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Laboratory Sciences |

Use of Mitochondrial Antioxidant Defenses for Rescue of Cells With a Leber Hereditary Optic Neuropathy–Causing Mutation FREE

Xiaoping Qi, MD; Liang Sun, PhD; William W. Hauswirth, PhD; Alfred S. Lewin, PhD; John Guy, MD
[+] Author Affiliations

Author Affiliations: Departments of Ophthalmology (Drs Qi, Sun, Hauswirth, and Guy), Molecular Genetics and Microbiology (Drs Hauswirth and Lewin), and Neurology (Dr Guy), College of Medicine, University of Florida, Gainesville.


Arch Ophthalmol. 2007;125(2):268-272. doi:10.1001/archopht.125.2.268.
Text Size: A A A
Published online

Objective  To explore a treatment paradigm for Leber hereditary optic neuropathy (LHON), we augmented mitochondrial antioxidant defenses to rescue cells with the G11778A mutation in mitochondrial DNA.

Methods  Cells homoplasmic for the G11778A mutation in mitochondrial DNA were infected with an adeno-associated viral vector containing the human mitochondrial superoxide dismutase (SOD2) gene. Control cells were infected with an adeno-associated viral (AAV) vector expressing the green fluorescent protein (GFP). Two days later, the high-glucose culture medium was exchanged for a glucose-free medium containing galactose. After 1 or 2days, cellular production of superoxide was assessed using the fluorescent probe dihydroethidium, and we used TUNEL (terminal deoxynucleotidyl transferase–mediated biotin–deoxyuridine triphosphate nick-end labeling) staining to detect apoptotic nuclei. The effect of SOD2 on LHON cell survival was quantitated after 2 or 3 days.

Results  Comparisons of AAV-SOD2–infected LHON cells relative to control cells infected with AAV–green fluorescent protein showed increased expression of mitochondrial SOD that attenuated superoxide-induced fluorescence by 26% (P = .003) and suppressed TUNEL-induced fluorescence by 21% (P = .048)after 2 days of growth in galactose medium, when cell survival increased by 25% (P=.05). After 3 days in galactose medium, SOD2 increased LHON survival by 89% (P = .006) relative to controls.

Conclusion  Protection against mitochondrial oxidative stress may be useful for treatment of LHON.

Clinical Relevance  Gene therapy with antioxidant genes may protect patients with LHON against visual loss.

Figures in this Article

A G-to-A transition at nucleotide 11778 in mitochondrial DNA (mtDNA)in the gene specifying the reduced form of nicotinamide adenine dinucleotide:ubiquinone oxidoreductase subunit 4 (ND4) of complex I was the first pathogenic point mutation linked to Leber hereditary optic neuropathy (LHON).1 At present, approximately 45 other mutations in mtDNA have been ascribed to LHON.2 Most LHON mutations affect the ND1, ND4, and ND6 complex I subunits in the oxidative phosphorylation pathway, where electrons first enter the electron transport chain.35

Although reductions in oxidative phosphorylation are invariably present in LHON, cell death appears to be mediated by oxidative stress via apoptotic mechanisms.611 Misdirected electrons from the electron transport chain may react with molecular oxygen,thus increasing generation of reactive oxygen species.1215 Increases in reactive oxygen species activity and diminished mitochondrial antioxidant defenses in LHON8 suggested to us that treatment of LHON may be possible by bolstering antioxidant defenses locally. In this report, we genetically increased mitochondrial defenses against superoxide to rescue LHON cells homoplasmic for the G11778A mutation in mtDNA because this mutation in the ND4 subunit of complex I is responsible for approximately half of all LHON cases, and visual loss in these patients has the worst prognosis for spontaneous recovery.

SUPEROXIDE DISMUTASE AND ADENO-ASSOCIATED VIRUS VECTORS

We constructed an adeno-associated virus (AAV) vector using the AAV vector plasmid pTR-UF12 regulated by the 381–base pair (bp) cytomegalovirus enhancer immediate early gene enhancer and the 1352-bp chicken β-actin promoter-exon1-intron1 driving expression of the human mitochondrial superoxide dismutase (SOD2) complementary DNA (Figure 1A and B). This plasmid was linked to green fluorescent protein (GFP) via a 637-bp poliovirus internal ribosomal entry site. The SOD2-containing plasmid and the parent pTR-UF12 plasmid were amplified and purified by means of cesium chloride gradient centrifugation and then packaged into AAV-2 capsids by transfection into human 293 cells using standard procedures.16 Genome titers of the recombinant AAV were determined using real-time polymerase chain reaction and assayed for infectious particles.17 Each virus preparation contained 1011 to 1012 vector genome particles/mL and 109 to 1010 infectious center U/mL.

