How mitochondria can vary, and consequences for human health

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

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

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

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

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

 

Investigating mitonuclear interactions in human admixed populations

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

Arslan A. Zaidi & Kateryna D. Makova

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

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

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

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

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

Memory of ancestral mitochondrial stress

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

Sarah-Lena Offenburg, Marcos Francisco Perez and Ben Lehner

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

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

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

THE RECENT FINDING

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

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

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

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

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

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

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

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

Quantitative 3D Mapping of the Human Skeletal Muscle Mitochondrial Network

https://www.cell.com/cell-reports/fulltext/S2211-1247(19)30018-X

Amy E. Vincent, Kathryn White, Tracey Davey, Jonathan Philips, R. Todd Ogden, Conor Lawess, Charlotte Warren, Matt G. Hall, Yi Shiau Ng, Gavin Falkous, Thomas Holden, David Deehan, Robert W. Taylor, Doug M. Turnbull,
and Martin Picard

• The authors investigate morphological differences of mitochondria in muscle between mice, humans and humans with mitochondrial disease.
• In all human and mouse muscle fibres analysed, the authors confirmed that the mitochondrial network is largely composed of distinct organelles, typically no more than a few microns in length. 
• The authors quantify "mitochondrial complexity" by taking the ratio of surface area to volume. Intuitively, a more "complex" organelle will have a higher surface area for a fixed volume, due to greater invagination. Naively taking the ratio yields a quantity with dimensions, so the authors raise the surface area to the power 3/2 to yield a dimensionless quantity. Phenomenologically, the authors find that squaring their metric increases its dynamic range. They name the resultant quantity the "mitochondrial complexity index" (MCI), which scales as MCI ~ SA^3/V^2 (SA=surface area, V=volume).
• The authors use a further metric, the "mitochondrial branching index" (MBI) which measured anisotropy. MBI > 1 denotes more branching in the transverse plane than the longitudinal direction of a muscle fibre. 
• The authors find that humans have smaller muscle mitochondria than mice, with comparable MCI.
• The authors found that, within cells, there is a large variability in mitochondrial volume and MCI (CV between 50-100%), although inter-cellular variability was smaller (CV < 50%). The authors also observed inter-individual variability in these metrics (CV ~ 50%).
• The authors studied a trio of genetically related patients carrying a tRNA-lys mutation at 40% (asymptomatic), 63% (mild myopathy) and 97% (severe myopathy). The patients with mild/severe myopathy had smaller mitochondria and lower MCI.
• Uncovering the correlation with single-cell heteroplasmy, respiratory chain function, and morphology remains a challenge for future studies.

Age-related declines in α-Klotho drive progenitor cell mitochondrial dysfunction and impaired muscle regeneration

A. Sahu, H. Mamiya, S. N. Shinde, A. Cheikhi, L. L. Winter, N. V. Vo, D. Stolz, V. Roginskaya, W. Y. Tang, C. St. Croix, L. H. Sanders, M. Franti, B. Van Houten, T. A. Rando, A. Barchowsky & F. Ambrosio

https://www.nature.com/articles/s41467-018-07253-3

• Aged muscle shows a decreased capacity to repair itself after acute injury.   Muscle stem cells (MuSCs) mediate muscle repair, which become activated when muscles are injured. MuSCs show increased apoptosis, decreased proliferation, impairment of mitophagy, senescence, and decreased resistance to stress, with age.
• The gene Klotho encodes a membrane-bound, circulating, hormonal protein in mice and humans; its deficiency is associated with ageing phenotypes including: decreased activity, gait disturbance, cognitive impairment, sarcopenia, and impaired wound repair. Declines in α-Klotho in tissues such as the skin, small intestine, and kidney, have been associated with senescence and stem cell dysfunction.
• In young skeletal muscle, the authors show that the α-Klotho promoter is transiently demethylated under acute muscle injury, which is associated with increased expression. In aged tissue, α-Klotho shows no significant change in methylation in response to muscle injury, and no significant expression. 
• Knockdown of α-Klotho in young animals results in an aged phenotype, with aberrant mitochondrial ultrastructure, decreased mitochondrial bioenergetics, mtDNA damage (perhaps mediated through cardiolipin peroxidation), and senescence.
• Sytematic delivery of exogenous α-Klotho to aged mice rejuvenates muscle progenitor stem cell (MPC) bioenergetics, enhances myofiber regeneration, and muscle function after acute injury.   

