Friday, 23 October 2015

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.





Thursday, 15 October 2015

New compounds to directly modulate mitochondrial ROS

Selective superoxide generation within mitochondria by the targeted redox cycler MitoParaquat
Ellen L. Robb, Justyna M. Gawel, Dunja Aksentijevińá, Helena M. Cochem√©,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
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.

Thursday, 8 October 2015

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.