Single Nucleotides in the mtDNA Sequence Modify Mitochondrial Molecular Function and Are Associated with Sex-Specific Effects on Fertility and Aging

http://www.cell.com/current-biology/abstract/S0960-9822%2815%2901091-X

M. Florencia Camus, Jochen B.W. Wolf, Edward H. Morrow, Damian K. Dowling 

In this study, the authors bred flies containing the same nuclear genome, but different mitochondrial genomes, corresponding to different mitochondrial haplotypes from across the world. In doing this, the authors were able to demonstrate how the mitochondrial genotype can affect the phenotype of the fly, without considering the effect of nuclear DNA.

In 7 of the 13 haplotypes considered, the authors found that mtDNA copy number was increased in females above males, with the effect becoming stronger with age. Conversely (and strikingly), across 9 of the 13 mitochondrial genes tested, mitochondrial gene expression was higher in males, in all 9 genes, across all 13 haplotypes.

The authors found that mean longevity was higher in female flies. This intriguing finding, although only correlative, suggests that differences in the expression of the mitochondrial genome between males and females, may result in increased female longevity, as this may provide a selective advantage for such haplotypes (as mtDNA is maternally inherited). As an additional curiosity, the authors found that the Brownsville haplotype in this nuclear background induces cytoplasmic male sterility (male infertility, due to an interaction between mtDNA and nucleus). This is the only known case in metazoans.

Cellular Heterogeneity in the Level of mtDNA Heteroplasmy in Mouse Embryonic Stem Cells

http://www.sciencedirect.com/science/article/pii/S2211124715011730


Jitesh Neupane, Sabitri Ghimire, Mado Vandewoestyne, Yuechao Lu, Jan Gerris, Rudy Van Coster, Tom Deroo, Dieter Deforce, Stijn Vansteelandt, Petra De Sutter, Björn Heindryckx

Recent work has shown that non-pathological mtDNA variants (haplotypes) can show preferential expansion, in vivo. This is contrary to the common belief that nonpathological mutations exhibit neutral genetic drift. The authors of this study sought to study this phenomenon at the single-cell level using Mouse Embryonic Stem Cells (ESCs).

The authors established sets of cell lines, with differing proportions of two mtDNA haplotypes (NZB and BALB). The parental mice were themselves heteroplasmic in these two mtDNA haplotypes. By successive passage of the cells, the authors measure the ratio of the two haplotypes (heteroplasmy) with time. They find that, regardless of the initial ratio, the NZB haplotype tends to dominate over BALB with time (~12% over 30 passages), in this system. Furthermore, upon differentiation, cells tended to become more heterogeneous in heteroplasmy, and tended to shift back in heteroplasmy towards BALB (~8% reduction).

The results are significant, as they show that apparently neutral haplotypes have some kind of selective pressure. These dynamics are not necessarily straightforward, and seem to have some dependence on the system under study.

Direct evidence of mitochondrial G-quadruplex DNA by using fluorescent anti-cancer agents

http://nar.oxfordjournals.org/content/early/2015/10/19/nar.gkv1061.full

Wei-Chun Huang, Ting-Yuan Tseng, Ying-Ting Chen, Cheng-Chung Chang, Zi-Fu Wang, Chiung-Lin Wang, Tsu-Ning Hsu, Pei-Tzu Li, Chin-Tin Chen, Jing-Jer Lin, Pei-Jen Lou and Ta-Chau Chang

When DNA has a high content of guanine (one of the bases of DNA), under certain conditions, it can form a more exotic structure than the well-known Watson-Crick base pairing, known as a G-quadruplex. This is where four guanine bases bond to form a square-planar structure. These planes can stack on top of each other, to form the G-quadruplex. These structures can form in vivo, and are thought to have a connection to telomeres.

In this paper, the authors explore the existence of G4-quadruplexes in mtDNA. They do this by using fluorescent compounds which are able to both bind to a G4-quadruplex, and also localise in mitochondria (rather than the cell nucleus). One such agent is called BMVC-12C-P. What is curious about this particular agent is that it is able to halt the proliferation of cells in a cancer-specific manner. The agent accumulates strongly in the mitochondria of HeLa cancer cells, whereas it mainly localises in the lysosome of MRC-5 normal fibroblasts. The anti-cancer effect of this agent is robust across 3 cancer cell lines, and 3 normal cell lines, that were tested. Tumour proliferation was also shown to be slowed in mice injected with cancer cells, and treated with BMVC-12C-P.

After 72 hours of treatment, the authors find that the expression of ND3 and COX1 transcripts were severely reduced. Thus, the authors suggest that the mechanism of cytotoxicity of this agent is to prevent mtDNA transcription of these core ETC components, by targeting G4-quadruplexes in mtDNA. As well as being an interesting agent in its own right, this study provides further evidence for mtDNA being a target to alter cell proliferation.

