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. 2012 Jun;23(6):647-57.
doi: 10.1089/hum.2011.177. Epub 2012 Apr 17.

Mitochondrial gene therapy improves respiration, biogenesis, and transcription in G11778A Leber's hereditary optic neuropathy and T8993G Leigh's syndrome cells

Shilpa Iyer  1 , Kristen Bergquist, Kisha Young, Erich Gnaiger, Raj R Rao, James P Bennett Jr
Affiliations

Mitochondrial gene therapy improves respiration, biogenesis, and transcription in G11778A Leber's hereditary optic neuropathy and T8993G Leigh's syndrome cells

Shilpa Iyer et al. Hum Gene Ther. 2012 Jun.

Abstract

Many incurable mitochondrial disorders result from mutant mitochondrial DNA (mtDNA) and impaired respiration. Leigh's syndrome (LS) is a fatal neurodegenerative disorder of infants, and Leber's hereditary optic neuropathy (LHON) causes blindness in young adults. Treatment of LHON and LS cells harboring G11778A and T8993G mutant mtDNA, respectively, by >90%, with healthy donor mtDNA complexed with recombinant human mitochondrial transcription factor A (rhTFAM), improved mitochondrial respiration by ∼1.2-fold in LHON cells and restored >50% ATP synthase function in LS cells. Mitochondrial replication, transcription, and translation of key respiratory genes and proteins were increased in the short term. Increased NRF1, TFAMB1, and TFAMA expression alluded to the activation of mitochondrial biogenesis as a mechanism for improving mitochondrial respiration. These results represent the development of a therapeutic approach for LHON and LS patients in the near future.

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Figures

FIG. 1.
FIG. 1.
Time-lapse confocal images to detect entry of donor mtDNA complexed with rhTFAM into LHON cybrid cells. Entry and colocalization (yellow overlay) of Cy5-human LHON mtDNA (red) complexed with rhTFAM was observed after 4 hr of incubation. Mitochondria within the cybrids have been stained with MitoFluor Green. Scale bar=10 μm.
FIG. 2.
FIG. 2.
Mitochondrial gene therapy increases respiration in LHON cells. Three weeks after exposure to mtDNA complexed with rhTFAM, LHON cybrid cells showed increases in (A) ROUTINE by ∼1.2-fold, (B) LEAK by ∼1.3-fold, and noncoupled (C) ETS capacity by ∼1.2-fold compared with CTL cells (left column). Respiration is expressed as pmol of O2/(sec/106 cells).
FIG. 3.
FIG. 3.
Mitochondrial gene therapy increases mtDNA copy numbers and gene expression in LHON cells. (A–C) Real-time qPCR analysis of candidate mitochondrial genes (ND2, COX3, ND4) showed a reversible increase in mtDNA copy numbers over CTL on treatment with mtDNA complexed with rhTFAM over 3 weeks (n=11). These results were not statistically significant. (D–F) RNA from the different mitochondrial genes was reverse-transcribed to cDNA and assayed by real-time qPCR. Shown are normalized mean values of significant increases in gene expression for ND4 (p<0.05; an increase by ∼1.8-fold) and COX3 (p<0.05; an increase by ∼1.7-fold) and a not so significant increase in gene expression for ND2 (p<0.07; an increase by ∼1.5-fold) over CTL for each gene comparison.
FIG. 4.
FIG. 4.
Mitochondrial gene therapy increases nuclear protein levels in LHON cells. Cells were passed, pellets were extracted, and total cellular protein was separated by SDS-PAGE. (A) The left panel is an immunoblot for members of ETS Complexes I–V and the outer membrane protein mitofilin using MitoSciences monoclonal antibodies. (B) The right panel shows ratios (n=11) of all protein bands normalized to β-actin loading control densities and expressed as percentage of buffer CTL cells for each time point. One-way ANOVA showed p<0.05 for Complexes I, II, and V and p>0.05 for Complexes III and IV for each comparison.
FIG. 5.
FIG. 5.
Mitochondrial gene therapy increases mitochondrial biogenesis in LHON cells. RNA from the different candidate biogenesis genes was reverse-transcribed to cDNA and assayed by real-time qPCR. Shown are geometric mean normalized cDNA values of mtDNA+rhTFAM treatment indicating significant increases in gene expression over CTL. (A) Increase in cDNA expression of TFAM1A by ∼1.5-fold over CTL. One-way ANOVA showed p<0.05. (B) Increase in cDNA expression of NRF1 by ∼1.6-fold over CTL. One-way ANOVA showed p=0.05. (C) Increase in cDNA expression of TFAMB1 by∼1.7 fold over CTL. One way ANOVA showed p<0.05. All CTLs are shown in gray and individual genes in black for each comparison.
FIG. 6.
FIG. 6.
Mitochondrial gene therapy increases respiration in LS cells. (A) Two weeks after exposure to mtDNA+rhTFAM (gray) or rhTFAM only (black), LS cells showed increased respiration, increased sensitivity to oligomycin, and lowered uncoupled respiration compared with untreated CTL (beige). Respiration is expressed as pmol of O2/(sec/106 cells). (B) A recording of the different aspects of the respiration profile of LS (CTL) cells after 2 weeks. (C) An overlay of the respiration recording of LS cells treated with mtDNA+rhTFAM (green) over CTL (red). (D) Two to 3 weeks after exposure to mtDNA+rhTFAM (gray), LS cells showed increased ROUTINE respiration compared with rhTFAM only (black). Respiration is expressed as pmol of O2/(sec/106 cells). (E) mtDNA+rhTFAM treatment but not rhTFAM alone increased oligomycin-sensitive LEAK respiration after 2 and 3 weeks.
FIG. 7.
FIG. 7.
Mitochondrial gene therapy increases mtDNA copy numbers in LS cells. (A) Increased trend of 18S rRNA normalized mtDNA gene copy numbers of ND2, ND4, and COX3 genes. (B) Increased trend of geometric mean values of 18S rRNA, GAPDH, and β-actin normalized cDNA gene expression of ND2, ND4, and COX3 genes. CTL, beige; TFAM, black; mtDNA+rhTFAM, gray. Color images available online at www.liebertonline.com/hum
FIG. 8.
FIG. 8.
Mitochondrial gene therapy increases mitochondrial biogenesis in LS cells. (A) Representative western blot of ETS complex proteins in LS fibroblast cells. (B) β-Actin-normalized levels of mitochondrial proteins. (C) Increased trends of normalized geometric mean of cDNA gene expressions of TFAM1A, TFAMB1, and PGC1-α genes. CTL, beige; TFAM, black; mtDNA+rhTFAM, gray. Color images available online at www.liebertonline.com/hum
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