Place holder to copy figure label and caption
Figure 1.

Illustrations of the control adeno-associated viral (AAV) vector plasmid (pTR-UF12) (A) and the AAV containing the superoxide dismutase gene (SOD2) (B). Immunoblots of mitochondrial SOD (C) show that, relative to uninfected Leber hereditary optic neuropathy cells (lane 1) or controls infected with AAV–green fluorescent protein (GFP) (lane 2), manganese SOD (MnSOD) (24 kDa) is increased in cybrid cell cultures infected with AAV-SOD2 (lane 3). Expression of β-actin (42 kDa) is relatively comparable in each of the 3 lanes.CBA indicates chicken β-actin; CMV, cytomegalovirus enhancer; IRES, internal ribosomal entry site; and iTR, inverted terminal repeat.

Graphic Jump Location
CELL CULTURE AND INFECTION

Homoplasmic 143B osteosarcoma cells (cybrids) containing 100% mutated (11778A) mtDNA were grown in Dulbecco modified eagle medium (Fisher Scientific,Hampton, NH) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin streptomycin (Sigma-Aldrich Corp, St Louis, Mo) at 37°C with 5% carbon dioxide. The cybrids were created by fusion of enucleated cells from patients with mutated mtDNA, in this case the G11778A mutation, with osteosarcoma (143B.TK)–derived human cells containing wild-type mtDNA cells that were depleted of their mtDNA by chronic exposure to ethidium bromide (ρ0 cells).8,18 The LHON cybrids were seeded in two 6-well or two 96-well dishes. For AAV infections, cybrid cells at approximately 50% confluency were infected at multiplicities of infection of 5000 viral particles per cell, one 6-well dish or one 96-well dish with AAV-SOD2, and one 6-well dish or one 96-well dish with AAV-GFP. Two days after the AAV infections, the high-glucose medium was replaced with glucose-free galactose medium as previously described.18 This selective medium forces the cells to use oxidative phosphorylation to produce adenosine triphosphate. After 2 days of growth in glucose-deficient galactose medium, the SOD2-infected cells from each of 6 wells and the GFP-infected cells from each of 6 wells were trypsinized and counted using an automated particle counter (Z-100; Coulter Diagnostics, Hialeah, Fla). After 3 days of growth in glucose-deficient galactose medium, the SOD2-infected cells from each of 10 wells and the GFP-infected cells from each of 10 wells were trypsinized and counted.

DETECTION OF SOD2 EXPRESSION

Two days after AAV infections, we harvested AAV-SOD2–transfected cybrids, control cells infected with AAV-GFP,19 or LHON cells that were not exposed to either AAV.Briefly, this involved washing the trypsinized cells in cold phosphate-buffered saline solution. Cells were then manually homogenized and stored at −80°C for later analysis. For immunodetection, 15 μg of total protein was separated on a 10% sodium dodecyl sulfate–polyacrylamide gel and electrotransferred to a polyvinylidene fluoride membrane (BioRad Laboratories, Hercules, Calif).The protein content of the samples was measured using a DC protein assay (BioRad Laboratories). We immunostained the membrane with polyclonal anti-SOD2 antibodies (Stressgen Bioreagents, Victoria, British Columbia) and then goat antirabbit IgG horseradish peroxidase–conjugated secondary antibodies (Sigma-Aldrich Corp). We detected complexes using the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ). Antimouse β-actin antibody was used as an internal control for protein loading.

DETECTION OF SUPEROXIDE

We used the fluorescent probe dihydroethidium (DHE) to detect intracellular superoxide (Molecular Probes, Eugene, Ore). Superoxide oxidizes the weakly blue fluorescent DHE to a bright red fluorescent signal. Cybrids were seeded into 48 wells of the 96-well plates. Cells in 24 wells were transfected with SOD2, and cells in the other 24 wells were transfected with GFP. Two days later, the medium was replaced with glucose-free galactose medium. After 24 or 48 hours, cells were incubated with 1μM DHE for 20minutes at 37°C. They were washed and then observed under a fluorescence microscope (Leitz, Wetzlar, Germany). The intensity of fluorescence was quantitated using a fluorophotometer (Eclipse; Varian Medical Systems, Palo Alto, Calif)with excitation at 480 nm and emission at 560 nm (red). Wells were counted in duplicate or greater. Protein content of the samples was measured using the DC protein assay (BioRad Laboratories), and the intensity of fluorescence was adjusted to the sample protein content.

