads coated with anti-HA antibody and performing immunoprecipitation.
  • The authors crossed worms containing the CS-MAP construct with animals containing mitochondrial DNA deletions, and analysed the heteroplasmy in the individual mitochondria purified from different cell types and compared this to homogenate heteroplasmy across the whole animal. The authors found that intestine and neurons had significantly lower heteroplasmy than the homogenate, whereas the germline had significantly higher heteroplasmy than the homogenate.
  • The authors also quantified mtDNA copy number per mitochondrion in a number of different tissues, finding that the germline had ~3.5 mtDNAs per mitochondrion whereas tissues such as neurons and the intestine had ~1.5 mtDNAs per mitochondrion.
  • Using three-dimensional reconstruction from fluorescence images, the authors found that individual germ cells contained 71.2+/-6.5 mtDNAs per cell, whereas neurons contained 14.4+/-0.5 mtDNAs per cell.
  • The authors suggest that mtDNA turnover is higher in germ cells, which may account for their observations of increased copy number and heteroplasmy in the germline

Check out a pre-print from our group with some ideas on how increases in copy number may be related to heteroplasmy, and thoughts about comparisons between homogenate heteroplasmy and cellular heteroplasmy

An energetic view of stress: Focus on mitochondria

Martin Picard, Bruce S McEwen, Elissa S Epel & Carmen Sandi

In this review, the authors discuss the link between mitochondria and mental stress. 

  • Allostasis is defined as the active (i.e. energy-requiring) process of achieving stability, or homeostasis, through physiological or behavioural change. This includes neuroendocrine, autonomic, epigenetic, metabolic and immune changes, and is generally a short-term adaptation when regulated in a healthy setting.
  • When allostatic mediators are not turned off, these same mediators can cause unhealthy changes in the brain and body: these are the pathophysiological consequences of stress. The authors refer to "allostatic load" as the pathophysiological consequences of chronic dysregulation of allostatic mediators.
  • Metabolic intermediates that are the substrates or co-factors for epigenetic modifications are all derived from the Krebs cycle and other metabolic pathways within mitochondria. Some examples are discussed within the review. Hence, both the addition and removal of epigenetic marks are metabolically/mitochondrially regulated.
  • Mitochondria are the site of synthesis for all steroid hormones, including glucocorticoids such as cortisol (the archetypal stress hormone), androgens such as testosterone and estrogens such as estriol. Norepinephrine and epinephrine are also hormones (called catecholamines) which are released in response to certain stressors. Enzymes involved in the degradation of catecholamines (MAO-A and MAO-B) are anchored to the outer mitochondrial membrane.
  • Glucocorticoids (GCs) increase blood glucose levels by acting on the liver, skeletal muscles and adipose tissue by targetting the glucocorticoid receptor (GR). In the liver, GR activation has been shown to induce chromatin remodelling (an epigenetic effect). In skeletal muscle, GCs antagonize several elements of insulin signalling, and inhibits the uptake of pyruvate by mitochondria.
  • Humans with higher circulating levels of cortisol under resting conditions also have higher levels of glucose, triglycerides and higher insulin resistance (essentially a pre-diabetic state). In mice, chronic GC administration results in glucose intolerance, elevated triglycerides, weight gain and depressive behaviour.
  • Under healthy conditions, GCs are associated with the proper maintainance of a diurnal cycle.
  • Some, but not all, synapses in many parts of the cerebral cortex turn over during the diurnal cycle. Interfering with the daily cycle of GCs can impair motor learning in humans.
  • An animal model of shift work caused dendrites to shrink in the prefrontal cortex and the animal to become cognitively rigid, as well as gaining weight and becoming insulin resistant.

Wednesday, 7 February 2018

Mitochondrial levels determine variability in cell death by modulating apoptotic gene expression

Silvia Márquez-Jurado, Juan Díaz-Colunga, Ricardo Pires das Neves, Antonio Martinez-Lorente, Fernando Almazán, Raúl Guantes & Francisco J. Iborra

Chemotherapies often leave a proportion of cancer cells behind. Even genetically identical cells grown in vitro show this effect, suggesting that there exists some level of non-genetic heterogeneity in cancer cells. 

Two well-known pathways are able to induce cell death: the intrinsic and extrinsic pathways. The intrinsic pathway (which does not involve signalling from outside of the cell) directly involves mitochondria. In contrast, the extrinsic pathway may be activated via the binding of specific ligands to cell death receptors on the plasma membrane of the cell and does not directly involve mitochondria. However, several proteins may participate in both the intrinsic and extrinsic pathways, meaning that these pathways have cross-talk. 

TNF-related apoptosis-inducing ligand (TRAIL) is a protein which may induce the extrinsic apoptosis pathway. When cells are treated with TRAIL, the authors observe that the fraction of cells which are killed saturates at 35% with the concentration of TRAIL, and that there is heterogeneity in the time to death at every concentration of TRAIL. The authors also observed (as noted previously by other authors) that sister cells tended to have the same fate and very similar times to death (Pearson correlation >0.8).

The authors investigated how mitochondrial content affected cell death propensity. They found that:
  • Cells with higher mitochondrial mass (as determined by Mitotracker Green with 24 hr live-cell imaging) were more likely to die under TRAIL (as well as other cell death inducing drugs such as CHX and DRB).
  • Cells show a weak correlation between time to death and mitochondrial mass (rho = -0.47) for intermediate TRAIL concentrations.
  • After sorting cells into mito-high and mito-low fractions, (a fold-change of ~x5 between fractions), mito-high fractions had ~x3 more RNA than mito-low.
  • Mitochondrial mass contributed to around 50% of the total variability observed in proteins which participate in apoptosis (both pro-apoptotic and anti-apoptotic).
  • Including the observed correlation between mitochondrial mass and protein levels of apoptosis genes in a pre-existing mathematical model of the extrinsic cell death pathway in HeLa cells was able to recapitulate many of the authors' experimental observations.

The authors also investigated whether these results hold in real tumours, as opposed to cell culture conditions where environmental noise is minimised. The authors stained sections from colon cancer biopsies with antibodies against Aconitase 2 (for mitochondrial mass) and various cell death proteins. Whilst the mitochondrial contribution to variability for some apoptotic proteins was lost, others were retained (where pro-apoptotic proteins tended to have a higher correlation with mitochondrial mass than anti-apoptotic proteins).
Does cell volume confound any of these observations?

