Are you TORCing tau me? Amyloid‐β blocks the conversation between lysosomes and mitochondria

http://emboj.embopress.org/content/early/2018/11/02/embj.2018100839

Juan Carlos Polanco, Jürgen Götz

INTRODUCTION

This EMBO News&views paper is about recent progress on how amyloid‐β (Aβ) and tau exacerbate Alzheimer's disease (AD) at a subcellular level.

A link has been established between type 2 diabetes and AD: insulin dysregulation occurs in brain tissue from AD patients and mouse models for AD.

mTOR (the protein kinase mammalian target of rapamycin) is a key molecule in the insulin signalling: its hyperactivation mediates insulin resistance, which is one cause of type 2 diabetes.

AD is characterized by the aggregation of two molecules: the peptide amyloid‐β (Aβ) and the protein tau, which impair neuronal function. This accumulation has been linked to lysosomal dysfunction.

Lysosomes and mitochondria both malfunction in various human diseases. This makes sense in the light of the discovery of mitochondria–lysosome membrane contact sites. This could mean that these two organelles also cooperate in physiological conditions.

RECENT DEVELOPMENTS

In the same issue of EMBO, Norambuena et al provide evidence that oligomeric forms of Aβ (Aβos) disrupt the functional crosstalk between lysosomes and mitochondria, thereby contributing to the early stages of AD.

It was already known that mitochondrial dysfunction impairs lysosomal structure and function in a manner that depends on the levels of mitochondrial ROS.

Norambuena et al have provided evidence that that there is also an opposite information flow, with mitochondria receiving signals from lysosomes. This mechanism is dependent on mTORC1, a multiprotein signalling complex nucleated by mTOR and bound to the cytosolic side of lysosomes. This mTORC1 was shown to be activated by insulin and amino acids, leading to what the authors have called “nutrient‐induced mitochondrial activation (NiMA)”,

This lysosomal signalling was strongly inhibited by Aβos and was found to depend on the activation of mTORC1 by a mechanism that requires tau. Indeed, NiMA was not blocked by Aβos in tau knockout neurons.

This reinforces the view that tau facilitates the formation of signalling complexes affecting neuronal function. Future research could be targeted at the isolation and characterization of these putative complexes.

Metformin Targets Mitochondrial Electron Transport to Reduce Air-Pollution-Induced Thrombosis

https://www.sciencedirect.com/science/article/pii/S1550413118305862?via%3Dihub

Saul Soberanes, Alexander V. Misharin,  Amit Jairaman, Luisa Morales-Nebreda, Alexandra C. McQuattie-Pimentel, Takugo Cho, Robert B. Hamanaka, Angelo Y. Meliton, James M. Walter, Ching-I Chen,  Monica Chi, Stephen Chiu, Francisco J. Gonzalez-Gonzalez, Matthew Antalek, Hiam Adbala-Valencia, Sergio E. Chiarella, Kaitlyn A. Sun, Parker S. Woods, Andrew J. Ghio, Manu Jain, Harris Perlman, Karen M. Ridge, Richard I. Morimoto, Jacob I. Sznajder, William E. Balch, Sangeeta M. Bhorade, Ankit Bharat, Murali Prakriya, Navdeep S. Chandel, Go¨ khan M. Mutlu  and G.R. Scott Budinger.

Intro: a word on macrophages.
Macrophages (big eaters, from Greek μακρός and φαγείν) are a type of white blood cell, of the immune system, that engulfs and digests cellular debris, foreign substances, microbes, cancer cells, and anything else that does not have the type of proteins specific to healthy body cells on its surface, in a process called phagocytosis. They are large phagocytes found essentially in every tissue, where they patrol for potential pathogens.

An alveolar macrophage (or dust cell) is a type of macrophage found in the pulmonary alveolus, near the pneumocytes, but separated from the wall.

The study
While air pollution exposure is associated with a variety of poor health outcomes, the major driver of mortality is an increased risk of death attributable to ischemic cardiovascular events, primarily heart attacks and ischemic/thrombotic strokes.

Urban PM air pollution induces the release of pro-inflammatory cytokines, including interleukin-6 (IL-6) from alveolar macrophages. Alveolar macrophage-produced IL-6 enters the circulation to induce the transcription of several coagulation factors in the liver, and augments the tendency toward arterial thrombosis in a murine model of stroke. This phenomenon was not observed in mice lacking IL-6.