We selected DHE not only because of its specificity for detection of intracellular superoxide20 but also because other commercially available fluorophores such as dichlorodihydrofluorescein have a green emission similar to that of GFP and may interfere with detection of the oxidized green fluorescence of dichlorodihydrofluorescein. In contrast,the peak of red fluorescent DHE oxidized by superoxide and used herein was easily distinguished from the other emission at 520 nm from the green fluorescence of GFP.

DETECTION OF APOPTOSIS

Cybrids were seeded into 48 wells of the 96-well plates. Cells in 24wells were transfected with AAV-SOD2, and cells in the remaining 24 wells were transfected with AAV-GFP. Two days later, the high-glucose medium was exchanged for glucose-free galactose medium. After 1 day (24 wells) and 2 days (24 wells) in this restrictive medium, apoptotic cell death was assessed with a TUNEL (terminal deoxynucleotidyl transferase–mediated biotin–deoxyuridine triphosphate nick-end labeling) reaction kit, according to the manufacturer's specifications (Roche Diagnostics Corp, Indianapolis,Ind). The red TUNEL-positive cells (emission, 560 nm) were visualized and quantitated as described for superoxide.

STATISTICAL ANALYSIS

We compared the AAV-SOD2–transfected cells with controls inoculated with AAV-GFP. Statistical analysis was performed by analysis of variance. P<.05 was considered significant.

INCREASE OF SOD2 AND DECREASE OF SUPEROXIDE WITH AAV-SOD2

Immunoblots of AAV-SOD2–infected LHON cells showed increased manganese SOD expression relative to the control uninfected cybrids and those infected with AAV-GFP (Figure 1C). Fluorescence micrographs confirmed a decrease in superoxide-induced fluorescence following AAV-SOD2 infection. Treatment with AAV-SOD2 decreased superoxide-induced DHE fluorescence in LHON cells after 1 day (Figure 2A)or 2 days (Figure 2C) in the restrictive medium, relative to infection with AAV-GFP (Figure 2B and D). After 1 day of growth in the glucose-free galactose medium,quantitative analysis of the emission at 560 nm that was distinct from the green emission of GFP at 520 nm revealed that superoxide-induced DHE fluorescence decreased 15% relative to AAV infection with AAV-GFP (Figure 2E). This difference was not statistically significant. However,after 2 days of growth in this restrictive medium, superoxide-induced DHE fluorescence decreased 26% relative to the LHON cells infected with the control AAV. This difference was significant (P = .003).Clearly, SOD2 suppressed cellular production of superoxide.

Place holder to copy figure label and caption
Figure 2.

Micrographs show decreased superoxide-induced dihydroethidium (DHE) fluorescence with adeno-associated viral vector containing the superoxide dismutase gene (AAV-SOD2) (A) relative to AAV–green fluorescent protein (GFP) infection (B), after 1 day in the galactose medium. After 2 days in galactose medium, decreased DHE fluorescence is also evident with AAV-SOD2 infection (C) relative to AAV infection (D) (original magnification ×100). The histogram (E)shows that the mean ± SD intensity of superoxide-induced DHE fluorescence is diminished with AAV-SOD2 infection relative to infection with AAV-GFP.

Graphic Jump Location
SUPPRESSION OF APOPTOSIS WITH AAV-SOD2

Because mitochondrial oxidative stress is closely linked to apoptotic cell death, we assayed for TUNEL-positive cells as early as 1 day after growth in the galactose medium. Treatment with AAV-SOD2 decreased TUNEL-positive LHON cells after 1 day (Figure 3A) or 2 days (Figure 3C)in the restrictive medium, relative to infection with AAV-GFP (Figure 3B and D). Quantitative analysis revealed that, relative to the control AAV infection, the intensity of TUNEL fluorescence was diminished by 34% (not significant) after 1 day and 21% (P = .048)with SOD2 infection after 2 days in the galactose medium (Figure 3E). Clearly, SOD2 infection protected LHON cells against apoptotic cell death.

Place holder to copy figure label and caption
Figure 3.