Friday, 19 January 2018

Identification of New Activators of Mitochondrial Fusion Reveals a Link between Mitochondrial Morphology and Pyrimidine Metabolism

Laia Miret-Casals, David Sebastián, José Brea, Eva M.Rico-Leo, Manuel Palacín, Pedro M.Fernández-Salguero, M. Isabel Loza, Fernando Albericio, Antonio Zorzano

  • The authors develop a high-throughput drug screen on HeLa cells to identify FDA-approved drugs which modulate the activity of the mitochondrial fusion protein MFN2, allowing the authors to find compounds which are able to upregulate MFN2 expression and induce mitochondrial fusion.
  • The authors identify leflunomide (a drug used for the treatment of arthritis) as the most potent modulator of MFN2 expression, inducing a 67% increase in MFN2 mRNA levels. The compound was also found to increase both MFN1 and MFN2 protein levels by a factor of ~x2. HeLa cells morphologically appeared to have higher fusion and mitochondrial membrane potential.
  • Leflunomide inhibits de novo synthesis of pyrimidines by inhibiting the mitochondrial inner membrane enzyme dihydroorotate dehydrogenase (DHODH).
  • As a consequence, leflunomide had anti-proliferative effects upon cells.
  • Uridine may be added to cells as an external source of pyrimidines. The authors found that addition of uridine to leflunomide-treated cells abolished the ability of leflunomide to induce MFN2 expression.
  • Another drug, brequinar sodium (BRQ), which is an inhibitor of DHODH also has similar properties to leflunomide.
  • Hence, inhibition of pyrimidine nucleotide synthesis may induce mitochondrial elongation via MFN induction.
  • DHODH uses ubiquinone as a substrate, which is converted to ubiquinol. Ubiquinol is substrate of complex III of the respiratory chain.
  • Inhibiting DHODH therefore inhibits the cycling of ubiquinone -> ubiquinol -> ubiquinone, and therefore inhibits the activity of complex III. 
  • The authors found that direct inhibition of complex III via the drug myxothiazol inhibited DHODH activity, reflecting the coupling between pyrimidine synthesis via DHODH and complex III activity.
  • Complex III inhibition via myxothiazol induced MFN induction and also elongation of mitochondria, even in MFN knockout cells. 
Overall, depletion of pyrimidine pools by complex III inhibition causes cell-cycle arrest and promotes mitochondrial elongation as an adaptive response to energetic stress.

Wednesday, 17 January 2018

Pervasive within-Mitochondrion Single-Nucleotide Variant Heteroplasmy as Revealed by Single Mitochondrion Sequencing

Morris J, Na YJ, Zhu H, Lee JH, Giang H, Ulyanova AV, Baltuch GH, Brem S, Chen HI, Kung DK, Lucas TH, O'Rourke DM, Wolf JA, Grady MS, Sul JY, Kim J, Eberwine J

This study looks at the prevalence of mutations in mitochondrial DNA within single mitochondria. The authors do this by collecting single mitochondria from cells with a micropipette, then perform PCR to amplify the copy number of DNA and finally illumina deep sequencing.

The authors collected 118 samples from the brains of lab mice (C57BL/6N strain), and found on average 3.9 single-nucleotide variants per mitochondrion with a standard deviation of 5.71 (although the mtDNA copy number per mitochondrion was not quantified). Some of the mutations observed are thought to be deleterious: for instance, a mutation found at position 9027 (G>A) encoding MT-CO3 (complex III of the respiratory chain) is a missense mutation, annotated to have moderate pathophysiological impact. The authors found 59 samples with this mutation. The intra-mitochondrial heteroplasmy was > 90% for 39 of these mitochondrial samples.

The authors also collected 21 samples of mitochondria from 8 different neurons from the brain of a 63-year-old female using residual tissue removed after surgery. From these samples, the authors found that within-mitochondrion heteroplasmy was ~50% less common in their human sample than in lab mice.  The authors also found that the within-mitochondrion heteroplasmy of different mitochondria in the same cell, and the inter-cellular heteroplasmy between cells, tended to be similar in their human sample but different in mouse.

The authors suggest that the differences between humans and mice are most likely due to the effect of only observing a single individual for their human experiment, but many individuals for mice. The authors found a large effect from the identity of the mother in determining the extent of within-mitochondrion heteroplasmy.

Wednesday, 10 January 2018

How cells adapt to progressive mitochondrial mutation

Mitochondria produce the cell's major energy currency: ATP. If mitochondria become dysfunctional, this can be associated with a variety of devastating diseases, from Parkinson's disease to cancer. Technological advances have allowed us to generate huge volumes of data about these diseases. However, it can be a challenge to turn these large, complicated, datasets into basic understanding of how these diseases work, so that we can come up with rational treatments.

We were interested in a dataset (see here) which measured what happened to cells as their mitochondria became progressively more dysfunctional. A typical cell has roughly 1000 copies of mitochondrial DNA (mtDNA), which contains information on how to build some of the most important parts of the machinery responsible for making ATP in your cells. When mitochondrial DNA becomes mutated, these instructions accumulate errors, preventing the cell's energy machinery from working properly. Since your cells each contain about 1000 copies of mitochondrial DNA, it is interesting to think about what happens to a cell as the fraction of mutated mitochondrial DNA (called 'heteroplasmy') gradually increases. We used maths to try and explain how a cell attempts to cope with increasing levels of heteroplasmy, resulting in a wealth of hypotheses which we hope to explore experimentally in the future.


The central idea arising from our analysis of this large dataset is that cells attempt to maintain the number of normal mtDNAs per cell volume as heteroplasmy initially increases from 0% mutant. We suggest they do this by shrinking their size. By getting smaller, cells are able to reduce their energy demands as the fraction of mutant mtDNA increases, allowing them to balance their energy budget and maintain energy supply = demand. However, cells can only get so small and eventually the cell must change its strategy. At a critical fraction of mutated mtDNA (h* in the cartoon above), we suggest that cells switch on an alternative energy production mode called glycolysis. This causes energy supply to increase, and as a result, cells grow larger in size again. These ideas, as well as experimental proposals to test them, are freely available in our latest publication in Biochemical Journal. Juvid, Iain and Nick.

Friday, 24 November 2017

Glucose feeds the TCA cycle via circulating lactate

Sheng Hui, Jonathan M. Ghergurovich, Raphael J. Morscher, Cholsoon Jang, Xin Teng, Wenyun Lu, Lourdes A. Esparza, Tannishtha Reya, Le Zhan, Jessie Yanxiang Guo, Eileen White & Joshua D. Rabinowitz

  • When oxygen is present, it is commonly thought that glucose (derived from the food we eat) is catabolised via glycolysis to pyruvate, which is then transported into mitochondria, fuelling the TCA cycle and oxidative phosphorylation. Alternatively, when oxygen is less available, glucose can be catabolised to lactate.
  • Although traditionally thought of as a waste product, it is becoming increasingly clear that lactate can itself be used as a fuel molecule
  • Here, the authors investigate the relative contribution of glucose and lactate to feeding the TCA cycle in mice, across various tissues
  • In fasting mice, the contribution of glucose to the TCA cycle is primarily via circulating lactate in all tissues except the brain
  • The circulatory turnover of lactate is the highest of all metabolites, exceeding that of glucose in both the fed and fasted state
  • In tumours, lactate is a primary TCA substrate

Wednesday, 22 November 2017

Structural Basis of Mitochondrial Transcription Initiation

Hauke S.Hillen, Yaroslav I.Morozov, Azadeh Sarfallah, Dmitry Temiakov, Patrick Cramer

  • Transcription of the mitochondrial DNA is a critical aspect of understanding mitochondrial physiology. Not only is this linked to the generation of protein, as per nuclear transcription, there is also a link with the actual replication of mtDNA. Many molecular players are involved in both of these processes. Yet, mitochondrial transcription is not well understood in its details.
  • The authors report crystal structures of the protein responsible for mtDNA transcription (mtRNAP) when attached to mtDNA.
  • TFAM tethers to mtRNAP to recruit the protein to the mtDNA promoter site.
  • TFB2M induces structural changes in mtRNAP to enable it to open mtDNA