New compounds to directly modulate mitochondrial ROS

Selective superoxide generation within mitochondria by the targeted redox cycler MitoParaquat

http://www.sciencedirect.com/science/article/pii/S0891584915005870  

Ellen L. Robb, Justyna M. Gawel, Dunja Aksentijević, Helena M. Cochemé,Tessa S. Stewart, Maria M. Shchepinova, He Qiang, Tracy A. Prime, Thomas P. Bright, Andrew M. James, Michael J. Shattock, Hans M. Senn, Richard C. Hartley, Michael P. Murphy

A mitochondria-targeted derivative of ascorbate: MitoC

http://www.sciencedirect.com/science/article/pii/S0891584915005894 

Peter G. Finichiu, David S. Larsen, Cameron Evans, Lesley Larsen, Thomas P. Bright, Ellen L. Robb, Jan Trnka, Tracy A. Prime, Andrew M. James, Robin A.J. Smith, Michael P. Murphy

When mitochondria malfunction, the components of the electron transport chain may leak electrons and generate reactive oxygen species (ROS). The role of ROS in physiological and pathophysiological settings is subtle, as low levels are thought to be important in cell signalling whereas high levels may cause damage to a variety of biomolecules. In order to test the causal link between ROS and pathology, it is important to be able to directly modulate their levels and test their outcome.

In the above two papers, the Murphy group fuse a particular cation (triphenylphosphonium lipophilic cation, TPP), to two agents (paraquat and ascorbate, which are oxidizing and reducing agents repectively) allowing their localisation to the mitochondrial matrix. The resulting compounds are named MitoPQ and MitoC respectively. Once in the matrix, MitoPQ is able to generate ROS, whereas MitoC is able to mop ROS up. Importantly, this occurs at the site of ROS generation (the mitochondrial matrix). Many known agents can only modulate ROS in the cytosol, so these compounds provide more direct control over mitochondrial ROS. Furthermore, due to their direct localisation, lower concentrations of the compounds are required relative to their non-mitochondrial counterparts, to achieve the same effect.

Consequences of zygote injection and germline transfer of mutant human mitochondrial DNA in mice

http://www.pnas.org/content/early/2015/09/30/1506129112
 
Hong Yu, Rajeshwari D. Koilkonda, Tsung-Han Chou, Vittorio Porciatt^, Arpit Mehta, Ian D. Hentall, Vince A. Chiodo, Sanford L. Boye, William W. Hauswirth, Alfred S. Lewin and John Guy

In trying to understand mutations of mitochondrial DNA, biologists tend to use cells from either: 1) biopsies directly from patients; 2) early cell culture (where a biopsy from a patient has been purified in some way, then grown in the lab for a small number of generations); or 3) immortal cell lines (these are often cancer cells, which have been manipulated to contain the mtDNA mutation of interest).

Whilst all of these model system have their pros and cons (e.g. cell purity vs similarity to a natural environment), what has been lacking in this field is the ability to study human mitochondrial mutations in an animal model. This has the advantage of being able to introduce further genetic/drug perturbations to a living biological system, in the background of a mtDNA mutation. Whilst introducing nuclear mutations into mice is commonplace, it has never been attempted for mitochondrial genomes, which are held in multiple copy number.

In this study, the authors use an adeno-associated virus, which usually targets the nucleus, and add a particular protein (MT-COX8) which causes the virus to localise to mitochondria. The virus then delivers mutated human mtDNA to a mitochondrion. By exposing a mouse zygote to the virus, the authors were able to generate a mouse with a mutation in complex I, associated with LHON. The inserted viral mtDNA existed separately from the endogenous mtDNA, and was able to replicate and be transferred between generations after cross-breeding with wild-type animals. 

In humans, LHON is typically associated with retinal degeneration. The mutated mtDNA was able to express the mutant form of complex I in the mice, and cause a visual deficiency. Using mice which showed a visual defect, the authors used ocular injection with a virus containing wild-type mtDNA, and show that this was partially able to restore the visual defect, 1 month after treatment. 

PKA Phosphorylates the ATPase Inhibitory Factor 1 and Inactivates Its Capacity to Bind and Inhibit the Mitochondrial H+-ATP Synthase

http://www.sciencedirect.com/science/article/pii/S2211124715009493

Javier García-Bermúdez, María Sánchez-Aragó, Beatriz Soldevilla, Araceli del Arco, Cristina Nuevo-Tapioles, José M. Cuezva

ATP synthase is the motor of the cell, generating most cellular ATP under normal conditions (watch a video of this here). The protein ATPase Inhibitory Factor 1 (IF1) is known to inhibit both hydrolysis and synthesis of ATP by ATP synthase, by blocking its rotation. This study investigates the mechanism of this protein's action, as well as its physiological and pathophysiological role.