Key findings of this model were recently confirmed in humans in an interventional trial of filtered air compared with ambient air conducted in a region of China with high levels of ambient PM.

Accordingly, molecules that can attenuate IL-6 release in response to PM predicted to lower the risk of arterial thrombosis in exposed populations.

Soberanes et al., find that metformin can attenuate PM-induced IL-6 release from alveolar macrophages in mice and reduce the resulting increase in the risk of arterial thrombosis.

The link to mitochondria
In alveolar macrophages from mice and humans, PM-induced mitochondrial ROS generation caused endoplasmic reticulum calcium store depletion, and the opening of store-operated calcium channels, which augmented IL-6 release.

Metformin acted as a complex I inhibitor in alveolar macrophages to reduce mitochondrial ROS from complex III of the mitochondrial electron transport chain in response to PM. That’s the mechanism behind his effectiveness in limiting IL-6 release in from alveolar macrophages.

Mutations of mitochondrial DNA are not major contributors to aging of fruit flies

http://www.pnas.org/content/early/2018/09/21/1721683115.long

Timo E. S. Kauppila, Ana Bratic, Martin Borch Jensen, Francesca Baggio, Linda Partridge, Heinrich Jasper, Sebastian Grönke, and Nils-Göran Larsson

 

• The authors engineered fruit flies expressing mutant versions of mitochondrial DNA polymerase as a means to introduce mtDNA mutations. This model is analogous to the 'mutator mouse', which shows premature ageing phenotypes.
• The authors did not observe an increase in levels of somatic mtDNA mutations with age in the thorax of these mutant fruit flies, suggesting low mtDNA turnover (relative to total lifespan) in this tissue. However, old female flies tranmit more mtDNA mutations than young female flies, suggesting that mtDNA mutations may accumulate in proliferative tissues such as the female gonads.
• Moderately increasing the somatic mtDNA mutation load did not have any impact on fly lifespan or physiology
• Even after multiple generations, flies showed high tolerance to mtDNA mutations. The authors suggest this is likely due to the relatively large mtDNA genetic bottleneck during fly development
• Very high levels of mtDNA mutation caused sensitivity to mechanical and starvation stress, intestinal stem cell dysfunction, and reduced lifespan
• Flies with 9.7×10⁻⁴ mutations per bp had similar lifespans to WT flies, whereas mice with 2.1×10⁻⁴ mutations per bp display shortened lifespan.

Increased TFAM binding to mtDNA damage hot spots is associated with mtDNA loss in aged rat heart

Chimienti G, Picca A, Sirago G, Fracasso F, Calvani R, Bernabei R, Russo F, Carter CS, Leeuwenburgh C, Pesce V, Marzetti E, Lezza AMS

https://www.sciencedirect.com/science/article/pii/S0891584918311614

• In aged rat hearts (27 months), the authors observe 25% decrease in mtDNA content, an anti-correlated 29% increase in the 4.8 kb mtDNA deletion relative content, and 38% decrease in TFAM levels.
• The TFAM-binding activity to specific mtDNA regions increased at the mtDNA replication origins, D-loop and Ori-L
• A marked increase in the relative content of mtDNA strand damage was found in the D-loop and Ori-L in aged animals, suggesting the existence of mutational hotspots in these regions

Mitochondrial Supercomplexes Do Not Enhance Catalysis by Quinone Channeling

Fedor JG and Hirst J

https://www.sciencedirect.com/science/article/pii/S1550413118303310?via%3Dihub

• Components of the electron transport chain have been observed to form respiratory units, called the "respirasome" consisting of 1 CI, 2 CIII and 1 CIV. It has been proposed that the respirasome serves to stabilize CI and mitigate the production of ROS.
• More controversially, the respirasome has been suggested to confer a kinetic advantage on respiration by trapping/channeling quinone to enhance its transfer between the enzymes in the supercomplex, creating an independent, local quinone pool that does not exchange with the quinone pool outside.
• The authors test the quinone channeling hypothesis by introducing an external enzyme which competes for quinone. If the substrate is truly channeled, flux through the competing pathway is negligible.
• The authors introduce the alternative oxidase (AOX) protein. The authors find that AOX competes effectively with the CIII/CIV pathway. Therefore, quinone is not channeled or sequestered by the respiratory-chain supercomplexes.