Thursday, 16 November 2017

Abrogating Mitochondrial Dynamics in Mouse Hearts Accelerates Mitochondrial Senescence

Song M, Franco A, Fleischer JA, Zhang L, Dorn II GW

  • Mitochondrial fragmentation is often considered as harmful. This is often determined by inhibiting fusion fission proteins such as Mfn2 and Opa1. However, such conclusions are confounded by Mfn2 functioning in mitophagy and Opa1 functioning in cristae organisation. The authors sought to determine the role of fusion/fission dynamics in maintaining healthy heart function
  • The authors overexpressed the pro-fission protein Drp1 in cardiomyocytes (10 & 25 wild-type expression levels). This induced fragmentation of the network without affecting the expression of other mitochondrial proteins. The fragmented mitochondria appeared healthy
  • Through 93 weeks of age, Drp1 overexpression resulted in no phenotype
  • Previous work by the authors found that overexpression of Mfn2 induces enlargement of mitochondria in the heart, without any other clear phenotype
  • The authors conclude that increased or decreased mitochondrial size alone is not necessarily a mechanism of heart dysfunction
  • The authors then investigated a mouse model where Mfn1, Mfn2 and Drp1 expression could be switched off in the adult heart (since knockouts of these proteins are embryonic lethal)
  • Abolishing mitochondrial dynamics resulted in: fragmentation of the network, partial depolarisation of mitochondria, parkin aggregation and impaired mitophagy
  • Surprisingly, such mice were able to survive 14 weeks after mitochondrial dynamics abrogation (whereas a cardiac knockout of any single one of the 3 genes is rapidly lethal in mice)
  • The hearts of such mice were enlarged, and had a mitophagy defect resulting in suppressed elimination of defective mitochondria

Tuesday, 14 November 2017

Inertial picobalance reveals fast mass fluctuations in mammalian cells

David Martínez-Martín, Gotthold Fläschner, Benjamin Gaub, Sascha Martin, Richard Newton, Corina Beerli, Jason Mercer, Christoph Gerber & Daniel J. Müller

  • Use a highly sensitive balance to measure the mass of single or multiple adherent cells in culture conditions over days with millisecond time resolution and picogram mass sensitivity 
  • The mass of living mammalian cells varies by around 1-4% over timescales of seconds throughout the cell cycle
  • These mass fluctuations are linked to ATP synthesis and water transport
  • The balance works by oscillating a microcantilever immersed in cell media at the microcantilever's natural frequency. A cell is grown at the tip of the microcantilever. The frequency and amplitude of the vibrations of the microcantilever can be measured using a laser. These data can be used to infer the mass of the cell growing at the tip.
  • Blocking aquaporins reduced the amplitude of slow mass fluctuations (period ~17s) by a factor of 4
  • Inhibition of ATP synthesis in starved cells reduced the amplitude of slow mass fluctuations by a factor of ~4, and reduced the amplitude of fast mass fluctuations by around 1/3. 

Tuesday, 26 September 2017

Live imaging reveals the dynamics and regulation of mitochondrial nucleoids during the cell cycle in Fucci2-HeLa cells

Taeko Sasaki, Yoshikatsu Sato, Tetsuya Higashiyama, Narie Sasaki

  •  Authors investigate the dynamics of mitochondrial DNA replication through the cell cycle by labelling HeLa cells with Fucci markers (a fluorescent probe which changes color according to the cell cycle stage) and SYBR Green I (which stains mtDNA)
  • Observe that mitochondrial nucleoids often attach/detach from each other
  • Mitochondrial replication occurs throughout the cell cycle, but peaks during the S-phase

Tuesday, 19 September 2017

Mitochondrial fission facilitates the selective mitophagy of protein aggregates

Jonathon L. Burman, Sarah Pickles, Chunxin Wang, Shiori Sekine, Jose Norberto S. Vargas, Zhe Zhang, Alice M. Youle, Catherine L. Nezich, Xufeng Wu, John A. Hammer, Richard J. Youle

  • This study focuses on the interplay between mitophagy and mitochondrial fission. Whilst many studies have suggested that fission is required for mitophagy, there is mixed evidence on the issue
  • Authors express a mutant form of ornithine transcarbamylase (ΔOTC) in HeLa cells, which creates insoluable protein aggregates which localise to the mitochondrial matrix, inducing the mitochondrial unfolded protein response
  • This induces PINK1-Parkin-mediated mitophagy, to clear the ΔOTC aggregates
  • Mitochondria associated with Parkin were observed to fragment and traffic away from their parental mitochondrion. These mitochondria remain coated in Parkin
  • The authors overexpressed a dominant-negative mutant of the fission protein Drp1, effectively inhibiting fission. They found that, for both wild-type and ΔOTC, inhibition of fission did not reduce protein aggregate clearance. In fact, inhibition of mitochondrial fission fosters excessive PINK1-Parkin-mediated mitophagy of entire fused mitochondrial networks, even in the wild-type case. [The authors showed that Drp1-independent mitophagy was not dependent upon mitochondrial-derived vesicles (MDVs) through knockout of syntaxin 17, a protein involved in MDVs]
  • The authors suggest that mitochondrial fission (via Drp1) restricts mitophagic activity (via PINK1-Parkin) to specific, dysfunctional, mitochondrial subdomains by localising the PINK1-Parkin positive feedback loop away from healthy mitochondria

Tuesday, 12 September 2017

Promoting Drp1-mediated mitochondrial fission in midlife prolongs healthy lifespan of Drosophila melanogaster

Rana A, Oliveira MP, Khamoui AV, Aparicio R, Rera M, Rossiter HB, Walker DW 

  • Transient induction of Drp1-mediated fission for 7 days during midlife of the fruit fly D. melanogaster is sufficient to extend lifespan. (Note that Drp-1 upregulation in early life had no significant impact on longevity)
  • Short-term midlife Drp1 induction gave increased day-time physical activity levels, suggesting an extension of healthy lifespan, rather than prolonging frailty. These flies also had improved starvation resistance, improved fertility, and delayed intestinal aging.
  • In flight muscle, elongated mitochondrial morphology was associated with lowered mitochondrial membrane potential, accumulation of dysfunctional mitochondria, lowered OXPHOS complex activity, increased ROS and lowered respiration with aging. These phenotypes are reversed upon short-term midlife Drp1 induction.
  • These effects were not mediated by the mitochondrial unfolded protein response. 
  • Short-term midlife Drp1 induction reduced levels of protein aggregates in aged muscle and aged brain tissue
  • Disruption of mitophagy, via Atg1 inhibition, inhibits the anti-aging effects of midlife Drp1 induction.