Mechanistically, Protein Kinase A phosphorylates IF1 (p-IF1), which inhibits its ability to interact with ATP synthase, and so is expected to allow OXPHOS to occur. As a consequence, the authors find that dephosphorylated IF1 (dp-IF1) causes inhibition of oxidative phosphorylation and increased glycolytic flux.

Studies in yeast have shown that different energy pathways are activated, depending on the stage of the cell cycle. G1 is believed to be OXPHOS dependent, whereas G2/M is largely independent of oxygen consumption and relies on aerobic glycolysis. Consistent with this picture, the authors show that cells arrested in G1 phase had mostly p-IF1 and high levels of OXPHOS, whereas cells that were arrested in G2/M had dp-IF1 and low OXPHOS levels.

The authors also found hypoxia to be able to induce dephosphorylation of IF1, and thus inhibition of ATP synthase.  Indeed, a number of carcinomas investigated by the authors have an abundance of dp-IF1.

In terms of its physiological significance, the authors investigated the phosphorylation status of IF1 in mouse heart, in vivo. Fascinatingly, ~50% of IF1 is found in its phosphorylated state. Administration of drugs which induce an adrenaline response, causes a sharp increase in p-IF1 and OXPHOS activity. This suggests that the protein has a physiological role of fine-tuning mitochondrial output, in response to variable energy demands.

----------------
Thoughts:
It is known that the maintenance of mitochondrial membrane potential (ΔΨ) is vital for cell viability, as mitochondria perform a plethora of functions besides energy production, which are ΔΨ dependent. If cells actively inhibit their ATP synthase during hypoxia (and so can't hydrolyse ATP and pump protons), and are unable to pump protons due to a lack of oxygen, how are the cells maintaining ΔΨ? 

Dissecting tumor metabolic heterogeneity: Telomerase and large cell size metabolically define a sub-population of stem-like, mitochondrial-rich, cancer cells

http://www.ncbi.nlm.nih.gov/pubmed/26323205

Rebecca Lamb, Bela Ozsvari, Gloria Bonuccelli, Duncan L. Smith, Richard G. Pestell, Ubaldo E. Martinez-Outschoorn, Robert B. Clarke, Federica Sotgia
and Michael P. Lisanti

Telomeres are regions of non-coding DNA, which protectively cap the ends of chromosomes. After successive rounds of replication, telomeres shorten because DNA polymerase does not duplicate DNA all the way to the end of a chromosome, and induces senescence after 50-70 divisions. Telomerase (hTERT) is an enzyme which lengthens nucleotides, the overexpression of which is sufficient to immortalize a cell.

Here, the authors fluorescently tag the promoter of hTERT with GFP, to select cancer cells with high telomerase transcriptional activity, and purify so-called cancer stem-like cells. The authors, studying breast cancer cells, found that cells in the top 5% of hTERT-expressing cells (GFP-high) formed ~2.5 times more mammospheres than the bottom 5% (GFP-low). GFP-high cells also showed a 1.7-fold increase in the median MitoTracker fluorescence, indicating a strongly increased mitochondrial content in these cells.

The authors also sorted their cells by size, taking the top ~15% as 'large' and the rest as 'small'. They found that larger cells possessed a ~2.7-fold increase in hTERT activity, and 1.6-fold increase in mitochondrial mass.

----------------------------
Thoughts:

Is the correlation between cell size and mitochondrial content surprising? Do ordinary cells possess a larger mitochondrial content, because they have a larger cytoplasmic volume and therefore greater energy demand? The finding that large cells have greater hTERT activity is, I think, surprising on its own terms because DNA content is independent of cell size. But disambiguating variation of mitochondrial content with cell volume, from cancer stemness is an interesting statistical question I think.

Mitochondrial reticulum for cellular energy distribution in muscle

 http://www.nature.com/nature/journal/v523/n7562/full/nature14614.html

Brian Glancy, Lisa M. Hartnell, Daniela Malide, Zu-Xi Yu, Christian A. Combs, Patricia S. Connelly, Sriram Subramaniam, and Robert S. Balaban


Cells need energy, especially muscle cells. Mitochondria generate part of this energy and to be able to do this they need to be supplied with various resources. Muscle cells can be quite big, so these resources (that enter the cell at its periphery) need to diffuse through the cell towards the mitochondria, which might take a long time. Is there a better, more efficient way of producing energy in large muscle cells?

In this paper they show that the mitochondrial network provides a conductive pathway for energy distribution.

