Assays for Measuring Mitochondrial Biogenesis

It has become increasingly important in studies of many diseases including genetic mitochondrial diseases, neurodegenerative disorders, diabetes, and cancer to determine the levels of biogenesis of mitochondria. Drugs are being fashioned to increase biogenesis while at the same time inhibition of biogenesis is an unwanted side effect of a wide range of otherwise good drug candidates.

Two biogenesis kits are available: one designed for patient studies and one designed for high-throughput screening. These novel kits measure the level of a mitochondrial protein that is encoded in part by mtDNA (cytochrome c oxidase) in relation to that of a mitochondrial nuclear-encoded protein (frataxin). They avoid cumbersome PCR determination of mtDNA and they measure protein levels without use of radioactivity, yet both are:

• Sensitive - Each test requires only a very small amount of material (a single drop of blood, a cheek swab, or as little as 1 µg of whole cultured cell extract).

• Comprehensive - By measuring protein levels the tests summate all possible biogenesis effects.

• Accurate - Test results correlate very positively with traditional methods (see below).

• Flexible - Both rapid and high-throughput options are available, and both formats use the same assay principle (see below).

The Kits:

MS631 MitoProfile® Biogenesis Dipstick Assay Kit
A rapid and simple assay suitable for clinical testing.

MS641 MitoProfile® Biogenesis Microplate Assay Kit
A 96-well assay suitable for high-throughput screening.


Mitochondrial biogenesis is a complex and highly regulated process involving both the nuclear and mitochondrial genomes, as well as cytosolic and mitochondrial ribosomes. When considering effects on mitochondrial biogenesis the focus is on the enzymes involved in oxidative phosphorylation, as these are multi-protein complexes that require essential sub-units encoded in mtDNA and synthesized by mitochondrial ribosomes along with their nuclear-encoded sub-units. Perturbations to the mitochondrial biogenesis machinery can thus have significant consequences, both positive as well as negative, for oxidative metabolism and thus overall organism health.

Inhibition of Mitochondrial Biogenesis

Some drugs, such as nucleoside reverse transcriptase inhibitors inhibit not only their primary target, the HIV reverse transcriptase, but can also inhibit DNA polymerase γ of mitochondria. This leads to decreased levels of mtDNA and, hence, to a decrease in the proteins encoded by mtDNA. Other drugs such as antibiotics which are bacterial protein synthesis inhibitors have a propensity to impair mtDNA-encoded protein synthesis due to the similarity in structure between the bacterial ribosome and the mitochondrial ribosome.

The figure above illustrates the relative inhibition of mitochondrial protein synthesis by a variety of anti-bacterial and anti-viral drugs in which HepG2 cells were grown for five population doublings, as compared to cells grown without drugs. Results can be confirmed by Western blotting or immunocytochemistry.

Activation of Mitochondrial Biogenesis

It has been long understood that endurance exercise results in mitochondrial biogenesis in skeletal muscle, but interesting new lines of research involve the up-regulation of mitochondrial biogenesis by new mechanisms such as activation of sirtuins, by caloric restriction, and by over-expression of phosphoenolpyruvate carboxykinase (PEPCK-C). The result of such up-regulated mitochondrial biogenesis in animal models is increased longevity, resistance to metabolic syndrome, and greatly increased exercise performance.


Traditional Methods for Measuring Mitochondrial Biogenesis

Inhibition of mtDNA replication is usually detected by PCR, and inhibition of mtDNA-encoded protein synthesis is usually detected by incorporation of a radioactive amino acid into the mitochondrial extracts. Neither of these methods is rapid, simple, nor amenable to both cell-based as well as tissue-based analyses.

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 the MitoProfile® Biogenesis Kits, 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.


MitoProfile® Biogenesis Assay Principle

The assays compare the levels of Complex IV (cytochrome c oxidase), a mitochondrial enzyme 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 changes predictably with alterations to mtDNA replication or mtDNA-encoded protein synthesis. Complex IV and frataxin are captured by their respective monoclonal antibodies on either a dipstick (lateral flow device) or in a well (96-well plate). In the case of the dipstick assays the detection is done using gold-conjugated anti-Complex IV and anti-frataxin monoclonal antibodies, and in the case of the microplate assay the monoclonal detectors are AP and HRP labeled (in both cases the capture and detector mAbs 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). Frataxin is used as the control for nDNA-encoded protein synthesis as it is the least sensitive among the many tested to changes in level due to signaling or compound toxic events that reduced cytochrome c oxidase levels.

N.B. Deciding on a perfect control protein for any assay is difficult if not impossible. All cellular proteins are under transcriptional control, often by several transcription factors. This is equally true of such commonly used control proteins as succinate dehydrogenase, citrate synthase, and GAPDH as it is for frataxin, which was used in these assays based on numerous internal studies, all of which have shown stable levels of frataxin in OXPHOS patients as well as in cell models after treatment with many different drugs. Conditions which alter iron metabolism are likely to affect frataxin levels and there may be other treatments about which we are as yet unaware.

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