Tuesday, 29 August 2017

Production of superoxide and hydrogen peroxide from specific mitochondrial sites under different bioenergetic conditions

Wong HS, Dighe PA, Mezera V, Monternier PA, Brand MD

  • Relative contributions of mitochondrial superoxide/ hydrogen peroxide production by different sites in the electron transport chain, under different conditions

Monday, 14 August 2017

Endocrine disruptors induce perturbations in endoplasmic reticulum and mitochondria of human pluripotent stem cell derivatives

Rajamani U, Gross AR, Ocampo C, Andres AM, Gottlieb RA, Sareen D

  • Study of the effect of common man-made chemicals (specifically endocrine distrupting chemicals, or EDCs)  on human-induced pluipotent stem cells
  • The authors suggest that exposure to perfluoro-octanoic acid (found in cookware), tributyltin (found in house dust), and butylhydroxytoluene (found in food additives) can induce endoplasmic reticulum stress, perturb inflammatory and cell-death signalling pathways (NF-kB and p53), diminish mitochondrial respiratory gene expression, spare respiratory capacity and ATP levels in stem cells.
  • Consequently, normal secretion of appetite control hormones is affected.
  • The authors provide this as mechanistic evidence that repeated exposure to these "obesogenic" endocrine distrupting chemicals in utero can alter some genetically pre-disposed individuals' normal metabolic control, setting them up for long-term obesity.

In vivo imaging reveals mitophagy independence in the maintenance of axonal mitochondria during normal aging

Cao X, Wang H, Wang Z, Wang Q, Zhang S, Deng Y, Fang Y

  • Study of mitophagy and aging in Drosophila
  • Mitochondria become fragmented in aged mitochondria
  • Lack of Pink1 or Parkin does not lead to the accumulation of axonal mitochondria or axonal degeneration
  • Knockdown of core mitphagy genes Atg12 or Atg17 has little effect on turnover of axonal mitochondria or axonal integrity suggesting that mitophagy is not necessary for axonal maintainence, regardless of whether it is Pink1-Parkin dependent
  • Adult onset of neuronal downregulation of fission-fusion but not mitophagy genes dramatically accelerated features of aging
  • Thought: Is this partly because Drosophila generally has a short lifespan and, in some sense, die before they have the chance to get old? Are these results observable in other, longer-lived, species?

Tuesday, 8 August 2017

Interesting papers

The Mitochondrial Basis of Aging
Nuo Sun, Richard J. Youle and Toren Finkel
Molecular Cell
  • An interesting review on the theory that mitochondrial decline contributes to ageing.
Mitochondrial Dysfunction Induces Senescence with a Distinct Secretory Phenotype
Christopher D. Wiley, Michael C. Velarde, Pacome Lecot, ..., Akos A. Gerencser, Eric Verdin, Judith Campisi
Cell Metabolism
  •  How mitochondrial dysfunction can induce senescence in proliferative cell types. Such cells have lower NAD+/NADH ratios. Progeroid mtDNA mutator mice accumulate sensescent cells with a mitochondrially-associated senescent secretory phenotype (MiDAS SASP).
Transit and integration of extracellular mitochondria in human heart cells
Douglas B. Cowan, Rouan Yao, Jerusha K. Thedsanamoorthy, David Zurakowski, Pedro J. del Nido, James D. McCully
  • Transplanting isolated mitochondria from healthy tissue into ischaemic heart tissue can be internalised within minutes, fuse to the mitochondrial network, decrease cell death, increase energy production and improve contractile function

Mitochondrial DNA heteroplasmy is shared between human liver lobes
Manja  Wachsmuth, Alexander  Hübner, Roland  Schröder, ..., Mark Stoneking
  • Heteroplasmy distributions in multiple liver lobes from the same individual suggest sharing of heteroplasmy
Segregation of mitochondrial DNA mutations in the human placenta: implication for prenatal diagnosis of mtDNA disorders
Pauline Vachin, Elodie Adda-herzog, Gihad Chalouhi, ..., Julie Steffann
Journal Medical Genetics
  • Distribution of heteroplasmy for multiple samples per individual in the placenta
Mammalian Mitochondria and Aging: An Update
Timo E.S. Kauppila, Johanna H.K. Kauppila, Nils-Göran Larsson 
Cell Metabolism
  • Review on the mitochondrial theory of ageing 
Optogenetic control of mitochondrial metabolism and Ca2+ signaling by mitochondria-targeted opsins 
Tatiana Tkatch, Elisa Greotti, Gytis Baranauskas, ... and Israel Sekler
  • Reversible, tunable, optogenetic control of mitochondrial membrane potential using channelrhodopsins
Age-Associated Loss of OPA1 in Muscle Impacts Muscle Mass, Metabolic Homeostasis, Systemic Inflammation, and Epithelial Senescence
Caterina Tezze, Vanina Romanello, Maria Andrea Desbats, Gian Paolo Fadin, ...,  Luca Scorrano, Marco Sandri
Cell Metabolism
  •   Disturbing the mitochondrial network through OPA1 deletion in muscle may induce faster ageing across distal organs

Mesenchymal stem cells sense mitochondria released from damaged cells as danger signals to activate their rescue properties
 Meriem Mahrouf-Yorgov, Lionel Augeul, Claire Crola Da Silva, Maud Jourdan, ..., Anne-Marie Rodriguez
Cell Death & Differentiation
  •   Mesenchymal cells can 'sense danger' by taking up and degrading mitochondria from stressed cells

The mitochondrial respiratory chain is essential for haematopoietic stem cell function
Elena Ansó,  Samuel E. Weinberg,  Lauren P. Diebold,  Benjamin J. Thompson, ..., Navdeep S. Chandel
Nature Cell Biology
  • OXPHOS is required for differentiation of haematopoietic stem cells 
Heteroplasmic Shifts in Tumor Mitochondrial Genomes Reveal Tissue-specific Signals of Relaxed and Positive Selection
Grandhi S, Bosworth C, Maddox W, Sensiba C, Akhavanfard S, Ni Y, LaFramboise T
Human Molecular Genetics
  •  Signs of positive selection for mitochondrial DNA mutations in certain cancers

Interesting papers

MitoNEET-dependent formation of intermitochondrial junctions
Alexandre Vernay, Anna Marchetti, Ayman Sabra, Tania N. Jauslin, Manon Rosselin, Philipp E. Scherer, Nicolas Demaurex, Lelio Orci, and Pierre Cosson 
  • MitoNEET, a factor which contributes to the formation of inter-mitochondrial junctions is knocked out. Network becomes more fragmented and there are fewer mitochondria.

Hypothalamic stem cells control ageing speed partly through exosomal miRNAs
Yalin Zhang, Min Soo Kim, Baosen Jia, Jingqi Yan, Juan Pablo Zuniga-Hertz, Cheng Han & Dongsheng Cai
  • Secretions from stem cells in the hypothalamus, consisting of exosomes containing miRNAs, can slow down ageing phenotypes. The hypothalamus becomes an inflammatory environment with age. Modifying stem cells to become resistant to inflammation (via the NF-kB pathway) and implanting them into brains of mid-aged mice can slow down ageing.

Increased mitochondrial fusion allows the survival of older animals in diverse C. elegans longevity pathways
Snehal N. Chaudhari & Edward T. Kipreos
Nature Communications
  • Mitochondrial fusion is permissive of extended lifespan, but not sufficient.

Selective removal of deletion-bearing mitochondrial DNA in heteroplasmic Drosophila
Nikolay P. Kandul, Ting Zhang, Bruce A. Hay & Ming Guo
Nature Communications
  • Mitophagy is able to alter heteroplasmy levels of a deleterious mtDNA deletion mutation in Drosophila. Overexpression of PINK1 and Parkin produce large reductions in the frequency of deleterious mutations.