Comprehensive Quantification of the Modified Proteome Reveals Oxidative Heart Damage in Mitochondrial Heteroplasmy

Bagwan N, Bonzon-Kulichenko E, Calvo E, Lechuga-Vieco AV, Michalakopoulos S, Trevisan-Herraz M, Ezkurdia I, Rodríguez JM, Magni R, Latorre-Pellicer A, Enríquez JA, Vázquez J

https://www.sciencedirect.com/science/article/pii/S2211124718308611?via%3Dihub

• Mass spectrometry is a powerful tool for proteomics. A large proportion of unassigned spectra are thought to arise from peptides containing sequence variants or unknown chemical/post-translational modifications.
• The authors present a suite of bioinformatics tools which extend the coverage which can be attained from mass spectrometry, which allow the location of the modified residues and quantitative analysis of post-translational modification.
• The authors test their approach in the context of mitochondrial heteroplasmy in 12 week old (i.e. young) mice carrying two non-pathological mtDNA variants. The first mtDNA variant is the original variant which co-evolved with the nucleus (C57BL/6J). The second mtDNA variant is NZB.
• The authors found evidence of protein alterations in the heart of heteroplasmic mice which were consistent with mitochondrial dysfunction. 
• The alterations in heteroplasmic mice were consistent with the decreased ATP synthesis and the abnormal increase in phosphocreatine/ATP ratio in the heart, shift to glycolysis, and with the increase in plasma creatine kinase which have been observed in this animal model (unpublished data).
• The authors detected signs of inflammation in the liver of heteroplasmic mice with aging; however, heteroplasmy mainly produces oxidative modifications of OXPHOS proteins in the heart, at this age.  

Mitochondria and aging: A role for the mitochondrial transition pore?

Mathieu Panel, Bijan Ghaleh, Didier Morin

https://onlinelibrary.wiley.com/doi/abs/10.1111/acel.12793

The mitochondrial permeability transition pore (mPTP) is a protein that is formed in the inner membrane of the mitochondria under certain pathological conditions such as traumatic brain injury and stroke. Induction of the permeability transition pore, referred to as the mitochondrial membrane permeability transition (mPT), can lead to mitochondrial swelling and cell death through apoptosis or necrosis depending on the particular biological setting.

Recently, the mPTP has been implicated in the development of the ageing process.

In this paper, the authors review the potential role of mPTP in normal aging and in age-associated diseases.

CALCIUM HOMEOSTASIS, mPTP AND AGEING                                                                    Elevated matrix calcium was the first factor described to activate mPTP opening, and aging alters cytosolic calcium handling. This has been sown in the heart, where aging impairs the myocardial calcium transport system and calcium storage capacities. This was also confirmed in myocytes isolated from human right atria.   

ROS GENERATION, mPTP AND AGEING                                                                              

It is well known that mitochondria are producers of reactive oxygen species (ROS). Evidence suggests that aging involves a change in ROS regulatory processes encompassing a decline in mitochondrial function and an increase in ROS generation. The possible link between ROS production and mPT during ageing is that ROS decrease the calcium concentration needed for mPTP opening and thus sensitize it. This was observed with cardiolipin, a phospholipid that is specific of mitochondria and is susceptible to lipid peroxidaztion by ROS. Oxidized cardiolipin was shown to sensitize heart mitochondria to mPTP opening, and the level of oxidised cardiolipin increases with aging.

MEMBRANE POTENTIAL, mPTP AND AGEING

Several studies have shown that the mitochondrial membrane potential is lower in aged cells. This may have consequences on mPTP opening, as mPTP is a voltage-dependent channel which tends to open upon depolarization. It has been shown in vitro that depolarization induces mPTP opening when mitochondria have been suitably loaded with calcium.

NAD+, mPTP AND AGEING

Several data suggest that aging reduces cellular nicotinamide adenine dinucleotide (NAD+). This was observed in mice, C. elegans and human tissues. Conversion of NAD+ to NADH plays a key role in mitochondrial metabolism. A drop in NAD+ cellular levels can therefore limit NADH generation.  This decreases mitochondrial membrane potential, which increases the frequency and duration of mPTP opening. In turn, mPTP opening causes the release of NAD+ from mitochondria and its depleiton, therefore inducing a vicious circle.
Another important consequence of mitochondrial NAD+ depletion is the inhibition of mitochondrial sirtuin (SIRT, a class of deacetylases) activity, especially SIRT3. This enzyme plays a critical role in the protection of mitochondria and, more particularly, it was shown to inhibit mPTP opening.

