Mice lacking the mitochondrial exonuclease MGME1 accumulate mtDNA deletions without developing progeria

https://www.nature.com/articles/s41467-018-03552-x


Stanka Matic, Min Jiang, Thomas J. Nicholls, Jay P. Uhler, Caren Dirksen-Schwanenland, Paola Loguercio Polosa, Marie-Lune Simard, Xinping Li, Ilian Atanassov, Oliver Rackham, Aleksandra Filipovska, James B. Stewart, Maria Falkenberg, Nils-Göran Larsson & Dusanka Milenkovic

• The authors developed a knockout mouse model for the gene MGME1.  Loss of MGME1 expression in siRNA treated cells, or patient fibroblasts, leads to an accumulation of 7S DNA.
• 7S DNA is a single-stranded, ~650 nt long, nascent DNA species that creates a characteristic triple-stranded DNA structure in mtDNA called the D-loop. 7S DNA is formed by premature replication termination of mtDNA. 
• Human patients with loss-of-function MGME1 mutations show depletions/ rearrangements of mtDNA, and a number of devastating phenotypes.
• MGME1 is not essential for embryonic development, but its loss leads to accumulation of multiple deletions and depletion of mtDNA.
• In the heart, these mice possessed ~50% less wild-type mtDNA (Southern blot analysis), and ~x5 more 7SDNA than mtDNA (which is around ~x5 higher ratio than seen in wild-type mice).
• The authors found severe wt-mtDNA depletion in: kidney, liver, brain and heart. Skeletal muscle was least severely affected (if at all). (NB: this is from visual inspection of the Southern blots in Fig 2D).
• Mice with both germline and tissue-specific knockout of Mgme1 are viable and appear healthy, despite the existence of deletions and rearrangements of mtDNA.
• The authors found that the deleted species was a linear mtDNA molecule of ~11kb (~67% mass of normal mtDNA) due to the stalling of mtDNA replication
• Mgme KO mice had similar levels of point mutations as wild-type mice
• There were no clear OXPHOS defects in Mgme KO mice, despite depletion of mtDNA, accumulation of linear subgenomic mtDNA, and replication stalling in young animals. The mice did not show premature ageing.
• In liver tissue, a prominent stalling site is observed, whereas in heart, a range of replication intermediates is observed. This shows the existence of tissue-specific stalling profiles in this system.
• The authors suggest that MGME1 might be part of a regulatory switch acting at the end of the D-loop region that controls mtDNA replication and heavy-strand transcription termination. 

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

• Do the authors still see that the mice are susceptible to arrhythmias, as observed here (blog here)? In other words, are there phenotypes in these mice which only become evident when the animals are challenged e.g. through stress?
• How do we resolve the observation that loss of function of MGME1 in humans causes devastating phenotypes, whereas in mice it does not? 
• If these deletions are all linear, can they still transcribe? If not, to what extent are these deletions representative of deletions which occur naturally, for instance the common deletion, which (as I understand it) are circular, see here?

Age-Associated Impairments in Mitochondrial ADP Sensitivity Contribute to Redox Stress in Senescent Human Skeletal Muscle

http://www.cell.com/cell-reports/abstract/S2211-1247(18)30264-X

Graham P. Holloway, Andrew M. Holwerda, Paula M. Miotto, Marlou L. Dirks, Lex B. Verdijk, and Luc J.C. van Loon

• The authors sought to determine whether there is an age-associated increase in mitochondrial reactive oxygen species (ROS) in vitro, using permeabilized muscle fibres.
• They find that the capacity of mitochondrial H2O2 emission does not increase with ageing
• However, ADP sensitivity does reduce with age. Consequently, H2O2 levels increase with age.
• Increasing muscle mass, strength, and maximal mitochondrial respiration through exercise in older individuals did not alter H2O2 emission rates, the fraction of electron leak to H2O2 or the redox state of muscle.
• In summary, reduction in mitochondrial ADP sensitivity increases mitochondrial H2O2 emission, which cannot be rescued through resistance training in later life (although there were other benefits to health of these individuals).

Optimized Mitochondrial Targeting of Proteins Encoded by Modified mRNAs Rescues Cells Harboring Mutations in mtATP6

http://www.cell.com/cell-reports/abstract/S2211-1247(18)30253-5

Randall Marcelo Chin, Tadas Panavas, Jeffrey M. Brown, and Krista K. Johnson

• Allotopic expression where a gene ordinarily encoded by mitochondrial DNA (mtDNA) is placed inside the nucleus, and modified such that the resultant protein is correctly transported into the mitochondria.
• It is hoped that allotopic expression may be able to rescue pathologies which arise due to mutations in mitochondrial DNA: indeed, allotopic expression-based gene therapy is in phase 3 clinical trials for the mitochondrial disease LHON. 
• mtDNA-encoded proteins are highly hydrophobic, causing them to often fold into import-incompetent states, thereby preventing them from entering the mitochondria. 
• Mitochondrial targeting sequences (MTSs) and 3' untranslated regions (3' UTRs) have been used to target proteins or mRNA to the mitochondria. 
• In this study, the authors performed a screen of 31MTSs and 15 UTRs in their ability to localize up to 9 allotopically expressed proteins to the mitochondrial DNA (note that mtDNA encodes 13 proteins, 22 tRNAs and 2 rRNAs).
• Cybrid cells harbouring the 8993T>G point mutation in the mtATP6 gene were transiently transfected with a construct which was able to allotopically express mtATP6 and rescue the mtATP6-deficient cells.
  