Saturday, 10 June 2017

Mitochondrial heterogeneity, metabolic scaling and cell death

Juvid Aryaman, Hanne Hoitzing, Joerg P. Burgstaller, Iain G. Johnston and Nick S. Jones

Cells need energy to produce functional machinery, deal with challenges, and continue to grow and divide -- these activities and others are collectively referred to as "cell physiology". Mitochondria are the dominant energy sources in most of our cells, so we'd expect a strong link between how well mitochondria perform and cell physiology. Indeed, when mitochondrial energy production is compromised, deadly diseases can result -- as we've written about before.

The details of this link -- how cells with different mitochondrial populations may differ physiologically -- is not well understood. A recent article shed new light on this link by looking at a measure of mitochondrial functionality in cells of different sizes. They found what we'll call the "mitopeak" -- mitochondrial functionality peaks at intermediate cell sizes, with larger and smaller cells having less functional mitochondria. The subsequent interpretation was that there is an “optimal”, intermediate, size for cells. Above this size, it was suggested that a proposed universal relationship between the energy demands of organisms (from microorganisms to elephants) and their size predicts the reduction in the function of mitochondria. Smaller cells, which result from a large cell having divided, were suggested to have inherited their parent's low mitochondrial functionality. Cells were predicted to “reset” their mitochondrial activity as they initially grow and reach an “optimal” size.

We were interested in the mitopeak, and wondered if scientifically simpler hypotheses could account for it. Using mathematical modelling, our idea was to use the observation that as a cell becomes larger in volume, the size of its mitochondrial population (and hence power supply) increases in concert. We considered that a cell has power demands which also track its volume, as well as demands which are proportional to surface area and power demands which do not depend on cell size at all (such as the energetic cost of replicating the genome at cell division, since the size of a cell's genome does not depend on how big the cell is). Assuming that power supply = demand in a cell, then bigger cells may more easily satisfy e.g. the constant power demands. This is because the number of mitochondria increases with cell volume yet the constant demands remain the same regardless of cell size. In other words, if a cell has more mitochondria as it gets larger, then each mitochondrion has to work less hard to satisfy power demand.

To explain why the smallest cells also have mitochondria which do not appear to work hard, we suggested that some smaller cells could be in the process of dying. If smaller cells are more likely to die, and if dying cells have low mitochondrial functionality (both of these ideas are biologically supported), then, by combining this with the power supply/demand picture above, the observed mitopeak naturally emerges from our mathematical model.

As an alternative model, we also suggested that the mitopeak could come entirely from a nonlinear relationship between cell size and cell death, with mitochondrial functionality as a passive indicator of how healthy a cell is. This indicates the existence of multiple hypotheses which could explain this new dataset.

Interestingly, we also found that the mitopeak could be an alternative to one aspect of a model we used some time ago to explain a different dataset, looking at the physiological influence of mitochondrial variability. Then, we modelled the activity of mitochondria as a quantity that is inherited identically by each daughter cell from its parent, plus some noise -- noting that this was a guess at the true behaviour because we didn't have the data to make a firm statement. We needed this relationship because observed functionality varied comparatively little between sister cells but substantially across a population. The mitopeak induces this variability without needing random inheritance of functionality, and may thus be the refined picture we've been looking for. These ideas, and suggestions for future strategies to explore the link between mitochondria and cell physiology in more detail, are in our new BioEssays article here. Juvid, Nick, and Iain.

Mirrored from here

Monday, 27 March 2017

Dynamin-Related Protein 1-Dependent Mitochondrial Fission Changes in the Dorsal Vagal Complex Regulate Insulin Action

Beatrice M. Filippi, Mona A. Abraham, Pamuditha N. Silva, Mozhgan Rasti, Mary P. LaPierre, Paige V. Bauer, Jonathan V. Rocheleau, Tony K.T. Lam
Type 2 diabetes is a condition where the body does not produce enough, or is resistant to, insulin. In this study, the authors investigated the role mitochondrial dynamics plays in insulin resistance and glucose regulation. As well as its clinical consequences, this study offers to shed light on the relationship between glucose homeostasis and mitochondrial functionality.
In healthy rodents, the hypothalamus and dorsal vagal complex (DVC) regulate glucose homeostasis in the liver (which is where excess glucose is stored). However, after a high fat diet (HFD) as short as 3 days, this regulation is disrupted. This link between the DVC and high-fat feeding has been poorly understood. 
The authors found that, after a HFD, DVC neuronal cells in rats had a higher density of mitochondria, and these mitochondria were less elongated, shorter and less branched. 
The authors tested the effect of providing the 3-day HFD rats with an infusion of  MDIVI-1, which is an inhibitor of the mitochondrial fission factor Drp-1 (by blocking its translocation from the cytosol into the mitochondria). The authors found that, upon infusion, mitochondrial morphology was restored to wild-type levels, the glucose infusion rate increased to normal levels, as well as the glucose production rate decreasing to normal levels. This was confirmed through molecular inhibition of Drp-1 via adenoviral-mediated inhibition. Furthermore, inducing overexpression of Drp-1 in the DVC of rats which were fed normally induced insulin resistance and recapitulated the effects of HFD.

The authors found that endoplasmic reticulum (ER) stress was necessary and sufficient  to induce DVC-mediated insulin resistance, and that ER stress was a consequence of mitochondrial fission.

Thoughts: Are these associations still observed on a long-term high-fat diet, rather than a 3-day alteration to diet?

Tissue-Specific Mitochondrial Decoding of Cytoplasmic Ca2+ Signals Is Controlled by the Stoichiometry of MICU1/2 and MCU

Paillard M, Csordás G, Szanda G, Golenár T, Debattisti V, Bartok A, Wang N, Moffat C, Seifert EL, Spät A, Hajnóczky G

Mitochondrial respiration is sensitive to the concentration of calcium in the cytoplasm, acting as an important control mechanism of respiration rate. It is known that different tissues have different responses to the presence of calcium. For instance, in the liver, calcium oscillations in the cytoplasm tend to be low frequency and are effectively propagated to intra-mitochondrial calcium concentrations. However, in the heart, oscillations are high frequency and are integrated into a more continuous intra-mitochondrial calcium signal.

Here, the authors investigated the difference in mitochondrial response to calcium concentration in different tissues by measuring the relative stoichiometry of two protein components of the mitochondrial calcium uniporter: MCU (a calcium pore unit) and MICU1 (a Calcium-sensing regulator). The authors found that, in heart tissue, a low MICU1 to MCU ratio is present, which results in a low cytoplasmic calcium threshold for mitochondrial accumulation of calcium, relative to liver tissue. Furthermore, heart tissue displayed a more shallow response curve to cytoplasmic calcium, suggesting lower cooperativity in cardiac tissue, relative to liver tissue. Therefore, the ratio of MICU1:MCU controls the tissue-specific response to cytoplasmic calcium.

Monday, 30 January 2017

Tunneling nanotubes promote intercellular mitochondria transfer followed by increased invasiveness in bladder cancer cells

Jinjin Lu, Xiufen Zheng, Fan Li, Yang Yu, Zhong Chen, Zheng Liu, Zhihua Wang, Hua Xu, Weimin Yang

In cell culture, cells have been observed to create long, thin, protrusions to connect to other cells and transfer material, including entire organelles such as mitochondria. These protrusions are called tunneling nanotubes (TNTs). In this study, the authors co-culture two kinds of urothelial bladder cancer cells: T24 (highly invasive) and RT4 (less invasive) cells. The authors observed the formation of TNTs between the two cell types and mitochondrial exchange between the cell types.