In conclusion, a large number of studies demonstrated that the mPTP is more sensitive to opening in aged animals and in aging-associated diseases. However, doubts persist  and definitive experimental proofs of mPTP involvement have to be provided to demonstrate whether it is a cause or a consequence of aging.

Quantification of subclonal selection in cancer from bulk sequencing data

Williams MJ, Werner B, Heide T, Curtis C, Barnes CP, Sottoriva A, Graham TA

https://www.nature.com/articles/s41588-018-0128-6

• The authors investigate intratumoral genetic heterogeneity by performing Bayesian inference on a population-genetics model of asexual evolution, using data from high-coverage bulk sequencing data. The model combines a generative model for tumour development with an error model for sequencing.
• Previous work by the authors showed that, under a neutral evolutionary model, the variant-allele fractions (VAFs) (i.e. the percentage of genomes which are mutated in a particular allele) follow a power-law distribution. Subsequent work by the authors showed that by modelling spatial effects and selection, the authors could infer whether a particular variant was neutral or non-neutral
• In this work, the authors use a stochastic branching process model, whereby cells divide and die with particular rates, and acquire de novo mutations upon division. Mutant subclones are assigned a fitness advantage, which is related to the ratio of replication rate of the mutant to the background host population. Clones which have a selective advantage induce an additional peak in the distribution of VAFs.
• The mean VAF of a particular cluster is a measure of its relative size within the tumour; the total number of distinct mutations in the cluster is a measure of its age, since older subclones have had more time to accrue mutations. These two pieces of information allow the replication rate of the particular subclone to be constrained, and its selective advantage to be inferred. 
• Note that mutations can hitchhike with the actual driver event, so it is not necessarily the case that all mutations with a surprisingly high VAF cause a selective advantage. The driver event may not necessarily even be genetic in origin.
• Using this framework, the authors discover detectable subclones which were consistently present, with a fitness advantage >20%.

Hematopoietic stem cell fate through metabolic control

https://www.sciencedirect.com/science/article/pii/S0301472X18302650

Kyoko Ito and Keisuke Ito

•  Hematopoitic stem cells (HSCs) exist in bone marrow and give rise to a number of different cell types, see here. These stem cells are usually non-dividing (quiescent) until there is a need to i) renew the pool of HSCs or ii) generate new differentiated cells. HSCs can divide symetrically (giving rise to two new HSCs), divide asymmetrically (creating 1 differentiated cell and 1 HSC) and divide to generate 2 differentiated cells (symmetric commitment).
• The authors discuss a study where single HSCs were purified. They found that activation of the PPAR (peroxisome proliferator-activated receptor)- fatty acid oxidation pathway promotes HSC symmetric division through enhanced Parkin recruitment in mitochondria. This pathway activates enhanced mitophagy in HSCs. The authors suggest that enhanced mitophagic clearance of damaged mitochondria is necessary for self-renewing expansion of HSCs.
• Impaired autophagy has been shown to result in HSC exhaustion, and conditional depletion of Atg7 can lead to lethal anemia
• Defective autophagy by the ablation of Atg12 accelerates blood aging phenotypes
• The authors discuss another study which found that loss of authophagy in HSCs causes accumulation of mitochondria and an activated metabolic state, which drives differentiation. These features are shown in HSCs from aged mice. They suggest that autophagy actively suppresses HSC metabolism by clearing active, healthy mitochondria, to maintain quiescence and stemness. [Thought. The idea of actively degrading healthy mitochondria for the purpose of slowing metabolism seems drastic/wasteful?]
• Excessive mitophagy is associated with enlarged HSC pools and blocked lineage commitment. The authors argue that mitophagy levels must be controlled to ensure maintenance of HSCs and appropriate differentiation.

Power grid protection of the muscle mitochondrial reticulum.

https://www.ncbi.nlm.nih.gov/pubmed/28423313

Brian Glancy, Lisa M. Hartnell, Christian A. Combs, Armel Femnou, Junhui Sun, Elizabeth Murphy,

Sriram Subramaniam and Robert S. Balaban.

THE DRAWBACK OF THE MITOCHONDRIAL NETWORK

Cellular mitochondrial networks allow for sharing of metabolites and proteins as well as mitochondrial DNA, and also provide a rapid conductive path for the distribution of potential energy.

However, this extensive coupling presents a major risk as local failures can also spread quickly over the entire network and compromise cellular energy conversion.

