Mitochondrial gene therapy augments mitochondrial physiology in a Parkinson's disease cell model

Abstract

Neurodegeneration in Parkinson's disease (PD) affects mainly dopaminergic neurons in the substantia nigra, where age-related, increasing percentages of cells lose detectable respiratory activity associated with depletion of intact mitochondrial DNA (mtDNA). Replenishment of mtDNA might improve neuronal bioenergetic function and prevent further cell death. We developed a technology ("ProtoFection") that uses recombinant human mitochondrial transcription factor A (TFAM) engineered with an N-terminal protein transduction domain (PTD) followed by the SOD2 mitochondrial localization signal (MLS) to deliver mtDNA cargo to the mitochondria of living cells. MTD-TFAM (MTD = PTD + MLS = "mitochondrial transduction domain") binds mtDNA and rapidly transports it across plasma membranes to mitochondria. For therapeutic proof-of-principle we tested ProtoFection technology in Parkinson's disease cybrid cells, using mtDNA generated from commercially available human genomic DNA (gDNA; Roche). Nine to 11 weeks after single exposures to MTD-TFAM + mtDNA complex, PD cybrid cells with impaired respiration and reduced mtDNA genes increased their mtDNA gene copy numbers up to 24-fold, mtDNA-derived RNAs up to 35-fold, TFAM and ETC proteins, cell respiration, and mitochondrial movement velocities. Cybrid cells with no or minimal basal mitochondrial impairments showed reduced or no responses to treatment, suggesting the possibility of therapeutic selectivity. Exposure of PD but not control cybrid cells to MTD-TFAM protein alone or MTD-TFAM + mtDNA complex increased expression of PGC-1alpha, suggesting activation of mitochondrial biogenesis. ProtoFection technology for mitochondrial gene therapy holds promise for improving bioenergetic function in impaired PD neurons and needs additional development to define its pharmacodynamics and delineate its molecular mechanisms. It also is unclear whether single-donor gDNA for generating mtDNA would be a preferred therapeutic compared with the pooled gDNA used in this study.

FIG. 1.

(a) Structure of recombinant MTD–TFAM when it is initially produced with the N-terminal (6 × His) SUMO group (top) and after removal of the SUMO group with SUMO protease (bottom). PTD, 11-arginine protein transduction domain; HA, hemagglutinin epitope; SOD2, mitochondrial matrix localization signal (MLS) of superoxide dismutase-2; HMG, high mobility group domains; TFAM, mitochondrial transcription factor A. Arrowheads indicate location of cleavage by mitochondrial endopeptidase of MLS after importation. (b) Agarose gel electrophoresis image of Roche human genomic DNA before (lane 2) and after (lane 1) incubation with Plasmid Safe ATP-dependent exonuclease. A 200-ng amount of DNA was added to each lane. (c) Diagram of proposed binding of MTD–TFAM to mtDNA. (d) Uptake of Cy5-labeled human mtDNA like that shown in (b) after binding to MTD–TFAM and incubation with SH-SY5Y cybrid cells made from platelets of a patient carrying a high abundance of the G11778A mutation causing Leber's hereditary optic neuropathy (LHON). Mitochondria are labeled with MitoFluor green. Top: Image was acquired after about 40 min. Bottom: Image was acquired after about 240 min of incubation with MTD–TFAM–Cy5 mtDNA complex. Color images available online at www.liebertonline.com/hum.

FIG. 2.

Mitochondrial DNA genes and gene expression in PD59 cybrid, 10 weeks after a single treatment with MTD–TFAM or MTD–TFAM complexed with mtDNA. Top: mtDNA gene copy numbers normalized to 18S rRNA gene levels for basal conditions and after treatment with MTD–TFAM alone or with MTD–TFAM complexed with human mtDNA. Total genomic DNA was analyzed by qPCR. Bottom: Same as above, except showing gene expression data from cDNA generated from total RNA samples. Data are normalized to levels of 18S rRNA. Color images available online at www.liebertonline.com/hum.

FIG. 3.

(a) Intact cell respiration of PD59 cybrid under basal conditions (top) and 10 weeks after a single 5-hr treatment with MTD–TFAM alone (middle) or complexed with human mtDNA (bottom). In each tracing the single blue lines show chamber oxygen levels and the red lines show respiration rates. The closely spaced vertical purple lines indicate repetitive injections of the protonophore carbonyl cyanide-p-(trifluoromethoxy)phenylhydrazone (FCCP) to disperse the proton gradient and increase respiration to its maximal uncoupled state. B, basal; o, +oligomycin; r, plus rotenone to inhibit complex I; am, plus antimycin A/myxothiazole to inhibit complex III. (b) Apparent oxygen K_m values (μM) determined by curve fitting of respiration versus oxygen level during normoxic–anoxic transitions in control (CTL) and PD cybrids under basal conditions and after treatment with MTD–TFAM or MTD–TFAM + mtDNA. Shown are mean ± SEM composite results from CTL (n = 5 lines, 22 K_m assays) and PD (n = 7 lines, 27 K_m assays) studied under basal conditions; from CTL (n = 2 lines, 4 K_m assays) and PD (n = 4 lines, 9 K_m assays) after treatment with MTD–TFAM; and from CTL (2 lines, 5 K_m assays) and PD (n = 4 lines, 12 K_m assays) after treatment with MTD–TFAM + mtDNA. ^*p < 0.05 compared with basal CTL values. (c) Mitochondrial movement velocities in processes of differentiated PD59 cybrid under basal conditions and 10 weeks after a single treatment with MTD–TFAM or MTD–TFAM complexed with human mtDNA. Shown are velocities of individual mitochondria that moved. Data were analyzed by one-way, nonparametric ANOVA with post-hoc Dunn's testing. Mitochondrial movement measured in five differentiated CTL cybrid lines was 0.232 ± 0.017 (SEM) μm/sec. Mitochondrial movement velocity in differentiated SH-SY5Y was 0.21 μm/sec. The calculated velocity for baseline PD59 was 0.178 μm/sec. ^*p < 0.05 compared with basal movement; ^#p < 0.01 compared with MTD–TFAM treatment. Color images available online at www.liebertonline.com/hum.

FIG. 4.

Effects of treatment with MTD–TFAM alone or complexed with mtDNA on cybrid cell TFAM levels. Total protein extracts from CTL and PD cybrids before and 9–11 weeks after a single treatment with MTD–TFAM or MTD–TFAM complexed with mtDNA were analyzed by Western blot for TFAM levels. Top: Levels of ≈25-kDa TFAM normalized to levels of β-actin. Bottom: 25-kDa:29-kDa TFAM ratios expressed as a percentage of baseline. Color images available online at www.liebertonline.com/hum.

FIG. 5.

Effects of treatment with MTD–TFAM alone or complexed with mtDNA on cybrid cell PGC (peroxisome proliferator-activated receptor [PPAR]-γ-related cofactor)-1α expression. cDNAs from CTL and PD cybrids before and 9–11 weeks after a single treatment with MTD–TFAM or MTD–TFAM complexed with mtDNA were analyzed by RT-qPCR for PGC-1α and results are normalized to 18S rRNA levels. Color images available online at www.liebertonline.com/hum.