The authors found that the RT4 cells became more motile after intercellular mitochondria trafficking from T24 cells (RT4-Mito-T24) by around a factor of 2 relative to RT4 cells. Xenograft tumours from RT4-Mito-T24 cells were also around twice as large as T24 cells after ~30 days of growth.

This shows that transfer of material from a highly invasive cell type to a less invasive cell type results in increased invasive ability. It suggests that mitochondrial content may be the causal variable in determining invasive ability in this system.

Thoughts: This study adds to a growing body of evidence that mitochondrial content contributes to determining metastatic potential of cancer cells. What is it about these mitochondria that causes the increase in invasiveness? Are there other factors which are transferred through the TNTs? Is mitochondrial transfer necessary, or indeed sufficient, to see these effects? Interesting to note that the nuclear background of these cell types are presumably not the same -- to what extent can the nuclei be different between these cell types to observe the increase in invasiveness?

Wednesday, 7 December 2016

Selective removal of deletion-bearing mitochondrial DNA in heteroplasmic Drosophila

Nikolay P. Kandul, Ting Zhang, Bruce A. Hay & Ming Guo

Mitophagy is the process by which mitochondria are engulfed and degraded within the cell. It has long been thought that mitophagy has a role to play in quality control in the mitochondrial population, however it has remained unclear whether the effect can selectively degrade faulty organelles, or instead is an unbiased process, in vivo. A potential confounding effect is cell division where, simply by dividing, mutants can be driven to fixation through stochastic effects.

To address this question, the authors developed a Drosophila model which can inducibly generate a mitochondrial mutation which would otherwise be lethal if present at birth in the whole-body. This is done through the inducible expression of a mitochondrially-targetted restriction enzyme which cleaves mtDNA in Drosophila in two places, creating a 2584 bp deletion which disrupts or removes several important mitochondrial genes. The authors were able to induce expression of this restriction enzyme in a non-essential, energy-intense, post-mitotic tissue, namely the indirect flight muscle. This is an ideal tissue to study mitophagy, since its energy requirements imply the need for a healthy mitochondrial population, and is non-dividing so does not suffer from confounding effects from the stochastic nature of mtDNA dynamics.

Flies tended to accumulate ~76% heteroplasmy in the mitochondrial deletion by day 10 after hatching, stabilizing thereafter, with no large difference in mtDNA copy number. The flies had similar flight performances to wild-type animals, suggesting that the tissue may withstand high levels of heteroplasmy without phenotypic consequences.

The authors probed the effects of modulating the expression of genes which have been thought to play a role in mitophagy, and measured the resultant heteroplasmy. They investigated Atg1, Atg8a, Pink1 and Parkin, which all had the expected effects on heteroplasmy. Parkin overexpression caused ~71% reduction in heteroplasmy, and Atg1 caused ~72% reduction, these genes having the largest effect sizes. The authors found that inhibition of mitochondrial fusion through MFN silencing had a modest effect on heteroplasmy reduction (37%), which is expected if defective mitochondria are not allowed to re-enter the mitochondrial network. Interestingly, inhibiting ATP synthase from hydrolysing ATP and therefore maintaining mitochondrial membrane potential, through expression of ATPIF1, had a synergistic effect with MFN silencing, resulting in a 64% effect size.  This suggests that mitochondria with mutated mtDNAs may attempt to cheat the mitophagic system by consuming ATP to maintain their membrane potential and avoid detection.


These results show that mitophagy can be a selective process, and may be induced to have greater effect sizes. The key question is, if mitophagy is able to clear mitochondrial mutants, why do we see them at all in the wild-type case? What is the tradeoff that keeps mitophagy low? It would be interesting to see the lifespan of these flies upon induction of mitophagy. Are they more susceptible to other pathologies e.g. cancer or aging?

Tuesday, 1 November 2016

Evolution of Cell-to-Cell Variability in Stochastic, Controlled, Heteroplasmic mtDNA Populations

Iain G. Johnston and Nick S. Jones
Mitochondrial DNA (mtDNA) contains instructions for building important cellular machines. We have populations of mtDNA inside each of our cells -- almost like a population of animals in an ecosystem. Indeed, mitochondria were originally independent organisms, that billions of years ago were engulfed by our ancestor's cells and survived -- so the picture of mtDNA as a population of critters living inside our cells has evolutionary precedent! MtDNA molecules replicate and degrade in our cells in response to signals passed back and forth between mitochondria and the nucleus (the cell's "control tower"). Describing the behaviour of these population given the random, noisy environment of the cell, the fact that cells divide, and the complicated nuclear signals governing mtDNA populations, is challenging. At the same time, experiments looking in detail at mtDNA inside cells are difficult -- so predictive theoretical descriptions of these populations are highly valuable.

Why should we care about these cellular populations? MtDNA can become mutated, wrecking the instructions for building machines. If a high enough proportion of mtDNAs in a cell are mutated, our cells struggle and we get diseases. It only takes a few cells exceeding this "threshold" to cause problems -- so understanding the cell-to-cell distribution of mtDNA is medically important (as well as biologically fascinating). Simple mathematical approaches typically describe only average behaviours -- we need to describe the variability in mtDNA populations too. And for that, we need to account for the random effects that influence them.
​In our cells, signals from the "control tower" nucleus lead to the replication (orange) and degradation (purple) of mtDNA. These processes affect mtDNA populations that may contain normal (blue) and mutant (red) molecules. Our mathematical approach -- extending work addressing a similar but simpler system -- describes how the total number of machines, and the proportion of mutants, is likely to behave and change with time and as cells divide.
In the past, we have used a branch of maths called stochastic processes to answer questions about the random behaviour of mtDNA populations. But these previous approaches cannot account for the "control tower" -- the nucleus' control of mtDNA. To address this, we've developed a mathematical tradeoff -- we make a particular assumption (which we show not to be unreasonable) and in exchange are able to derive a wealth of results about mtDNA behaviour under all sorts of different nuclear control signals. Technically, we use a rather magical-sounding tool called "Van Kampen's system size expansion" to approximate mtDNA behaviour, then explore how the resulting equations behave as time progresses and cells divide.

Our approach shows that the cell-to-cell variability in heteroplasmy (the potentially damaging proportion of mutants in a cell) generally increases with time, and surprisingly does so in the same way regardless of how the control tower signals the population. We're able to update a decades-old and commonly-used expression (often called the Wright formula) for describing heteroplasmy variance, so that the formula, instead of being rather abstract and hard to interpret, is directly linked to real biological quantities. We also show that control tower attempts to decrease mutant mtDNA can induce more variability in the remaining "normal" mtDNA population. We link these and other results to biological applications, and show that our approach unifies and generalises many previous models and treatments of mtDNA -- providing a consistent and powerful theoretical platform with which to understand cellular mtDNA populations. The article is in the American Journal of Human Genetics here and a preprint version can be viewed here. (crossed from Evolution, Energetics & Noise)

Sunday, 23 October 2016

Cellular Allometry of Mitochondrial Functionality Establishes the Optimal Cell Size

Teemu P. Miettinen and Mikael Björklund

Cells in a population, despite having the same genetic content, are often very different from each other due to the stochastic nature of biological processes. An example is cellular size: some cells are big, some are small and some have an intermediate size. How does the size of a cell affect its functionality? Is there an optimal cell size? This paper focusses on how mitochondrial functionality changes with cell size.