Like many power networks that physically segment elements with circuit breakers, similar strategies may be in place to protect cells with coupled mitochondrial networks from propagating local failures.

EXISTENCE OF SUBNETWORKS

Using 2-to-4 month old mice, the authors demonstrate the existence a physically and electrically connected mitochondrial reticulum arranged into longitudinal subnetworks within the cardiac cell.

Each subnetworks comprises many mitochondria and subnetworks are linked through abundant contact sites at highly specific intermitochondrial junctions, IMJs.

(A junction is defined by the close apposition of both the inner and outer membranes of two adjacent mitochondria with high electron density).

PROTECTIVE FUNCTION OF THE SUBNETWORKS

This arrangement of mitochondria into several regional subnetworks as opposed to a single, cell-wide network limits the spread of localized mitochondrial dysfunction to within defined volumes.

In both cardiac and Skeletal muscle subnetworks, a rapid electrical and physical separation of malfunctioning (depolarised) mitochondria occurs, consistent with detachment of IMJs, allowing the remaining mitochondria to resume normal function within seconds. This limits the impact of mitochondrial dysfunction.

These rapid alterations in mitochondrial connectivity allow muscle cells to respond to local dysfunction and restore the energy distribution systems to the remainder of the cell.

Mitochondrial Translation Efficiency Controls Cytoplasmic Protein Homeostasis

https://www.sciencedirect.com/science/article/pii/S1550413118302547?via%3Dihub

Suhm T, Kaimal JM, Dawitz H, Peselj C, Masser AE, Hanzén S, Ambrožič M, Smialowska A, Björck ML, Brzezinski P, Nyström T, Büttner S, Andréasson C, Ott M

• As mitochondria possess their own DNA, they also possess their own transcription and translation machinery. 
• This study focuses on the mitochondrial translation machinery. There exists a tradeoff between the accuracy with which mRNAs are translated into protein, and the speed at which proteins can be formed.
• The mitoribosome is structurally similar to the bacterial ribosome. Several mutations are known for the bacterial ribosome which can increase or decrease the fidelity of translation. The authors sought to determine the effects of such mutations in yeast. 
• The authors investigated two mutations in particular: P50R which caused reduced fidelity of translation, and K71T which increased fidelity (likely at the cost of slower translation). Both mutants caused growth defects on galactose (a respiratory substrate which forces oxidative phosphorylation). 
• The authors observed that the K71T hyperaccurate mutant displayed extended lifespan. These mutants also had lower levels of reactive oxygen species and fewer protein aggregates. The authors suggest that hyperaccurate translation inside mitochondria mitigates ROS-induced aggregation of proteins.

Restoring mitochondrial DNA copy number preserves mitochondrial function and delays vascular aging in mice

https://onlinelibrary.wiley.com/doi/abs/10.1111/acel.12773

Kirsty Foote, Johannes Reinhold, Emma P. K. Yu, Nichola L. Figg, Alison Finigan, Michael P. Murphy, Martin R. Bennett.

INTRODUCTION

Ageing of the large conduit arteries is a major cause of morbidity and mortality, contributing to hypertension. Arterial ageing is associated with multiple structural and functional changes, including vessel dilatation and wall thickening, loss of elastin and deposition of collagen.

Several invasive and noninvasive parameters of vascular stiffness can reliably predict cardiovascular events. However, furthering research in mouse models is not easy. Improved animal welfare means that laboratory mice can now live more than 2 years. This complicates ageing research, since such aged animals might be too frail for functional analyses, and makes ageing studies very long and expensive to perform. Furthermore, it is unclear what the earliest time points that constitute vascular ageing in laboratory mice are, which physiological measures of large artery stiffness correspond most closely to humans.

It is unclear whether decreased mitochondrial function promotes vascular ageing directly or is just a consequence of ageing.

The authors examine multiple parameters of vascular function, histological markers, and markers of mitochondrial damage and function during normal vascular ageing, and the effects of reducing or augmenting mitochondrial function on the onset and progression of vascular ageing.