Micrographs of TUNEL (terminal deoxynucleotidyl transferase–mediated biotin–deoxyuridine triphosphate nick-end labeling) fluorescence show decreased TUNEL-positive cells with adeno-associated viral vector containing the superoxide dismutase gene (AAV-SOD2) (A) relative to AAV–green fluorescent protein (GFP) infection (B) after 1 day in the galactose medium. After 2 days in galactose medium,a decrease in TUNEL-positive cells is also evident with AAV-SOD2 infection (C) relative to AAV infection (D) (original magnification ×100). The histogram (E) shows that the mean ± SD intensity of TUNEL-induced fluorescence is diminished with AAV-SOD2 infection relative to infection with AAV-GFP.

Graphic Jump Location
AAV-SOD2 INCREASES LHON CELL SURVIVAL

Reducing apoptotic cell death by protection against mitochondrial oxidative stress with AAV-SOD2 increased the survival of LHON cybrids. After 2 days of growth in the galactose medium, we found that LHON cell survival increased by 25% with AAV-SOD2 infection relative to the control infection with AAV-expressing GFP (P = .05) (Figure 4A-C).Although the population of cells dwindled relative to 2 days of growth in the galactose medium, after 3 days of growth in this restrictive medium, we found that AAV-SOD2 increased LHON cell survival by 89% relative to the controls (P = .006)(Figure 4C). Clearly, increasing mitochondrial antioxidant defenses rescued LHON cells.

Place holder to copy figure label and caption
Figure 4.

Micrographs show an increase in Leber hereditary optic neuropathy (LHON) cell survival with adeno-associated viral vector containing the superoxide dismutase gene (AAV-SOD2) treatment (A) relative to AAV–green fluorescent protein (GFP)infection (B) after 2 days in galactose medium (original magnification ×100).The histogram (C) shows that the mean ± SD LHON cell survival is increased with AAV-SOD2 relative to AAV-GFP infection after 2 and 3 days of growth in the galactose medium (C).

Graphic Jump Location

Our findings show that the superoxide anion is involved in LHON cell death and suggest that increasing mitochondrial antioxidant defenses may be a potential treatment for LHON. Reactive oxygen species that include superoxide anion, hydrogen peroxide, nitric oxide, and peroxynitrite are major initiators of the apoptotic pathway leading to cell death in LHON cells.7,8 Although tissue levels of SOD2 expression and activity in the optic nerves of patients with LHON have yet to be determined, a decrease in mitochondrial SOD activity has been detected in the LHON cybrid cell line.8 Mitochondria mitigate oxygen toxicity predominantly via enzymatic antioxidants that include SOD and glutathione peroxidase. Lowered levels of mitochondrial SOD activity likely increase cellular injury and induce optic neuropathy in mitochondrial disorders, particularly those like LHON that are related to a loss of complex I activity.9,14,21,22

Bolstering anti–reactive oxygen species defenses may suppress the death of retinal ganglion cells in LHON.8 Rescue of our animal model of complex I deficiency with SOD2 suggests that antioxidant gene therapy may be useful for patients with complex I deficiencies such as LHON.23 In that model system, suppression of reactive oxygen species inhibited apoptotic death of retinal ganglion cells,a phenomenon that is also involved in the pathogenesis of disease caused by the mutated human ND4 complex I subunit gene. Apoptotic cell death associated with complex I impairment induced by rotenone can also be blocked by overexpression of SOD2, further supporting our work described in this report.11

Treatment options for patients with LHON and those with other mitochondrial disorders are limited at present.24 The most direct approach to treatment would be to correct the mutated mitochondrial DNA. Although genes have been inserted into the nucleus and cytoplasm through the use of vectors, the technology to introduce a gene into the mitochondria is not yet possible.25 Because it is expression of the mutant complex I subunit at the protein level that causes the biochemical defect of LHON, an alternative and feasible approach is to import a normal protein allotopically into the mitochondria to complement the defective protein encoded by the mutated mtDNA.18,2628 Our previous study showing allotopic rescue of this same LHON cell line with mutated G11778A mtDNA supports this form of intervention.18 However,a different allotopic construct would be needed for the 3 mitochondrial genes containing mutations in ND1, ND4, or ND6 responsible for 85% of LHON cases.