It is known that if a cell is twice as big, it will approximately have twice as many mitochondria, keeping the mitochondrial density roughly constant. However, the expression of mitochondrial genes becomes less than twice as high, meaning that bigger cells express relatively less mitochondrial genes. This may mean that there is a particular cell size corresponding to optimal mitochondrial functionality.

In the paper, they use single cell flow cytometry to measure the size of about 10^5-10^6 cells. Additionally, the mitochondrial membrane potential per unit cell size (ΔΨ) is measured. The relationship between cell size and ΔΨ can then be investigated.

Some of the findings are:
  • Consistent with previous studied, mitochondrial mass increases linearly with cell size
  • ΔΨ first increases as cells get larger, but then decreases again as cells get very large.
  • Mitochondrial respiration is highest in intermediate-sized cells
  • Intermediate-sized cells show the lowest variation in mitochondrial membrane potential
  • A higher ΔΨ variation is correlated with a higher rate of apoptosis (cell death)
  • Intermediate-sized cells showed (on average) the fastest growth

These results strongly indicate that mitochondrial functionality is largest in intermediate-sized cells in a population. Cells also seem to try to maintain the size at which mitochondrial functionality is largest, meaning that this is probably an optimal cell size.

Tuesday, 18 October 2016

Mitochondrial Dysfunction Prevents Repolarization of Inflammatory Macrophages

Jan Van den Bossche, Jeroen Baardman, Natasja A. Otto, Saskia van der Velden, Annette E. Neele, Susan M. van den Berg, Rosario Luque-Martin, Hung-Jen Chen, Marieke C.S. Boshuizen, Mohamed Ahmed, Marten A. Hoeksema, Alex F. de Vos, Menno P.J. de Winther

Macrophages are types of white blood cell. They engulf and digest bodies which do not possess the correct protein markers which mark healthy cells. Whilst these cells play a crucial role in immunity, their dysfunction is associated with a number of auto-immune diseases such as asthma and rheumatoid arthritis. Macrophages exist in a spectrum of states, ranging from pro-inflammatory (M1) to anti-inflammatory (M2). In this study, the authors wished to investigate the mechanisms which prevent the transition from M1 to M2, so that we may better understand the mechanisms of inflammation.

Previous studies have shown that M1 cells are reliant upon glycolysis whereas M2 cells use mitochondrial oxidative phosphorylation (OXPHOS). These modes of energy production have also been associated with promoting the activation of these macrophage states. The authors find here that when macrophages are induced (using LPS + IFN-γ) to become M1 (pro-inflammatory) cells, this process inhibits OXPHOS. The signal (IL-4) which induces M2 (anti-inflammatory) cells cannot reverse this suppression of OXPHOS, and so they remain trapped in the pro-inflammatory state. The authors found that nitric oxide production by M1 cells, which is used as an antimicrobial mechanism and inhibits mitochondrial function, prevents the ability of M1 macrophages to be reprogrammed as non-inflammatory M2 cells.

Tuesday, 11 October 2016

Loss of Dendritic Complexity Precedes Neurodegeneration in a Mouse Model with Disrupted Mitochondrial Distribution in Mature Dendrites

López-Doménech G, Higgs NF, Vaccaro V, Roš H, Arancibia-Cárcamo IL, MacAskill AF, Kittler JT

Miro proteins link mitochondria to motor proteins, allowing them to be trafficked through neurons. In this study, the authors disrupted the expression of Miro proteins in neurons to understand the role of mitochondrial trafficking in neurodegeneration. The authors found that Miro1-KO caused the distribution of mitochondria in dendrites (the branched extensions of nerve cells which receive electrochemical signals from other neurons) to become more accumulated around the soma, and more sparse along dendrites. Miro1-KO cells also appeared smaller and less developed than wild-type neurons; this was also shown to be the case in an inducible Miro1-KO system in mature neurons of the forebrain of mice. The deletion of this gene was associated with neurodegeneration 12 months after induction of Miro1-KO.

Tuesday, 20 September 2016

Prediction of multidimensional drug dose responses based on measurements of drug pairs

Anat Zimmer, Itay Katzir, Erez Dekel, Avraham E. Mayo, and Uri Alon

*Not mitochondrial, but very cool

Cocktail therapies are common to treatments of a diverse array of diseases, in order to combat effects such as: antibiotic resistance in bacterial infections; persister cells in tuberculosis; and chemotheraputic resistance in cancer. It is, however, incredibly difficult to optimize the dose of multiple theraputic agents because of combinatorial explosions. For instance, if we have 10 possible doses for 3 different drugs, then we must test 10x10x10 = 1000 different dose combinations to find the optimal treatment. This becomes 10,000 if we wish to use 4 drugs. What makes this problem even more difficult is that drugs often show antagonism: it is not simply the case that using higher and higher doses of each drug is more effective, the optimum is often found at intermediate doses.

Here, the authors use mathematics to try and approximate the optimal dose of a cocktail of three or more drugs (N in general) whilst avoiding the problem of combinatorial explosion. They model the survival of cells (g) versus drug concentration (Di), for each drug (i), using Hill curves. They approximate g(D1, ... ,DN) using information only from single-drug dose response curves g(Di) and two-drug data g(Di, Dj) for all pairs of drugs. Their computation therefore only scales quadratically with the number of drugs N, rather than exponentially if we were to brute-force compute the global optimum. The authors show that their method is able to well-describe dose-response curves for a case study of six triplet and two quadruplet combination therapies, with 0.85 < R^2 < 0.93 for all of the examples tested.

These methods not only allow us to find the most effective doses, but also has the potential to minimize side effects by optimizing with the assumption that side-effects increase with higher dose. More realistic predictions could be made with more accurate models for side-effects.

Friday, 16 September 2016

Lactate metabolism is associated with mammalian mitochondria

Ying-Jr Chen, Nathaniel G Mahieu, Xiaojing Huang, Manmilan Singh, Peter A Crawford, Stephen L Johnson, Richard W Gross, Jacob Schaefer, Gary J Patti

Lactate is sometimes referred to as "metabolic waste" but this has been established as a misnomer for quite some time, with it being shown as early as the 1920s that lactate can be transformed back into glucose via gluconeogenesis in the liver. There are multiple other examples where shuttling of lactate between tissues allows it to be metabolised. However,  it remains controversial whether individual cells are able to metabolise this apparent metabolic by-product.

Here, the authors show that lactate is able to enter mitochondria and participate in mitochondrial energy metabolism. By culturing cells in radiolabelled lactate, the authors show that carbon from lactate can be found in intermediate metabolites of the TCA. They show that mitochondria are able to metabolize lactate, suggesting that they are able to import the metabolite, and that mitochondria possess the necessary enzyme (lactate dehydrogenase B) to convert lactate into the better-known mitochondrial substrate, pyruvate. The authors suggest this may be particularly relevant in cancer cells, which have particularly high glycolysis and lactate production.

Thoughts: It seems like a pretty important follow-up question to ask how much lactate is used by mitochondria relative to secretion in vivo. If the effect is big, doesn't this mean extracellular acidification rate is not a good metric of glycolysis levels?

