Overview
Mitochondria contain their own DNA, DNA replication machinery, and protein synthesis machinery. There are 13 proteins encoded by mitochondrial DNA (mtDNA), all of which are subunits of 4 of the oxidative phosphorylation complexes. Drug-induced mitochondrial toxicity can occur as a result of impairment of mitochondrial DNA replication, as when some antiretrovirals, for example, cause off-target inhibition of mtDNA polymerase γ. Toxicity can also occur due to impairment of mtDNA-encoded protein synthesis. Antibiotics that are protein synthesis inhibitors can inhibit mtDNA-encoded synthesis due to the similarity between the bacterial protein repliction machinery and mitochondrial protein replication machinery.

The MT-BG1 MitoBiogenesis™ assay is used in cultured cells to compare the levels of Complex IV (cytochrome c oxidase), a mitochondrial protein which has 3 subunits encoded by mtDNA and made by mitochondrial ribosomes, with that of frataxin, a mitochondrial protein encoded by nuclear DNA and made by cytosolic ribosomes. The ratio of Complex IV : frataxin decreases when a drug inhibits mtDNA replication or mtDNA-encoded protein synthesis.

Assay Details
Complex IV and frataxin are captured by their respective monoclonal antibodies on a dipstick (lateral flow device) and visualized by gold-conjugated anti-Complex IV and anti-frataxin detector monoclonal antibodies (the capture and detector antibodies identify different epitopes of Complex IV and frataxin, and in the case of Complex IV they only form an antibody-Complex IV-antibody sandwich with fully assembled Complex IV).

Standard curves of Complex IV and frataxin levels are generated by the user with a dilution series of control cells. The signal intensities of the Complex IV and frataxin bands on the dipsticks are measured by a dipstick reader (MitoSciences' MS1000 is recommended) or may be analyzed by another imaging system. The levels of Complex IV and frataxin in drug-treated cells are then measured. The Complex IV : frataxin ratio of the drug-treated cells is then compared with that of the control cells.

MT-BG1 is suitable for in vitro screening in cells as well clinical monitoring using blood (a single drop is all that is required), or tissue (needle biopsy or cheek swabs provide sufficient material). Therefore the same assay can be used to monitor inhibition of mitochondrial biogenesis all the way through clinical trials.

Frequently Asked Questions

Q1. How does the protein-based MT-BG1 assay stack-up against a PCR-based mtDNA assay?
A1. MT-BG1 provides more relevant information when considering the effects of a compound on mitochondrial biogenesis. Identifying and characterizing altered mitochondrial biogenesis requires consideration of the loss (or gain) of copies of mtDNA, translation and transcription rates of the encoded genes, and stability and assembly of the 13 mitochondrially-encoded subunits into the 5 OXPHOS complexes. Measuring protein levels as done with MT-BG1 summates all of these effects, and is equally informative of compounds that alter mtDNA replication, mitochondrial protein synthesis, or both.

The figure above illustrates the loss of mtDNA, as measured by PCR, and the loss of mitochondrially-encoded protein (cytochrome c oxidase), as measured by MT-BG1, in the presence of 4µM ddC in cultured fibroblasts. Not surprisingly the reduction in levels of protein trails the loss of mtDNA as the protein complexes, including cytochrome c oxidase, have a significant half-life before degradation, but the correlation of the rate of depletion of mtDNA to mitochondrially-encoded protein is clear in this example.

The MT-BG1 assay also has several important technical advantages over a PCR-based mtDNA assay:
• More cost effective
• Does not require expensive equipment
• Can be performed very easily and does not require specially trained personnel
• Faster - 15 minutes versus several hours
• Can be done at the point-of-care in clinical studies.


Q2. Why use frataxin as a control?
A2. All cellular proteins are under transcriptional and many under post-translatonal control of their levels, and there is no true "control" protein for use as a standard against which changes in levels of other cellular components can be compared. We have examined a number of mitochondrial proteins as possible reference proteins and find that frataxin is the least sensitive among the ones tested to changes in level due to signaling or compound toxic events that reduced cytochrome c oxidase levels.


Q3. Can MT-BG1 be used for in vivo studies?
A3. Yes. The assay can detect altered mitochondrial biogenesis in human tissues, including blood and cheek swabs, as well as in biopsies of muscle and fat. This should make the assay particularly useful in following patients on antiviral and antibiotic drugs. In the case of patients with HIV infection on HAART, early indication of adverse effects would be a signal to change the therapeutic regimen. The table below shows the very small amount of blood and cheek swab material needed for such measurements.

Sample	MT-BG1 Working Range
PBMC	0.5 - 20 µg
Cultured cells	0.5 - 20 µg
Fat	2 - 30 µg
Buccal swab	5 - 30 µg

Q4. Can MT-BG1 be used to identify genetic defects involving cytochrome c oxidase?
A4. Yes. As shown in the figure below, our biogenesis assay readily identifies patients with altered cytochrome c oxidase levels whether due to mtDNA mutations, or to mutations in the several assembly factors needed to insert prosthetic groups and/or to combine the mitochondrial and cytosol-synthesized units into the functional complex.