RESULTS
The authors show that:

• Vascular ageing in mice can be demonstrated by changes in a variety of physiological parameters, with multiple robust reproducible markers appearing as early as 44 wk (earlier than previously thought, allowing for shorter vascular ageing protocols).
• Mouse vascular ageing is associated with characteristic structural changes over the same time, confirming that these changes in physiological parameters represent structural changes associated with ageing.
• Mitochondrial copy number (mtCN), the proteins that regulate it, and mitochondrial respiration are all reduced at the same age that changes in functional and structural parameters were observed.
• Finally, using gain- and loss-of-mitochondrial-function mouse models, we identify that mtCN and mtDNA integrity directly regulate the onset and progression of vascular ageing in mice. In other words, manipulations that result in increased or decreased respiration delay or accelerate changes associated with ageing, respectively.
• The mouse models used were mice overexpressing the helicase Twinkle, to increase mtCN and mitochondrial respiration, and the PolG mutator mice, to compromise mtDNA integrity. These mice do not show change of ROS (at least in early life for the PolG mice, which is when these mice were examined). This suggests that the role of mitochondria in vascular ageing goes beyond ROS.

Linear mitochondrial DNA is rapidly degraded by components of the replication machinery

https://www.nature.com/articles/s41467-018-04131-w

Peeva V, Blei D, Trombly G, Corsi S, Szukszto MJ, Rebelo-Guiomar P, Gammage PA, Kudin AP, Becker C, Altmüller J, Minczuk M, Zsurka , Kunz WS

• Tools such as mitoTALENs and mitochondrial ZFNs are able to cut mtDNA in a sequence-specific manner. Whilst these methods are known to be able to reduce the proportion of mutant mtDNAs, the mechanism by which linear mtDNAs are degraded is unknown.
• The authors show that linear mtDNA is eliminated within hours
• Inactivation of the mitochondrial exonuclease MGME1; inhibition of the exonuclease activity of POLG (see also this complementary study); or knockdown of the mitochondrial DNA helicase TWNK leads to severe imediment of mtDNA degradation. 
• Failure to remove damaged mtDNA leads to the accumulation of abnormal linear and rearranged molecules.
• Degradation of linearized mtDNA is performed by the same machinery that is responsible for mtDNA replication.

Mitochondrial nicotinamide adenine dinucleotide reduced (NADH) oxidation links the tricarboxylic acid (TCA) cycle with methionine metabolism and nuclear DNA methylation

http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.2005707

Lozoya OA, Martinez-Reyes I, Wang T, Grenet D, Bushel P, Li J, Chandel N, Woychik RP, Santos JH

• The authors use an inducible dominant-negative mutant of the mtDNA polymerase POLG in human embryonic kidney cells (HEK293), see here (DN-POLG cells).
• The authors perform RNA-seq on cells with induced DN-POLG at 0, 3, 6 and 9 days after treatment, with approximately 100%, 30%, 10% and 0% mtDNA copy number respectively.
• Loss of mtDNA resulted in DNA hypermethylation. 57% of differentially expressed genes showed significant alteration in their promoter methylation compared with day 0.

• The authors expressed two non-mammalian proteins (NDI1/AOX, see here) which by-pass the mitochondria allowing a normal NAD+/NADH balance to be maintained. In doing this, the authors found far fewer differentially expressed genes when comparing day 0 to day 9 cells. Furthermore, no significant changes in DNA methylation were observed in cells expresing NDI1/AOX. 
• The authors suggest that mtDNA depletion induces imbalance in the NAD+/NADH pool, causing DNA methylation and pathological gene transcription

Amelioration of premature aging in mtDNA mutator mouse by exercise: the interplay of oxidative stress, PGC-1 a, p53, and DNA damage. A hypothesis

Safdar A, Annis S, Kraytsberg Y, Laverack C, Saleem A, Popadin K, Woods DC, Tilly JL, Khrapko K

https://www.sciencedirect.com/science/article/pii/S0959437X16300855?via%3Dihub

(Not a new article, but thought-provoking)

