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

http://www.sciencedirect.com/science/article/pii/S2451945617304282?via%3Dihub

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

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

Overall, depletion of pyrimidine pools by complex III inhibition causes cell-cycle arrest and promotes mitochondrial elongation as an adaptive response to energetic stress.

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

http://www.cell.com/cell-reports/references/S2211-1247(17)31668-6

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

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

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

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

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

How cells adapt to progressive mitochondrial mutation

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

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

 

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