Recent studies showing subtle retinal and optic nerve injury in families harboring the G11778A mtDNA mutation29,30 suggest that treatment may be necessary before symptoms actually develop. Nevertheless,many patients with LHON are found at the initial examination to have optic disc edema and predominantly unilateral visual loss. Thus, there is a window of opportunity of several months for prophylactic intervention in the fellow eye31 with SOD2 gene therapy before it too loses vision. Still, the early retinal changes detected in LHON carriers before apoplectic visual loss29 suggest that this approach may have the best chance for success if it is initiated at the earliest stages of disease. The aim would be to reduce the accumulation of optic nerve damage so that injury does not progress to a point beyond which loss of function becomes irreversible.

Correspondence: John Guy, MD, Box 100284,Department of Ophthalmology, College of Medicine, University of Florida, Gainesville,FL 32610-0284 (johnguy@eye.ufl.edu).

Submitted for Publication: June 9, 2006; final revision received August 14, 2006; accepted August 30, 2006.

Financial Disclosure: Dr Hauswirth and the University of Florida have a financial interest in the use of AAV vectors for treating retinal diseases associated with their involvement with Applied Genetic Technologies Corporation.

Funding/Support: This study was supported by grant EY 12355 from the National Eye Institute (Dr Guy).

Acknowledgment: We thank Valerio Carelli, MD,PhD, for the generous gift of the cybrids and Mabel Wilson for editing the manuscript.

Wallace  DCSingh  GLott  MT  et al.  Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy. Science 1988;2421427- 1430
PubMed Link to Article
Mayorov  VBiousse  VNewman  NJ  et al.  The role of the ND5 gene in LHON: characterization of a new, heteroplasmic LHON mutation. Ann Neurol 2005;58807- 811
PubMed Link to Article
Wallace  DC Mitochondrial diseases in man and mouse. Science 1999;2831482- 1488
PubMed Link to Article
Chinnery  PFJohnson  MAWardell  TM  et al.  The epidemiology of pathogenic mitochondrial DNA mutations. Ann Neurol 2000;48188- 193
PubMed Link to Article
Carelli  VGhelli  ABucchi  L  et al.  Biochemical features of mtDNA 14484 (ND6/M64V) point mutation associated with Leber's hereditary optic neuropathy. Ann Neurol 1999;45320- 328
PubMed Link to Article
Baracca  ASolaini  GSgarbi  G  et al.  Severe impairment of complex I–driven adenosine triphosphate synthesis in Leber hereditary optic neuropathy cybrids. Arch Neurol 2005;62730- 736
PubMed Link to Article
Perier  CTieu  KGuegan  C  et al.  Complex I deficiency primes Bax-dependent neuronal apoptosis through mitochondrial oxidative damage. Proc Natl Acad Sci U S A 2005;10219126- 19131
PubMed Link to Article
Floreani  MNapoli  EMartinuzzi  A  et al.  Antioxidant defences in cybrids harboring mtDNA mutations associated with Leber's hereditary optic neuropathy. FEBS J 2005;2721124- 1135
PubMed Link to Article
Wong  ACavelier  LCollins-Schramm  HE  et al.  Differentiation-specific effects of LHON mutations introduced into neuronal NT2 cells. Hum Mol Genet 2002;11431- 438
PubMed Link to Article
Danielson  SRWong  ACarelli  V  et al.  Cells bearing mutations causing Leber's hereditary optic neuropathy are sensitized to Fas-induced apoptosis. J Biol Chem 2002;2775810- 5815
PubMed Link to Article
Li  NRagheb  KLawler  G  et al.  Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. J Biol Chem 2003;2788516- 8525
PubMed Link to Article
Esposito  LAMelov  SPanov  A  et al.  Mitochondrial disease in mouse results in increased oxidative stress. Proc Natl Acad Sci U S A 1999;964820- 4825
PubMed Link to Article
Brown  MD The enigmatic relationship between mitochondrial dysfunction and Leber's hereditary optic neuropathy. J Neurol Sci 1999;1651- 5
PubMed Link to Article
Barrientos  AMoraes  CT Titrating the effects of mitochondrial complex I impairment in the cell physiology. J Biol Chem 1999;27416188- 16197
PubMed Link to Article
Kussmaul  LHirst  J The mechanism of superoxide production by NADH: ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. Proc Natl Acad Sci U S A 2006;1037607- 7612
PubMed Link to Article
Hauswirth  WWLewin  ASZolotukhin  S  et al.  Production and purification of recombinant adeno-associated virus. Methods Enzymol 2000;316743- 761
PubMed
Warrington  KH  JrGorbatyuk  OSHarrison  JK  et al.  Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus. J Virol 2004;786595- 6609
PubMed Link to Article
Guy  JQi  XPallotti  F  et al.  Rescue of a mitochondrial deficiency causing Leber hereditary optic neuropathy. Ann Neurol 2002;52534- 542
PubMed Link to Article
Fernandez-Vizarra  ELopez-Perez  MJEnriquez  JA Isolation of biogenetically competent mitochondria from mammalian tissues and cultured cells. Methods 2002;26292- 297
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Lieven  CJVrabec  JPLevin  LA The effects of oxidative stress on mitochondrial transmembrane potential in retinal ganglion cells. Antioxid Redox Signal 2003;5641- 646
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Beretta  SMattavelli  LSala  G  et al.  Leber hereditary optic neuropathy mtDNA mutations disrupt glutamate transport in cybrid cell lines. Brain 2004;1272183- 2192
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Ghelli  AZanna  CPorcelli  AM  et al.  Leber's hereditary optic neuropathy (LHON) pathogenic mutations induce mitochondrial-dependent apoptotic death in transmitochondrial cells incubated with galactose medium. J Biol Chem 2003;2784145- 4150
PubMed Link to Article
Qi  XLewin  ASSun  L  et al.  SOD2 gene transfer protects against optic neuropathy induced by deficiency of complex I. Ann Neurol 2004;56182- 191
PubMed Link to Article
Schon  EADiMauro  S Medicinal and genetic approaches to the treatment of mitochondrial disease. Curr Med Chem 2003;102523- 2533
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Zullo  SJ Gene therapy of mitochondrial DNA mutations: a brief, biased history of allotopic expression in mammalian cells. Semin Neurol 2001;21327- 335
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Gray  RELaw  RHDevenish  RJ  et al.  Allotopic expression of mitochondrial ATP synthase genes in nucleus of Saccharomyces cerevisiaeMethods Enzymol 1996;264369- 389
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Barboni  PSavini  GValentino  ML  et al.  Retinal nerve fiber layer evaluation by optical coherence tomography in Leber's hereditary optic neuropathy. Ophthalmology 2005;112120- 126
PubMed Link to Article
Savini  GBarboni  PValentino  ML  et al.  Retinal nerve fiber layer evaluation by optical coherence tomography in unaffected carriers with Leber's hereditary optic neuropathy mutations. Ophthalmology 2005;112127- 131
PubMed Link to Article
Newman  NJBiousse  VDavid  R  et al.  Prophylaxis for second eye involvement in Leber hereditary optic neuropathy:an open-labeled, nonrandomized multicenter trial of topical brimonidine purite. Am J Ophthalmol 2005;140407- 415
PubMed