• The PolG mutator mouse accumulates single nucleotide polymorphisms in its mtDNA and shows premature ageing phenotypes
• The threshold effect states that heteroplasmy must reach very high levels before a phenotype is shown. Although each molecule of mtDNA will possess several mutations, the activity of several mitochondrial enzymes are unaffected (whilst others such as Complex IV show activities of about 35%). In this sense, mtDNA mutations can be thought of as recessive.
• The authors here suggest that an accumulation of reactive oxygen species from a diversity of different mutations could explain how the mutator mouse displays premature ageing phenotypes.
• Endurance exercise is able to rescue the mutator phenotype in large part
• Somatic tissues and the stem cell pool suffer from oxidative stress in PolG mice. Anti-oxidants have been shown to ameliorate some phenotypes of the mutator mouse.
• Mitochondrial ROS induces telomere erosion
• ROS (in particular, hydroxyl free radicals) can react with the nucleotide guanine to form 8-OHdG. This is a marker of oxidative stress, and is referred to by the authors as "non-mutational oxidative DNA damage). In the PolG mutator mouse, endurance exercised mice have lower 8-OHdG levels by ~x3.
• A muscle-specific knockout of p53 abolishes the amelioration of the PolG phenotype by exercise. Importantly, p53 is a mtDNA repair protein, and can repair oxidative damage.
• The authors also highlight that non-mutational mtDNA damage (e.g. 8-OHdG) can be converted into spurious mutations during PCR amplification
• Note that the transcription machinery of the cell is also prone to making mistakes at sites of oxidative DNA damage
• The authors revive the idea of the classical "vicious cycle" of mtDNA damage (mtDNA damage -> ROS -> more mtDNA damage) but instead of nucleotide substitutions the authors suggest that non-mutational mtDNA damage could be a potentially explanatory hypothesis.
• Furthermore, the authors suggest that mitochondria compete with the nucleus for p53 during oxidative stress: mtDNA damage -> ROS -> nuclear DNA damage -> translocation of p53 to nucleus -> prevention of mtDNA repair -> mtDNA damage. The authors label this a "malicious cycle"
• The authors suggest that exercise promotes PGC-1a, which promotes the expression of antioxidants, which lowers the rate of nuclear DNA damage, allowing p53 to leave the nucleus. 

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

• In the "malicious cycle" hypothesis, the authors speculate that ROS induces nuclear DNA damage, causing p53 to translocate to the nucleus, meaning that mtDNA is repaired less. So implicit to this assumption is that the nucleus takes higher precedence over mtDNA in the context of oxidative stress. If so, that's interesting. Why not upregulate p53 so that it is not limiting, and both the nucleus and mtDNA can be repaired?

So Happy Together: The Storied Marriage Between Mitochondria and the Mind.

http://www.biologicalpsychiatryjournal.com/article/S0006-3223(18)31316-7/fulltext

Ruth F. McCan, David A. Ross

Many neuronal functions need mitochondria. Therefore, one would expect mitochondrial damage to to affect the nervous system. Indeed, we see a higher than normal incidence of psychiatric illnesses in people with genetic mitochondrial disorders and depressive episodes have been observed in mouse model of genetic mitochondrial diseases.

Another area of research is exploring the other direction of causality: can psychological stress and depression cause mitochondrial dysfunctions?

One proposed mechanism involves glucocorticoids. Experiments with cultured mouse neurons suggest that mitochondria are impaired by long-term exposure to glucocorticoids, which may be overproduced in states of stress and depression.

Another hypothesis involves oxidative stress (OS), caused by reactive oxygen species (ROS), which are produced by mitochondria. Biomarkers of OS are increased in people with depression, and other mood and anxiety disorders seem associated to OS. It might be that stress leads to a hyper-metabolic state in which mitochondria produce more ROS. These are toxic to mitochondria themselves, which are very vulnerable to oxidative damage, potentially causing more ROS production in a vicious cycle.

A study highlighting potential connections between stress, depression and mitochondria was published in 2015 by Cai et al., who collaborated with more than 60 scientists to look at a cohort of 11,670 women from China, through whole-genome sequencing of saliva samples.

It was found that women who had experienced stressful life events and depression had shortened telomeres, something which can be seen in settings of OS. It might be that stress acts on mitochondria, triggering a cascade which leads to depression. However, another possible explanation can be that stress take people who are more prone to depression and triggers an overdrive state in which mitochondria become overwhelmed, leading to OS. Therefore, it is not clear whether mitochondrial dysfunctions cause stress or the other way around.

Another question which is attracting interest concerns our mitochondria are involved in synaptic health and dysfunction in depression. It is thought that in depression neurons atrophy, synapses vanish and dendrites shrink. Although it is not clear whether or not (and how) these morphological changes are causally connected to the disease, it is worth considering the underlying mechanism. It is easy to imagine that large amounts of energy are required to create new neurons and synapses, so it is likely that mitochondria play a role. Moreover, mitochondria are involved in the regulation of intracellular calcium leves, which is crucial at synapses, since calcium stimulates neurotransmitter release.

It is clear that the more we learn about mitochondria, the more they can help unravel the connections between neurotransmitters, mood states, genetic diseases and psychiatric symptoms, life experiences and mental health.

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).

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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.