Mitochondria use their Electron Transport Chain protein complexes to pump protons across their membrane, which creates an electrochemical gradient in which energy is stored. Their ATP synthase then uses this stored energy to make ATP. If many mitochondria are connected in the cell (i.e. if they all have an electrically continuous inner membrane) then a mitochondrion at one end of the cell can use the potential energy created by a mitochondrion at the other end of the cell, to generate ATP. The conduction of electric potential along the mitochondria can be faster than all kinds of resources needing to diffuse through the cell. This idea was first proposed by a Russian scientist Владимир Скулачёв (Vladimir Skulachev).

Here they show that the proteins involved in generating the electrochemical gradient are mainly found at the cell periphery (where the resources enter the cell), while proteins involved in using this energy to create ATP are found in the cell's interior. They also show that the mitochondria are indeed electrically connected to each other. The mitochondria in muscle cells are organized in a way that facilitates energy conduction.

The question remains whether this mitochondrial conductivity plays a role in all cells, or only in cells that are very energy demanding. Skin cells for example, hardly seem to need a fast conducting mitochondrial network. Nevertheless, mitochondrial fusion and network forming is seen in a variety of cells, so the fusion of mitochondria probably has other uses as well.

Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid

http://www.pnas.org/content/early/2015/08/20/1512131112.short?rss=1

Christian Kuka, Karen M. Davies, Christian A. Wurm, Henrik Spåhr, Nina A. Bonekamp, Inge Kühl, Friederike Joos, Paola Loguercio Polosa, Chan Bae Park, Viktor Posse, Maria Falkenberg, Stefan Jakobs, Werner Kühlbrandt and Nils-Göran Larsson

Mitochondrial DNA is found to exist in protein-DNA complexes called nucleoids.  The number of mtDNA molecules per nucleoid is an important quantity to know, as it has consequences for how mtDNA is distributed amongst successive generations. The compactness of mtDNA also determines its ability to be transcribed and replicated, so the way it is packaged is also important to understand.

In their first experiment, the authors use electron microscopy in vitro, to observe how mtDNA is compacted, with increasing concentrations of the molecule TFAM (the only protein known to package mtDNA). They find that upon binding to mtDNA, TFAM causes the DNA to bend by 180°. At intermediate concentrations 1 TFAM/30 bp, dense protein-DNA spots were dispersed amongst regions of naked DNA (perhaps being a concentration appropriate for translation or replication of mtDNA). At concentrations of 1 TFAM/6 bp, mtDNA was completely compacted, and a further increase in TFAM concentration had no further effect. This may be the density of TFAM required for storage of mtDNA.

They then continued their analysis in vivo, by studying TFAM-overexpressing mouse embryonic fibroblasts (OE MEFS), which had ~2.5-fold higher mtDNA copy number. Nucleoids can cluster in cells, and confocal microscopy cannot always resolve individual nucleoids. The number of nucleoids detected in wild-type cells using superresolution STED microscopy / confocal was 1.36+/-0.76. By counting nucleoids using STED microscopy, and mtDNA copy number with rt-qPCR, they found that wild-type cells had ~1.1 mtDNA molecules / nucleoid and OE MEFS had ~1.5 mtDNA molecules / nucleoid. Thus, overexpressing TFAM has only minor changes in the mean amount of mtDNA per nucleoid. Instead, the number of nucleoids was observed to increase, relative to wild-type cells.  

Mitotic redistribution of the mitochondrial network by Miro and Cenp-F

http://www.ncbi.nlm.nih.gov/pubmed/26259702

Kanfer G, Courthéoux T, Peterka M, Meier S, Soste M, Melnik A, Reis K, Aspenström P, Peter M, Picotti P, Kornmann B


If a cell divides, how do its mitochondria get segregated into the two daughter cells?  In general, mitochondria often move along microtubules with the help of molecular motors. Specialized adaptors on the mitochondrial outer membrane  recruit these motors, one of them being the GTPase Miro. But how exactly the segregation of mitochondria during mitosis is coordinated, is not well understood.

In this paper, they identify centromeric protein F (Cenp-F) which interacts with Miro. Cenp-F is a large protein that binds to the microtubules. It was found to be strongly recruited to mitochondria at the end of mitosis.  During S/G2, a fraction of Cenp-F is found on mitochondria, located mainly at the mitochondrial tips projecting away from the cell centre. The mitochondrial Cenp-F puncta that were visible in the S/G2 phase were all colocalized with microtubules.

They further find that Cenp-F directly interacts with the GTPase domains of both Miro1 and Miro2, leading to its recruitment to mitochondria. In wildtype cells, during cytokinesis mitochondria were parallel and extended away from the division plane, whereas in cells without Miro (or Cenp-F) the mitochondria were all clustered together around the nucleus.
 