Thoughts
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In Figure 6a, the authors show that when aged  MPC are isolated from old mice, and cultured with α-Klotho for 48h, the authors observe a decreased number of mtDNA lesions relative to cells which are not treated with α-Klotho (a difference of about 1 mutation per molecule of mtDNA). This is a pretty huge number of mutations over 48 hrs! It would be fascinating to infer the mtDNA mutation rate from these data.

MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

Erol C. Bayraktar, Lou Baudrier, Ceren Özerdem, Caroline A. Lewis, Sze Ham Chan, Tenzin Kunchok, Monther Abu-Remaileh, Andrew L. Cangelosi, David M. Sabatini, Kıvanç Birsoy, and Walter W. Chen

https://www.pnas.org/content/116/1/303?ijkey=eedc3db1701d7607eb1389c8dc45fe6c9903294c&keytype2=tf_ipsecsha

• The authors establish MITO-Tag mice, which allow cell type specific isolation of mitochondria from different tissues. These mice express a mitochondrially-localised epitope, whose expression is driven by a Cre-recombinase. Therefore, mice engineered such that Cre-recombinase is under the control of a promoter which is active in a particular cell type, allows isolation of mitochondria from particular cell types via immunoprecipitation.
• Purified mitochondria can be subsequently analysed through e.g. proteomic, lipidomic, and metabolomic analyses. The authors demonstrate this for hepatocytes.

Mind your mouse strain

José Antonio Enríquez
 
https://www.nature.com/articles/s42255-018-0018-3

• Many commonly used inbred mouse strains carry random mutations which can affect the interpretation of results derived from these strains
• Mice of a single strain is still susceptible to random genetic drift. Whilst some animal providers have implemented a genetic stability program, this is not common practice across all animal facilities in research institutions
• The most commonly used lab strain is BL6. There are two prominent mutations which are likely present in all substrains (Cdh23, causing age-related hearing loss, and COX7A2L which involves mitochondrial supercomplex formation). 
• It is important to be aware of the nuclear genetic differences between substrains because they can result in different molecular and phenotypic signatures.
• Correct reporting of animal substrains may go some way towards explaining contradictory observations between laboratories when they occur

Reversing wrinkled skin and hair loss in mice by restoring mitochondrial function

https://www.nature.com/articles/s41419-018-0765-9

Bhupendra Singh, Trenton R. Schoeb, Prachi Bajpai, Andrzej Slominski & Keshav K. Singh

• The authors created an inducible mouse expressing a dominant negative mutant of POLG1 which induces mtDNA depletion (approximately x2 depletion) in the whole animal (mtDNA-depleter mouse). 
• The depletion of mtDNA caused widespread reduction in activity of components of the electron transport chain.
• Skin wrinkles and hair loss were amongst the earliest and most predominant phenotypic changes, along with a reduction in body weight and height, and skin inflammation
• The authors induced mtDNA depletion for 2 months, resulting in the above phenotypes, then restored mtDNA copy number. After 1 month at normal mtDNA copy number, skin wrinkles and hair loss reverted, and the animals displayed relatively normal cutaneous structures. Skin inflammation reduced, although was not returned fully to wild-type levels. 
• This study is further evidence for the causal role of mtDNA perturbations in mammalian ageing, and is amongst the first studies to demonstrate that mtDNA-induced ageing may be reversible.