Mitochondrial DNA as an inflammatory mediator in cardiovascular diseases

Hiroyuki Nakayama and Kinya Otsu

http://www.biochemj.org/content/475/5/839.long


In this review, the authors discuss the role of mitochondria, and especially mitochondrial DNA (mtDNA) in triggering and maintaining cardiac inflammation. In this blost post we only summarise some parts of the review directly related to mtDNA.

We all know that the immune system provides protection against microorganisms such as bacteria, viruses, and fungi. This is achieved by sensing both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs).

One major mechanism for activating the innate immune system is the sensing of pathogen-derived nucleic acids, and this is where mitochondria come into play. Due to their bacterial origin, mtDNA shares similarities with bacterial DNA (e.g. it contains cardiolipin and a predominantly unmethylated CpG motif). Mitochondria also release other DAMPs which can bind and activate multiple pattern recognition receptors similar to those activated by PAMPs.

MtDNA can leave the mitochondria and enter the cytoplasm or leave the entire cell. Opening of the mitochondrial transition pore plays an important role of mtDNA release from mitochondria, as inhibition of pore opening reduced levels of mtDNA in the cyotosol. MtDNA release is also controlled by other regulatory proteins such as the voltage dependent anion channel, Bax, and Bak.

MtDNA released after cell death functions as a DAMP. The mechanism of releasing mtDNA from non-nectrotic cells remains unclear, though exosomal release is proposed to be involved in this mechanism.  MtDNA enters the endocytic pathway by endocytosis and stimulates pattern recognition receptors which eventually leads to inflammasome formation.

It is important to degrade extracellular mtDNA to inhibit unnecessary inflammatory responses. It could be that mtDNA, like other non-host DNA in circulation, is digested in part by circulating nucleases. It is, however, unclear whether this occurs in physiological conditions, especially when mtDNA exists in microvesicles such as exosomes. Inside cells, DNasell plays an important role in mtDNA degradation


Levels of circulating mtDNA increase with age and correlate with levels of pro-inflammatory cytokines. Therefore, mtDNA-induced inflammatory responses can be involved in age-related cardiovascular disease, heart failure and atherosclerosis.

During heart failure, multiple endogenous DAMPs (including mtDNA) are released and recognized to induce an inflammatory response. However, no associated was found between the severity of heart failure and mtDNA levels in serum of patients (patients do show much higher serum mtDNA levels compared to controls).

.

Efficient termination of nuclear lncRNA transcription promotes mitochondrial genome maintenance

https://elifesciences.org/articles/31989

Dorine Jeanne Mariëtte du Mee, Maxim Ivanov, Joseph Paul Parker, Stephen Buratowski, Sebastian Marquardt

• Most of the DNA of eukaryotes does not code for protein, yet many such regions of non-coding DNA are still transcribed into RNA (these are called lncRNA). Understanding the biological functions of these large expanses of non-coding DNA is an active area of current research.
• Here, the authors show that a particular non-coding RNA in budding yeast (CUT60) is required for the proper transcription of its neighbouring gene ATP16. Mutations in CUT60 could result in the fusion of the lncRNA with the RNA of ATP16, and consequently the ATP16 protein could not be produced.
• Interestingly, this had the consequence of yeast cells losing their mitochondrial DNA. ATP16 constitutes a subunit of ATP-synthase, which perhaps explains this striking phenotype.
• The authors speculate that loss of mtDNA triggered by controlled, transient, transcription termination efficiency of CUT60 could allow cells to detoxify themselves of deleterious mtDNA 

Thoughts
-----------------------
The authors raise an interesting idea that yeast cells may be able to cleans their mtDNA through reducing CUT60 transcription termination efficiency. They suggest that cells may shed their mtDNA, and then gain healthy copies of molecules through mating. We know from animals that, during development, the mtDNA bottleneck serves to cleanse the developing embryo of deleterious mtDNA mutations. I wonder whether yeast cells could use this mechanism to a gentler extent (not completely losing their mtDNA, just reducing their mtDNA copy number) to bottleneck their mtDNA?

Hallmarks of Cellular Senescence

https://www.sciencedirect.com/science/article/pii/S0962892418300205

Alejandra Hernandez-Segura, Jamil Nehme, Marco Demaria

A senescent cell is one which permanently stops dividing. In vitro, this can be caused by various stimuli, although it is unclear which amongst these cause senescence in vivo. The accumulation of senescent cells is observed through ageing, and a growing body of evidence is pointing towards the removal of senescent cells as a strategy to combat ageing.