Tuesday, 13 September 2016

The repopulating cancer cells in melanoma are characterized by increased mitochondrial membrane potential

Don G. Lee, Beom K. Choi, Young H. Kim, Ho S. Oh, Sang H. Park, Young Soo Bae, Byoung S. Kwon

In this study, the authors investigate whether mitochondrial membrane potential (ΔΨ) serves as a biomarker of higher proliferative potential in melanoma cells. The authors found that tumour cells which survived stressors such as serum starvation and cisplatin treatment had substantially higher ΔΨ. Furthermore, upon sorting cells into categories of low and high ΔΨ, the authors found that high-ΔΨ cells tended to induce tumour growth whereas low-ΔΨ could not, for doses of 10^5 cells/mouse: this was demonstrated for cells grown in vitro as well as sorted tumour cells grown in vivo.

Thursday, 8 September 2016

Homeostatic Responses Regulate Selfish Mitochondrial Genome Dynamics in C. elegans

Bryan L. Gitschlag, Cait S. Kirby, David C. Samuels, Rama D. Gangula, Simon A. Mallal, Maulik R. Patel

Why haven't deleterious mutations in mitochondrial DNA gone extinct? Naively, if a mutation has a negative impact on the fitness of an organism, then that organism may be less likely to reproduce and, in time, we expect to see fewer organisms with the mutation in nature. And yet deleterious mutations in mtDNA are still seen and passed down from generation to generation (albeit that this is often through carriers who bear such mutations at lower loads). 

The authors of this study explore this simple, but fundamental, question by establishing a slightly deleterious mtDNA variant in C. elegans, called uaDF5. This is a deletion mutation which removes four protein-coding genes and seven tRNAs. The authors show that worms are still viable with a mutant load as high as 80% (but lethal at 100%), and that mtDNA copy number tended to increase in individuals with large mutant load (suggesting expansion of the total mtDNA population so that there are enough wild-type mtDNA molecules to fulfil the metabolic needs of the animal). The authors also determined that it is unlikely that the mutation has a proliferative advantage by virtue of its smaller size, through comparison with another deletion mutant which was much smaller.

In addition to mtDNA copy number control, the authors suggest an additional mechanism whereby mutant mtDNAs may proliferate. The authors find that silencing of the mitochondrial unfolded protein response (mt-UPR) causes a substantial reduction in mutant load. The mt-UPR is thought to provide a protective role against adverse conditions for mitochondria; the authors suggest here that the process inadvertently allows mutants to proliferate as it suppresses mitophagy: the mechanism by which faulty mitochondria are recycled by the cell. They show this by blocking mt-UPR and parkin-mediated mitophagy to show a recovery in mutant load.

Thoughts: A natural question to ask is, given that these heteroplasmic animals are less fit, why do cells not have a larger mitophagy rate if this allows quality control? Is there some tradeoff where wild-type mtDNAs are also consumed? Perhaps with a lower probability?

Thursday, 10 March 2016

Selective Vulnerability of Cancer Cells by Inhibition of Ca2+ Transfer from Endoplasmic Reticulum to Mitochondria

César Cárdenas, Marioly Müller, Andrew McNeal, Alenka Lovy, Fabian Jaňa, Galdo Bustos, Felix Urra, Natalia Smith, Jordi Molgó, J. Alan Diehl, Todd W. Ridky, J. Kevin Foskett

Mitochondria are often found to be tethered to another organelle of the cell, called the endoplasmic reticulum (ER). The ER releases calcium into the mitochondria, which stimulates energy metabolism by increasing the rate of several catalysts in the metabolic network. A basal level of calcium release is necessary for mitochondrial ATP production in many cell types. Without this, cells tend to start recycling themselves through autophagy, as a survival mechanism.

In this study, the authors probe the difference in response to normal and cancer cells, to blocking calcium release (using the drug XeB, as well as genetic knockdown of the gene InsP3R) from the ER to mitochondria. The authors compared the cell death response of non-tumourigenic MCF10A cells to three tumorigenic cell lines (MCF7, T47D and HS578T). At 5uM XeB, the authors find that breast tumour cell lines experienced significant cell death (43, 53 and 22%) whereas normal cells were less sensitive to the drug (5% cell death). Similar effect sizes were seen in prostate cancer cells, at the same drug concentration. It was also the case that XeB did not induce large cell death in primary human fibroblasts, when compared to transformed cells.

The authors found that providing the tumour cells with additional pyruvate rescued their proliferation rate. Calcium stimulates the enzyme pyruvate dehydrogenase, which takes pyruvate from glycolysis and converts it into acetyl-coa, for use in mitochondrial metabolism. Therefore it is reasonable to conclude that providing additional pyruvate pushes flux through the network, to compensate for lower enzymatic activity (by mass-action kinetics).

The authors hypothesized that nucleoside supplementation may also rescue the effect of calcium import inhibition, since mitochondrial metabolism is intertwined with nucleoside synthesis (a necessity for DNA synthesis). Indeed, the authors found that nucleoside supplementation ameliorated cytotoxicity by ~50%. This indicates that nucleoside production of mitochondria is more important than their energy production, in this model. The authors show mechanistically that, when cancer cells are blocked in calcium uptake, they progress through the cell cycle normally, but their progression into mitosis results in necrotic cell death. In contrast, normal cells halt their cell cycle at G1 phase.

Thursday, 4 February 2016

Segregation of naturally occurring mitochondrial DNA variants in a mini-pig model

Gael Cagnone, Te-Sha Tsai, Kanokwan Srirattana, Fernando Rossello, David R. Powell, Gary Rohrer, Lynsey Cree, Ian A. Trounce, Justin St. John
Recent work in mice has shown that different mitochondrial sequences (haplotypes) will tend to accumulate in different tissues, and this segregation depends on the sequence in question.

In this study, the authors study four mtDNA mutations, over three generations of mini-pigs. These mutations were: Del A376 affecting 12 rRNA; Del A1302 affecting 16s rRNA; Del A1394 affecting 16s rRNA; and Del A9725 affecting NADH3 (a protein of the electron transport chain). The authors investigated which tissues had variable mutant load, and found that mutant load is significantly reduced in diaphragm (4/4 mutants), muscle (3/4 mutants), brain (2/4 mutants) and fat (2/4 mutants). 
They then go on to correlate the variation with mtDNA copy number, across all tissues, and all generations. In one of the four cases, no correlation was observed. However, in Del A376 and Del 1302, variant load had a fairly strong negative correlation with mtDNA copy number; in Del 1394, the correlation was weaker and also negative.
The authors therefore suggest that mutant load is lowered in high-respiratory tissue, such as brain, diaphragm, muscle, liver, heart and fat.

Thoughts: Are these mutants deleterious to respiratory activity? I imagine so, since they are all deletion mutations, so cause frameshifts. If that is the case, I wonder if the mutations could be ranked by how much they inhibit OXPHOS? I wonder whether the strongest inhibitors either have i) the strongest gradient with mtDNA copy number or ii) lowest overall abundance in all tissues?

I also find it pretty surprising that frameshift mutations can be found in healthy pig tissues, even 2-15%. I wonder whether there is a complementation effect happening here: several different mutants producing transcription products that the others cannot?