Figures

Place holder to copy figure label and caption
Figure 1.

Illustrations of the control adeno-associated viral (AAV) vector plasmid (pTR-UF12) (A) and the AAV containing the superoxide dismutase gene (SOD2) (B). Immunoblots of mitochondrial SOD (C) show that, relative to uninfected Leber hereditary optic neuropathy cells (lane 1) or controls infected with AAV–green fluorescent protein (GFP) (lane 2), manganese SOD (MnSOD) (24 kDa) is increased in cybrid cell cultures infected with AAV-SOD2 (lane 3). Expression of β-actin (42 kDa) is relatively comparable in each of the 3 lanes.CBA indicates chicken β-actin; CMV, cytomegalovirus enhancer; IRES, internal ribosomal entry site; and iTR, inverted terminal repeat.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Micrographs show decreased superoxide-induced dihydroethidium (DHE) fluorescence with adeno-associated viral vector containing the superoxide dismutase gene (AAV-SOD2) (A) relative to AAV–green fluorescent protein (GFP) infection (B), after 1 day in the galactose medium. After 2 days in galactose medium, decreased DHE fluorescence is also evident with AAV-SOD2 infection (C) relative to AAV infection (D) (original magnification ×100). The histogram (E)shows that the mean ± SD intensity of superoxide-induced DHE fluorescence is diminished with AAV-SOD2 infection relative to infection with AAV-GFP.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