The conclusion was that Cenp-F, recruiting by Miro, connects mitochondria to the tips of growing microtubules. The mitochondria then track the tips of the microtubules. Mitochondria were also seen to influence local microtubule dynamics, it can be that dragging forces caused by the attached mitochondria play a role in this.

Maternal transmission, sex ratio distortion, and mitochondria

http://www.pnas.org/content/112/33/10162.full

Steve J. Perlman, Christina N. Hodson, Phineas T. Hamilton, George P. Opit, Brent E. Gowen

Most (but not all) multicellular organisms have uniparental inheritance of mitochondrial DNA, which is thought to prevent invasion by a more competitive lineage, and maintain compatibility between mitochondrial and nuclear genomes. Mitochondrial DNA is also subject to evolutionary pressures, and in this review the authors discuss potential (deleterious) side-effects of uniparental inheritance. 

An immediate consequence of uniparental inheritance is that one sex is an evolutionary dead-end. Thus, any selective pressure on mtDNA is principally exerted on the transmitting sex (females), and so deleterious mutations for the non-transmitting (males) sex can accumulate. This explains why male infertility is often attributed to mutations in mtDNA. A study by Innocenti et al. [1], established fly lines with variation in their mtDNA. A large fitness variation was observed, but only in males; this caused significant differential expression across the nucleus of male flies. 

The authors suggest three possible consequences of maternal inheritance of mtDNA:

1. MtDNA mutations which are detrimental to males may fixate, if they do not affect female fitness
2. MtDNA mutations which cause the frequency of females to increase, may fixate
3. Nuclear symbionts carried on the nuclear female chromosome which fixate, will also cause their associated mtDNA haplotype to fixate

[1] Innocenti P, Morrow EH, Dowling DK (2011) Experimental evidence supports a sex-specific selective sieve in mitochondrial genome evolution. Science 332(6031):845–848.

An Essential Role of the Mitochondrial Electron Transport Chain in Cell Proliferation Is to Enable Aspartate Synthesis

http://www.sciencedirect.com/science/article/pii/S0092867415008533

Kıvanç Birsoy, Tim Wang, Walter W. Chen, Elizaveta Freinkman, Monther Abu-Remaileh, David M. Sabatini

When mitochondrial respiration is inhibited, it is observed that a cell's ability to proliferate is diminished. It is also known that cells with inhibited oxidative phosphorylation (OXPHOS) are still able to proliferate, if cultured in high concentrations of pyruvate (a metabolite which is the by-product of glycolysis, and feeds the tricarboxylic acid cycle). In this study, the authors screen a library of ~3,000 metabolic enzymes, treated with a low dose of complex I inhibitor phenformin, whose loss causes a severe anti-proliferative phenotype. This would reveal genes which are required in an adaptive response to mild OXPHOS inhibition.

The best scoring gene in their screen was GOT1, a component of the malate-aspartate shuttle, which transfers substrates for OXPHOS into the mitochondrial matrix. They find that GOT1-null cells are hypersensitive to phenformin, causing their proliferation to halt, at doses where wild-type cells do not.

Under normal conditions, GOT1 shuttles the amino acid aspartate, into the mitochondrial matrix. They find that, upon inhibition of complex I, GOT1 reverses its flux and exports aspartate. Aspartate is an amino acid, required for the synthesis of proteins, purines and pyramidines. Normally, it is generated in the mitochondrial matrix; the authors propose that during ETC inhibition, a drop in NAD+/NADH ratio causes aspartate synthesis to shut down, using normal pathways. Thus GOT1 reverses its flux, to become a source of this amino acid.

It is also known that supplementation of pyruvate can overcome the inhibitory effects of several ETC inhibitors. By culturing GOT1-null cells with/without pyruvate, the authors show that there is no benefit to supplementing cells with pyruvate under ETC inhibition, if cells lack GOT1. Thus a key mechanism of pyruvate supplementation is GOT1-catalyzed aspartate synthesis. Interestingly, these conclusions appear to hold in cybrid cell lines harbouring mtDNA mutations (homoplasmic mutations in CYTB and tRNA lysine were tested). When these cells overexpress an aspartate transporter SLC1A3, and cultured in high aspartate medium, their proliferation rate recovers to similar levels as wild-type cells.

Reduced mitochondrial Ca 2+ transients stimulate autophagy in human fibroblasts carrying the 13514A>G mutation of the ND5 subunit of NADH dehydrogenase

http://www.nature.com/cdd/journal/vaop/ncurrent/full/cdd201584a.html

Granatiero V, Giorgio V, Calì T, Patron M, Brini M, Bernardi P, Tiranti V, Zeviani M, Pallafacchina G, De Stefani D, Rizzuto R

Many layers of compensation potentially exist, to compensate for mutations in mitochondrial DNA: mtDNA biogenesis, modifications to transcription, translation, diffusive complementation and so on.

