MTD-TFAM () was produced initially as a N-terminal 6XHis-SUMO derivative to increase its intracellular solubility and with a rapid induction approach to minimize toxicity. The initial protein extract was treated with benzonase to remove contaminating DNA; 6XHis-SUMO-MTD-TFAM was isolated on a nickel column, eluted and treated with SUMO protease. Subsequent passage through a nickel column isolated the 6XHis-SUMO, and the eluted MTD-TFAM was purified further and shown to bind and retard electrophoresis of mtDNA by EMSA ().
We then labeled and purified MTD-TFAM with Alexa488 dye according to manufacturer’s instructions (Alexa Fluor®488 Protein Labeling Kit, InVitrogen) and incubated the Alexa488-MTD-TFAM with SH-SY5Y neuroblastoma cybrid cells carrying a G11778A mtDNA mutation in the ND4 gene from a patient afflicted with Leber’s Hereditary Optic Neuropathy (LHON), a cause of retinal ganglion cell degeneration and blindness in young adults (Wallace, Singh et al. 1988
; Yen, Wang et al. 2006
). Incubation with Alexa488-labeled MTD-TFAM revealed rapid entry of MTD-TFAM into the mitochondrial compartment ().
We next investigated if MTD-TFAM could alter the mitochondrial physiology in these LHON cybrid cells. Three consecutive independent experiments were carried out over several months in which LHON cybrid cells at the same initial passage numbers were treated with MTD-TFAM or buffer control (CTL). The two groups of cells in each of the three independent experiments were passed in parallel to generate adequate cell densities to carry out multiple, simultaneous “high-resolution” oximetry-respiration experiments using intact cells metabolizing glucose (Hutter, Unterluggauer et al. 2006
). In this approach, basal, ETC complex-dependent and incrementally uncoupled respiration rates were assayed in real time where all metabolic control systems were otherwise intact. Nine million living cells from individual experiments and their CTL were added to each 2 ml. respiration chamber. We expanded CTL and MTD-TFAM treated cells in culture in each experiment and studied cells at equivalent passage numbers (P2, P3, P4) after initial treatment. The basal respiration values were depicted as a function of the same number of live cells expressed at each of the same three passages after MTD-TFAM exposure. We observed that exposure to MTD-TFAM caused a time-dependent, reversible increase in basal respiration rates that reached a maximal ~2.5-fold increase over control samples at the second passage around 2 weeks (). The resulting effect of MTD-TFAM on respiration across passage number was highly significant (p=0.044) by ANOVA (). shows that MTD-TFAM treatment did not alter basic respiratory parameters related to respiratory chain coupling, indicating that basic respiratory chain physiology was not altered by MTD-TFAM exposure.
Figure 2 Effects of MTD-TFAM on respiration of intact cells metabolizing glucose. In three independent, consecutive experiments G11778A LHON cells were incubated with 32 ng of MTD-TFAM in 4 ml of DMEM media (or DMEM media with buffer salts) for 4 hours, then rinsed (more ...)
Effects of MTD-TFAM Treatment on Respiratory Parameters of G11778A LHON Cybrid Cells
Because TFAM is a recognized essential factor for mitochondrial genome replication and transcription, one possible explanation for this result is that MTD-TFAM exposure was increasing mitochondrial gene replication, transcription and translation into respiratory proteins. We used primers for the D-loop with SyberGreen detection and multiplex qPCR for several mitochondrial genes (12S rRNA, ND2, ND4, CO3) to monitor alterations in mitochondrial gene copy numbers in genomic DNA samples or mitochondrial gene expression in cDNA samples (primer and probe sequences are in Supplemental Table 1). shows that mtDNA copy number from the averages of D-loop, ND2, ND4 and CO3 qPCR for each sample did not change over the time course examined (one-way ANOVA p=0.56). Comparison between data for days 8–9 compared to days 11–12 approached significance (p=0.092). Mitochondrial gene expression in RNA samples, normalized to 12S rRNA expression in each sample, were highest at the earliest time points examined and declined subsequently (). Changes in ND2 and ND4 expression across time approached significance (one-way ANOVA p=0.061, 0.095, respectively). Restriction analysis using SfaN1 treatment of an ND4 PCR product followed by automated electrophoresis revealed that MTD-TFAM treatment did not alter the near-homoplasmic distribution (>97%) of the G11778A mutation in the LHON cybrid cells (not shown).