Micrographs of TUNEL (terminal deoxynucleotidyl transferase–mediated biotin–deoxyuridine triphosphate nick-end labeling) fluorescence show decreased TUNEL-positive cells with adeno-associated viral vector containing the superoxide dismutase gene (AAV-SOD2) (A) relative to AAV–green fluorescent protein (GFP) infection (B) after 1 day in the galactose medium. After 2 days in galactose medium,a decrease in TUNEL-positive cells is also evident with AAV-SOD2 infection (C) relative to AAV infection (D) (original magnification ×100). The histogram (E) shows that the mean ± SD intensity of TUNEL-induced fluorescence is diminished with AAV-SOD2 infection relative to infection with AAV-GFP.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Micrographs show an increase in Leber hereditary optic neuropathy (LHON) cell survival with adeno-associated viral vector containing the superoxide dismutase gene (AAV-SOD2) treatment (A) relative to AAV–green fluorescent protein (GFP)infection (B) after 2 days in galactose medium (original magnification ×100).The histogram (C) shows that the mean ± SD LHON cell survival is increased with AAV-SOD2 relative to AAV-GFP infection after 2 and 3 days of growth in the galactose medium (C).

Graphic Jump Location

Tables

References

Wallace  DCSingh  GLott  MT  et al.  Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy. Science 1988;2421427- 1430
PubMed Link to Article
Mayorov  VBiousse  VNewman  NJ  et al.  The role of the ND5 gene in LHON: characterization of a new, heteroplasmic LHON mutation. Ann Neurol 2005;58807- 811
PubMed Link to Article
Wallace  DC Mitochondrial diseases in man and mouse. Science 1999;2831482- 1488
PubMed Link to Article
Chinnery  PFJohnson  MAWardell  TM  et al.  The epidemiology of pathogenic mitochondrial DNA mutations. Ann Neurol 2000;48188- 193
PubMed Link to Article
Carelli  VGhelli  ABucchi  L  et al.  Biochemical features of mtDNA 14484 (ND6/M64V) point mutation associated with Leber's hereditary optic neuropathy. Ann Neurol 1999;45320- 328
PubMed Link to Article
Baracca  ASolaini  GSgarbi  G  et al.  Severe impairment of complex I–driven adenosine triphosphate synthesis in Leber hereditary optic neuropathy cybrids. Arch Neurol 2005;62730- 736
PubMed Link to Article
Perier  CTieu  KGuegan  C  et al.  Complex I deficiency primes Bax-dependent neuronal apoptosis through mitochondrial oxidative damage. Proc Natl Acad Sci U S A 2005;10219126- 19131
PubMed Link to Article
Floreani  MNapoli  EMartinuzzi  A  et al.  Antioxidant defences in cybrids harboring mtDNA mutations associated with Leber's hereditary optic neuropathy. FEBS J 2005;2721124- 1135
PubMed Link to Article
Wong  ACavelier  LCollins-Schramm  HE  et al.  Differentiation-specific effects of LHON mutations introduced into neuronal NT2 cells. Hum Mol Genet 2002;11431- 438
PubMed Link to Article
Danielson  SRWong  ACarelli  V  et al.  Cells bearing mutations causing Leber's hereditary optic neuropathy are sensitized to Fas-induced apoptosis. J Biol Chem 2002;2775810- 5815
PubMed Link to Article
Li  NRagheb  KLawler  G  et al.  Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. J Biol Chem 2003;2788516- 8525
PubMed Link to Article
Esposito  LAMelov  SPanov  A  et al.  Mitochondrial disease in mouse results in increased oxidative stress. Proc Natl Acad Sci U S A 1999;964820- 4825
PubMed Link to Article
Brown  MD The enigmatic relationship between mitochondrial dysfunction and Leber's hereditary optic neuropathy. J Neurol Sci 1999;1651- 5
PubMed Link to Article
Barrientos  AMoraes  CT Titrating the effects of mitochondrial complex I impairment in the cell physiology. J Biol Chem 1999;27416188- 16197
PubMed Link to Article
Kussmaul  LHirst  J The mechanism of superoxide production by NADH: ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. Proc Natl Acad Sci U S A 2006;1037607- 7612
PubMed Link to Article
Hauswirth  WWLewin  ASZolotukhin  S  et al.  Production and purification of recombinant adeno-associated virus. Methods Enzymol 2000;316743- 761
PubMed
Warrington  KH  JrGorbatyuk  OSHarrison  JK  et al.  Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus. J Virol 2004;786595- 6609
PubMed Link to Article
Guy  JQi  XPallotti  F  et al.  Rescue of a mitochondrial deficiency causing Leber hereditary optic neuropathy. Ann Neurol 2002;52534- 542
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