This study aims to determine how one such mutation is compensated for. The authors use primary skin fibroblasts from patients carrying a (phenotypically mild) heteroplasmic point mutation in a mitochondrially-encoded subunit of complex I (13514A>G, affecting the ND5 subunit).

They observed a higher rate of mitochondrial recycling in mutant cells, via mitophagy. In addition, the mutants had a lowered calcium uptake into the mitochondria when exposed to histamine (which causes calcium release from the endoplasmic reticulum into both the cytoplasm and mitochondria), which was associated with higher AMPK levels. Using split GFP technology, they showed that the mutants possessed fewer mitochondrial-ER contact sites.

To determine causality, the authors manipulated mitochondrial calcium uptake by overexpressing components of the mitochondrial calcium uniporter, and also treating cells with Kaempferol. These interventions recovered the calcium response to histamine, reduced AMPK levels and reduced autophagosome number. This intervention, however, reduced cell viability; presumably because of a lowered mitophagy rate.

Thus, the authors propose that, in this system, the cell reduces mitochondrial calcium uptake to increase mitochondrial recycling. They suggest this is mediated via AMPK, which senses the AMP/ATP ratio. This acts as a pro-survival mechanism, in mutated cells.

---------------------------
Thoughts: AMPK correlates with the recycling rate, but it would be interesting to see whether an intervention altering AMPK levels, alters mitophagy rate in the expected way, to demonstrate this causal link?

I wonder whether these cells have lowered respiration and generate less ATP, because of their mutation...? This could then explain their increased AMPK levels, and perhaps also their increased mitophagy rate? Maybe perturbing cells to uptake more calcium, causes more flux through the respiratory chain and so alleviates energy deficiency and lowers mitophagy, but a mutated complex I causes higher levels of ROS to give a pro-apoptosis signal? Interesting system!

Mitochondrial genomes are retained by selective constraints on protein targeting

http://www.pnas.org/content/early/2015/07/17/1421372112

Björkholm P, Harish A, Hagström E, Ernst AM, Andersson SG

Mitochondria originated through the fusion of two early bacterial forms of life: an endosymbiont giving rise to mitochondria, and a host to modern-day eukaryotic cells. Each of these original organisms contained their own genomes; however, over evolutionary time, there has been genetic transfer from mitchondrial DNA to nuclear DNA.

Several competing hypotheses exist to explain why any particular gene may be retained by mitochondria. One hypothesis is that a core set of genes must be retained by these organelles to retain local control over respiration (a similar argument exists for chloroplasts in photosynthesis, this is the CoRR hypothesis).

In this study, the authors provide evidence for the hydrophobicity of the gene products to determine gene retention. They predict that mitochondrially-encoded proteins have a larger insertion free energy than nuclear-encoded proteins. They show experimentally that when these proteins are expressed in the nucleus, they tend to be recognised by the signal recognition particle (SRP), and targeted to the endoplasmic reticulum, rather than mitochondria. This is due to the SRP's ability to bind to a hydrophobic domain. This may be problematic for gene therapies attempting to alleviate mitochondrial genetic diseases, by expression of such genes in the nucleus.

The authors discuss that hydrophobic proteins are unlikely to fold properly in the cytoplasm, and their import into double-membraned organelles like mitochondria, would be difficult (and potentially toxic if unfolded proteins were to accumulate). The authors emphasise that the hydrophobicity hypothesis is not mutually exclusive to the CoRR hypothesis, and many selective pressures are likely to operate on organelle genomes.

Describing the randomness in populations of mtDNA (and other stuff) within and over cell cycles

http://rspa.royalsocietypublishing.org/content/471/2180/20150050.full

Cell biology is a unpredictable world*! Molecules in the cell undergo diffusive motion, constant jostling, and interference from other molecules, meaning that precisely describing the motion of every atom is very hard and rarely useful. Instead, it's often more useful to consider biological processes as occurring "randomly", forgetting the precise details of all these complicating effects and just thinking about a reasonable "coarse-grained" model for their influence. In this (demonstrably powerful) picture, important machines in our cells -- including mitochondria, and particularly mtDNA -- replicate and degrade in processes that can be described as random; and when cells divide, the partitioning of these machines between the resulting cells also looks random. The number of machines we have in our cells is important, but how can we work with numbers in this unpredictable environment?

In our cells, elements including mtDNA are produced (red), replicate (orange), and degrade (purple) randomly with time, as well as being randomly partitioned when cells split and divide (blue). Our mathematical approach describes how the total number of machines is likely to behave and change with time and as cells divide.

