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 in the very high percentages of cells showing positive staining for both complex subunits.

Time course of effects of MTD-TFAM exposure on mitochondrial DNA copy number. Aliquots of cells used for respiration experiments were extracted for genomic DNA and analyzed for mtDNA copy number using qPCR and primers that amplified portions of the D-loop and 12S rRNA, ND2, ND4, and CO3 genes. Shown are averages of all determinations for each sample. Data are presented as mean +/− SEM of CTL values at each time point. One-way ANOVA was not significant (P=0.56).

Effects of MTD-TFAM exposure on mitochondrial gene expression. Aliquots of cells used for respiration experiments were extracted for RNA; 1 ug RNA was reverse transcribed to cDNA and assayed using qPCR and multiplex TaqMan probes for 12S rRNA, ND2, ND4 and CO3 genes with a linear complete mtDNA PCR product serving as external standard. Shown are 12S rRNA normalized mean +/− SEM copy numbers (ND2, ND4, CO3) for MTD-TFAM expressed as percentages of CTL at each time point. One-way ANOVA’s showed P=0.061 for ND2 and P=0.095 for ND4.

Effects of MTD-TFAM exposure on electron transport chain protein levels. Aliquots of cells from the third MTD-TFAM experiment were analyzed for ETC proteins using a panel of mouse monoclonal antibodies from MitoSciences. Beta actin served as loading control. Shown on the left are blot images for the complex I antibodies (top) and complexes II-V (bottom). Shown on the right are quantitations of Western blot data from for the cells exposed to MTD-TFAM and expressed as percentage of buffer CTL.

(a) schematic of MTD-TFAM. PTD=11 arginine protein transduction domain; HA=hemagglutinin epitope; SOD2=mitochondrial localization sequence for matrix enzyme SOD2; HMG=high mobility group domain. (b) (left) Coumassie stained gel showing purification of recombinant MTD-TFAM. Lanes 1=SUMO-TFAM; 2= after removal of SUMO; 3, 4= dialysis; 5, 6=further column purification. (right) agarose gel EMSA of circular human mtDNA incubated with increasing amounts of purified MTD-TFAM. (c) MTD-TFAM was labeled with Alexa488 and incubated with LHON cybrid cells where mitochondria were stained with MitoTracker Red. Shown are images obtained 39 minutes after exposing the cells to Alexa488-MTD-TFAM.

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 and passed in parallel in regular DMEM growth media. At varying passages after MTD-TFAM incubation, aliquots of cells were harvested with trypsin and utilized for intact cell respiration at densities of 4.5 million live cells/ml. Shown are mean +/− SEM basal respiration rates in glucose for CTL or MTD-TFAM exposed cells. P1=combined CTL respirations (n=8). P2, P3, P4= passage numbers after initial treatment with MTD-TFAM (n=3 each) or buffer CTL (n=3 for P2, P4; n=2 for P3). One-way parametric ANOVA across passage number for MTD-TFAM treatment p=0.0044.

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 motor endurance training and testing as described in Methods. Once week after the last (fourth) injection mice were sacrificed and organs from MTD-TFAM or buffer CTL injected mice were processed in equal weights in parallel to make P2 pellets, which were studied for substrate-specific State 3 (+ADP) respiration. Equal weights of tissue were studied in each respiration chamber. (a) Shown are mean +/− SEM times on the rotarod at 30 rpm for CTL and MTD-TFAM treated mice. (* P<0.05 by t-test compared to CTL). (b) Shown are mean +/− SEM respiration rates for MTD-TFAM and CTL injected mice for brain, heart and skeletal muscle mitochondria expressed as pmol O₂/ml-sec. CI=complex I (rotenone-sensitive malate/glutamate); CII=complex II (antimycinA/myxothiazole-sensitive succinate); CIV=complex IV (azide-sensitive ascorbate/TMPD/cyt C). (*P<0.05 by t-test compared to CTL).