Bennett MTD-TFAM and mitochondrial respiration
Bennett MTD-TFAM and Mitochondrial Respiration
In the third experimental series of LHON cybrid cell samples exposed to MTD-TFAM, we examined the levels of multiple individual ETC proteins with Western blots. We also studied assembly of ETC macrocomplexes with immunohistochemistry using antibodies directed against mtDNA-encoded catalytic subunits of complexes I and IV, compared to that of an antibody against a nuclear genome-encoded component of complex V (ATP synthase) as a marker for general mitochondrial distribution. Our Western blot analyses () revealed that multiple complex I proteins, all encoded by nuclear genes, increased many fold at the earliest time point examined and then declined to near control values afterwards. The relative mitochondrial mass in cells, expressed as a ratio of the outer mitochondrial membrane protein mitofilin to that of cytosolic beta actin, ~doubled (1.9-fold) in MTD-TFAM treated cells at the earliest time point examined (9 days) and was slightly below control cells by the last time point (20 days). The levels of a mtDNA-encoded (CIV, subunit 2) and multiple nuclear genome-encoded ETC proteins from several complexes also increased substantially and reversibly in the MTD-TFAM treated cells (). Confocal microscopy did not reveal any effects of exposure of the LHON cybrid cells to MTD-TFAM on the proportions of cells (97–100%) with intact ETC complex I or complex IV macroassemblies ().
Bennett MTD-TFAM and Mitochondrial Respiration
Figure 6 Exposure to MTD-TFAM does not alter ETC macrocomplex assembly. Shown are confocal images of LHON cells without and with MTD-TFAM exposure that were immunostained with MitoSciences antibodies against complex I and complex V subunits. There were no differences (more ...)
Because our results with MTD-TFAM treatment of LHON cybrid cells showed increases in several aspects of mitochondrial physiology, we next wished to determine if similar changes could be observed in vivo. We treated normal adult male mice with tail vein I.V. injections of MTD-TFAM or buffer control and assayed motor endurance and respiration in mitochondrial preparations from brain, heart and muscle. Mice were injected once a week for four weeks with MTD-TFAM sufficient to bind ~100 ug of DNA in each injection. Motor endurance was quantitated by the time each mouse could remain on a rotarod that rotated at a constant velocity, which was slowly increased in 10 rpm increments up to 30 rpm. We observed in both treatment groups a substantial effect of conditioning alone on motor endurance, with a 3–4 fold increase in time spent at 20 rpm over the four week testing period (not shown). After 3 weeks of treatment, mice receiving MTD-TFAM injections showed a ~3-fold increase in 30 rpm rotarod endurance that was statistically significant (, top). After 4 weeks of treatment, MTD-TFAM treated mice showed a non-significant ~2-fold increase in 30 rpm rotarod endurance.
Figure 7 Effects of MTD-TFAM injected in vivo on motor endurance and mitochondrial respiration in mouse organs. MTD-TFAM was dialyzed against 5% glycerol in PBS, concentrated to ~0.5 ml using an Amicon filter and injected IV once a week for 4 weeks. Mice underwent (more ...)
The mice were sacrificed 7 days after the last MTD-TFAM injection and rotarod test, organs were harvested, equal weights homogenized, centrifuged and studied with respirometry in parallel from the buffer CTL and MTD-TFAM injected mice. Crude mitochondrial P2 pellets from each organ pair (MTD-TFAM and CTL) were resuspended in mitochondrial respiration buffer and sequentially exposed to substrates that provide electrons to complex I (glutamate/malate), complex II (rotenone/succinate) and complex IV (antimycinA-myxothiazole/ascorbate/TMPD/cytochrome C) with ADP present to estimate State 3 respiration.
We observed significant increases in complex I-driven respiration in brain and skeletal muscle mitochondria isolated from MTD-TFAM treated mice (, bottom). We observed variable and non-significant increases in respiration among the mitochondrial preparations from different organs and other ETC complex substrates in the MTD-TFAM treated mice (, bottom).