Tools called "generating functions" are useful in this situation. A generating function is a mathematical function (like G(z) = z², but generally more complicated) that encodes all the information about a random system. To find the generating function for a particular system, one needs to consider all the random things that can happen to change the state of that system, write them down in an equation (the "master equation") describing them all together, then use a mathematical trick to push that equation into a different mathematical space, where it is easier to solve. If that "transformed" equation can be solved, the result is the generating function, from which we can then get all the information we could want about a random system: the behaviour of its mean and variance, the probability of making any observation at any time, and so on.

We've gone through this mathematical process for a set of systems where individual cellular machines can be produced, replicated, and degraded randomly, and split at cell divisions in a variety of different ways. The generating functions we obtain allow us to follow this random cellular behaviour in new detail. We can make probabilistic statements about any aspect of the system at any time and after any number of cell divisions, instead of relying on assumptions that the system has somehow reached an equilibrium, or restricting ourselves to a single or small number of divisions. We've applied this tool to questions about the random dynamics of mitochondrial DNA in cells that divide (like our cells) or "bud" (like yeast cells), but the approach is very general and we hope it will allow progress in many more biological situations.

* See, for example, http://onlinelibrary.wiley.com/doi/10.1111/j.1740-9713.2012.00586.x/abstract ( free version http://arxiv.org/abs/1208.2250 )

Elucidating the mechanism of the mtDNA bottleneck

http://elifesciences.org/content/early/2015/06/04/eLife.07464

Our mitochondrial DNA (mtDNA) provides instructions for building vital machinery in our cells. MtDNA is inherited from our mothers, but the process of inheritance -- which is important in predicting and dealing with genetic disease -- is poorly understood. This is because mitochondrial behaviour during development (the process through which a fertilised egg becomes an independent organism) is rather complex. If a mother's egg cell begins with a mixed population of mtDNA -- say with some type A and some type B -- we usually observe hard-to-predict mtDNA differences between cells in the daughter. So if the mother's egg cell starts off with 20% type A, egg cells in the daughter could range (for example) from 10%-30% of type A, with each different cell having a different proportion of A. This increase in variability, referred to as the mtDNA bottleneck, is important for the inheritance of disease. It allows cells with higher proportions of mutant mtDNA to be removed; but also means that some cells in the next generation may contain a dangerous amount of mutant mtDNA. Crucially, how this increase in variability comes about during development is debated. Does variability increase because of random partitioning of mtDNAs at cell divisions? Is it due to the decreased number of mtDNAs per cell, increasing the magnitude of genetic drift? Or does something occur during later development to induce the variability? Without knowing this in detail, it is hard to propose therapies or make predictions addressing the inheritance of disease.

We set out to answer this question with maths! Several studies have provided data on this process by measuring the statistics of mixed mtDNA populations during development in mice. The different studies provided different interpretations of these results, proposing several different mechanisms for the bottleneck. We built a mathematical framework that was capable of modelling all the different mechanisms that had been proposed. We then used a statistical approach called approximate Bayesian computation to see which mechanism was most supported by the existing data. We identified a model where a combination of copy number reduction and random mtDNA duplications and deletions is responsible for the bottleneck. Exactly how much variability is due to each of these effects is flexible -- going some way towards explaining the existing debate in the literature.  We were also able to solve the equations describing the most likely model analytically. These solutions allow us to explore the behaviour of the bottleneck in detail, and we use this ability to propose several therapeutic approaches to increase the "power" of the bottleneck, and to increase the accuracy of sampling in IVF approaches.

A "bottleneck" acts to increase mtDNA variability between generations. But how is this bottleneck manifest? Our approach suggests that a combination of copy number reduction (pictured as a "true" copy number bottleneck), and later random turnover of mtDNA (pictured as replication and degradation), is responsible.

Our excellent experimental collaborators, lead by Joerg Burgstaller, then tested our theory by taking mtDNA measurements from a model mouse that differed from those used previously and which, could in principle have shown different behaviour. The behaviour they observed agreed very well with the predictions of our theory, providing encouraging validation that we have identified a likely mechanism for the bottleneck. New measurements also showed, interestingly, that the behaviour of the bottleneck looks similar in genetically diverse systems, providing evidence for its generality.

High-fat diet and FGF21 cooperatively promote aerobic thermogenesis in mtDNA mutator mice

http://www.pnas.org/content/early/2015/06/23/1509930112.full.pdf

Christopher E. Wall, Jamie Whyte, Jae M. Suh, Ronald M. Evans et al.

The POLG mutator mouse is a well-known model of premature aging. They express a proofreading-deficient version of POLG, causing them to introduce point-mutations and deletions in their mitochondrial genome. The aging phenotype is visible from 9 months onwards, and yet most mutations accumulate during embryogenesis. This study sought to characterise younger mutator mice, which bare much of the mtDNA damage of older mice, but relatively little respiratory chain dysfunction, and without a progerioid phenotype.