Types of senescence currently known:

- DNA damage-induced senescence. This can be induced in vitro through radiation or drugs
- Oncogene-induced senescence. Activation of oncogenes (e.g. Ras or BRAF) or inactivation of tumour suppressors (e.g. PTEN) can induce senescence
- Chemotherapy-induced senescence. Drugs such as bleomycin or doxorubicin induce DNA damage. Drugs such as abemaciclib and palbociclib can inhibit cyclin-dependent kinases which regulate the cell cycle
- Mitochondrial dysfunction-associated senescence. The so-called "senescence associated secretory phenotype" (SASP) appears to be characteristic of this kind of senescence
- Epigenetically induced senescence. Inhibitors of DNA methylases or histone deacetylases can cause senescence
- Paracrine senescence. Senescence can be induced via the SASP produced by primary senescent cells

The senescence phenotype is often characterised by:

- Activation of a chronic DNA damage response
- Engagement of various cyclin-dependent kinase inhibitors
- SASP (which comprises, in part, various proinflamatory and tissue-remodelling factors)
- Induction of anti-apoptotic genes
- Altered metabolic rates
- Endoplasmic reticulum stress
- Consequent to the above, senescent cells are: enlarged and more flattened; have altered plasma membrane composition; accumulate lysosomes and mitochondria.

Current methods which are used to detect senescent cells include:

- DNA damage response: Immunostaining for γ-H2AX, p53
- Cell cycle arrest: Measurement of colony-formation potential or DNA synthesis rate via BrdU/EdU-incorporation. Expression level of the cyclin-dependent kinase inhibitors p16 and p21.
- Secretory phenotype: Cytokines (IL-1a, IL-6 and IL-8) , chemokines (CCL2) and metalloproteinases (MMP-1, MMP-3). However, the SASP is heterogeneous.
- Apoptosis resistance: Upregulation of BCL-proteins, BCL-2, Bcl-w or Bcl-xL.
- Cell size: Enlarged cell body and irregular shape using bright-field microscopy. Immunofluorescence targetting vimentin, actin or other cytoplasmic proteins have been used.
- Increased lysosomal content: e.g. SA-βgal, SSB, GL13, LysoTrackers, orange acridine
- Accumulation of mitochondria: MitoTrackers

Transitional correlation between inner-membrane potential and ATP levels of neuronal mitochondria

https://www.nature.com/articles/s41598-018-21109-2

R. Suzuki, K. Hotta and K. Oka

In this paper, the authors simultaneously measure mitochondrial membrane potential (Δψ) and mitochondrial ATP production (ATPmito) in dorsal root ganglion neurons from rat embryos. They measure the dynamics of Δψ and ATPmito, as well as their correlation, during physiological neuronal activity and focus on the following questions:

Is there a relation between Δψ, ATPmito and 

• mitochondrial size?
• mitochondrial transport velocity?
• mitochondrial transport direction?

 Furthermore, they ask the question

• What happens to Δψ and ATPmito during mitochondrial fusion and fission events? 20 fusion events and 20 fission events were investigated.

Some of the Results:

• Δψ and ATPmito were compared among anterogradely transported, retrogradely transported, and stationary mitochondria in axons. Retrogradely transported mitochondria had slightly lower Δψ compared to anterogradely transported mitochondria.
• No correlation was found between Δψ, ATPmito and mitochondrial velocity and transported distance.
• Post-fusion mitochondrial membrane potential Δψ seemed to be higher than the average of the pre-fusion potentials (i.e. Δψfused > 0.5 (Δψpre-1 + Δψpre-2)).*
• ATPmito was higher in the post-fusion mitochondrion compared to the average of the two pre-fusion mitochondria
•  The two post-fission mitochondria tended to have different values for
  Δψ and their average was typically lower than the pre-fission potential (again, no information on size was provided as discussed below*).
• No changes in ATPmito were observed upon fission
• Mitochondrial density was higher in growth cones (an extension of a developing or regenerating neurite seeking its synaptic target) compared to axons. 
• Average ATPmito levels were slightly lower in growth cones, though integrated ATP levels (over all mitochondria) were higher. 
• Average Δψ was higher in growth cones compared to axons
• Higher ATP levels in growth cones led to faster elongation of the axon, though no correlation between elongation speed and Δψ was found.
• ATPmito tends to follow a change in Δψ (i.e. the change in Δψ occurs first). 
  ATPmito and Δψ are not necessarily always correlated.
• Various other results were obtained which you can find by reading the paper!

*We note that no information was provided regarding mitochondrial size. If one of the pre-fusion mitochondria is much larger than the other, we might expect the membrane potential of the former to have more influence on the final potential of the post-fusion mitochondrion. In this case, one would not expect  Δψfused to be the arithmetic average of the two pre-fusion potentials.