To do this, the authors challenged the mice with a high-fat diet (HFD). The expectation was that these mice would fair poorly under such a diet, but surprisingly the mice appeared healthier than controls. POLG mice were highly resistant to weight gain, and had much lower insulin levels relative to controls. These mice also had a substantially higher mitochondrial content and oxygen consumption rate in their brown adipose tissue, once given a HFD. POLG mice on a normal diet have an abnormally low body temperature (by ~4°C), but HFD allowed the mice to recover normal core temperature, through aerobic thermogenesis.  

The gene FGF21, which is thought to mediate the benefits of caloric restriction (but also signals mitochondrial stress), was substantially upregulated in POLG mice in both HFD and normal diets. Thus, the authors suggest that young POLG mice are in a metabolic state of starvation. Since calorie restriction is associated with longevity, they suggest that these observations indicate a compensatory response, to oppose their mutated mtDNA. However, as the mice age, they eventually succumb to the progerioid phenotype. 

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Thoughts:

The authors suggest in their discussion that lipids from a HFD are able to function as a preferential metabolic substrate. From this reasoning, does it follow that mice supplemented with a HFD should have a delayed/ameliorated progerioid phenotype? The discussion suggests not, but I wonder why this isn't the case...

Physical exercise improves brain cortex and cerebellum mitochondrial bioenergetics and alters apoptotic, dynamic and auto(mito)phagy markers.

 http://www.ncbi.nlm.nih.gov/pubmed/26116519?dopt=Abstract

Marques-Aleixo I, Santos-Alves E, Balça MM, Rizo-Roca D, Moreira PI, Oliveira PJ, Magalhães J, Ascensão A

Physical exercise does not only trigger the release of endorphins, it is good for your brain mitochondria!

Eighteen male rates were divided in three groups, 1) a group without physical activity, 2) a group with voluntary free wheel activity, and 3) a group with treadmill endurance training, 5 days a week for 12 weeks.
Some behavioural tests were performed, and eventually the brains of the decapitated rats were washed and analysed.

What were the results? From the behavioural point of view, the mice from group 3 (the most active group) showed a general increase in activity and more willingness to explore new spaces. What about the condition of the brains? In both group 2 and 3, they found:

• an increase in state III mitochondrial respiration
• an increase in efficiency of ATP synthesis in brain cortex and cerebellum mitochondria
• an increased content of complex I, III and V subunits in brain cortex and cerebellum mitochondria
• increases in complex I and V activity in brain cortex mitochondria
• a decrease in lipid peroxidation
• less oxidative stress
• reduced apoptosis in cerebellum mitochondria
• increased PGC1alpha and TFAM (which stimulate mitochondrial replication) in the brain cortex
• Increased mitochondrial fusion (Mfn1,2 were increased, and Drp1 decreased)
• Increased autophagy markers

It therefore appears that exercise improves mitochondrial health in the brain. A recent review discusses how important healthy mitochondrial functioning is in the brain, and how impairment of mitochondrial fusion dysregulates neuronal function.

In conclusion, take some time off work and go do some exercise.

Selfish mitochondrial DNA proliferates and diversifies in small, but not large, experimental populations of Caenorhabditis briggsae

http://gbe.oxfordjournals.org/content/early/2015/06/23/gbe.evv116.short?rss=1

Wendy S. Phillips, Anna L. Coleman-Hulbert, Emily S. Weiss, Dana K. Howe, Sita Ping, Riana I. Wernick, Suzanne Estes and Dee R. Denver

Mitochondrial DNA (mtDNA) with deletion mutations can occur during aging in humans. These mutants are energetically compromised, and can accumulate at damaging concentrations in tissues. It is widely believed that these molecules have a replicative advantage, although the mechanism for this remains controversial.

Here, the authors study the nematode worm C. briggsae, and the dynamics of mtDNA deletions with both age and colony size, to probe the effect of competition in large populations of worms. For N = 1, 10, 100 and 1000 colony sizes, the colonies were propagated from generation to generation by transferring N worms to a new plate every 48 hours. The abundance of mtDNA deletions were then measured every 5 generations. This was repeated in nematode strains with low (0-5%), medium (5-20%) and high (40-50%) initial mutant loads.

The authors found that small populations (N = 1, 10) showed an accumulation of mtDNA mutants with time. However, for large populations (N = 100, 1000), deleterious mutations tended to die away with the number of generations. The interpretation is, for large populations, there exists a strong selective pressure against deleterious mutants (and therefore energetic inefficiency),  due to competition with other worms. This is strong enough to overcome any proliferative advantage of the deletion mutations.