Role of Reactive Oxygen Species-elicited Apoptosis in the Pathophysiology of Mitochondrial and Neurodegenerative Diseases Associated With Mitochondrial DNA Mutations

REVIEW ARTICLE

Role of Reactive Oxygen Species-elicited Apoptosis in the Pathophysiology of Mitochondrial and Neurodegenerative Diseases Associated With Mitochondrial DNA Mutations Chun-Yi Liu, Cheng-Feng Lee, Yau-Huei Wei* A wide spectrum of pathogenic mutations of mitochondrial DNA (mtDNA) has been demonstrated to cause mitochondrial dysfunction and overproduction of reactive oxygen species (ROS), in relation to mitochondrial and neurodegenerative diseases. Our previous studies have shown that large-scale deletions of mtDNA not only serve as an indicator of oxidative damage, but also result in greater susceptibility of human cells to apoptosis triggered by UV irradiation and other apoptotic stimuli. In this review, we focus on the involvement of mtDNA-mutation-associated oxidative stress and susceptibility to apoptosis in the pathophysiology of mitochondrial and neurodegenerative diseases. Different lines of research have provided concordant data to suggest that the mtDNA-mutation-elicited energy insufficiency and enhanced oxidative stress and damage lead to cell dysfunction, and increase the susceptibility of affected cells to apoptosis in patients with these diseases. Moreover, accumulating experimental evidence has shown that antioxidant therapy is a good strategy for decreasing intracellular ROS and alleviating oxidative-stressinduced apoptosis in cells of patients that harbor pathogenic mtDNA mutations. [J Formos Med Assoc 2009;108(8):599–611] Key Words: apoptosis, mitochondrial diseases, mitochondrial DNA mutation, neurodegenerative diseases, reactive oxygen species

accumulate with age in post-mitotic tissues of elderly subjects.2 It has been established that pathogenic mtDNA mutations can cause mitochondrial dysfunction and enhance oxidative stress, which in turn lead to apoptosis in affected tissues and primary culture of human cells that harbor mtDNA mutations. We contend that reactive oxygen species (ROS)-elicited susceptibility of mutantmtDNA-containing human cells to apoptosis may play a role in the pathogenesis and progression of

More than 100 mutations of mitochondrial DNA (mtDNA) are associated with human diseases, including mitochondrial myopathy, encephalomyopathy, deafness, and mitochondrial diabetes.1 Most of these mtDNA mutations are present at high levels (> 80%) in the affected tissues of patients with one of these mitochondrial diseases. On the other hand, some of these pathogenic mutations of mtDNA, especially the 4977-bp deletion and A3243G transition, have been found to

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Institute of Biochemistry and Molecular Biology, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan. Received: January 31, 2009 Revised: March 18, 2009 Accepted: March 30, 2009

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*Correspondence to: Professor Yau-Huei Wei, Institute of Biochemistry and Molecular Biology, National Yang-Ming University, 155 Li-Nong Street, Section 2, Peitou, Taipei 112, Taiwan. E-mail: [email protected]

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some types of mitochondrial and neurodegenerative diseases.

Mitochondria as a Power House and ROS Generator Mitochondria are responsible for production of ATP, through respiration and oxidative phosphorylation, to supply the energy needs for cellular functions.3 Under normal physiological conditions, 1–2% of molecular oxygen consumed by mammalian cells is metabolized to ROS via electron leakage from the mitochondrial electron transport chain. Therefore, mitochondria are a major source of endogenous ROS in human and animal cells.4,5 It has been demonstrated that the production or accumulation of ROS is increased when the mitochondrial respiratory chain is impaired by chemical compounds (e.g. respiratory inhibitors),6,7 or pathogenic mutations in mitochondrial genes that are involved in the biosynthesis of polypeptides that constitute respiratory enzymes.1,8 The overproduction of ROS from dysfunctional mitochondria may directly cause oxidative damage to cellular macromolecules, such as lipids, proteins and nucleic acids,8 and in turn, lead to membrane structural instability, accumulation of unfolded and misfolded proteins, and DNA mutations. On the other hand, ROS can activate numerous signaling pathways that result in alteration of gene expression profiles and culminate in different outcomes.9

Mitochondrial Role in Apoptosis Besides serving as the major energy producer, mitochondria also play a critical role in the regulation of apoptosis, and act as a major switch to initiate apoptosis in mammalian cells.10 The switch is believed to involve the release of several apoptotic proteins, such as apoptosis-inducing factor (AIF), Smac/DIABLO and cytochrome c, from the mitochondria through the opening of mitochondrial permeability transition pores (mtPTPs). 600

After being released from mitochondria, cytochrome c forms a multimeric complex with Apaf-1 (apoptotic protease activating factor-1), which activates procaspase-9 to activate procaspase-3 and other cytosolic enzymes that lead the cells to apoptosis. Smac/DIABLO is another mitochondrial protein that can inhibit the function of inhibitor of apoptotic proteins, and thereby allows the activation of caspase-9 on the apoptosome.11–13 AIF is translocated to the nucleus and participates in the destruction of chromatin, and DNA fragmentation.14 The dynamic change in antiapoptotic and proapoptotic proteins of the Bcl-2 family on the mitochondrial outer membrane is also involved in the mitochondrial permeability transition and thereby regulates the apoptotic process.15 Mitochondria are also an important mediator in the amplification of the apoptotic signals from the death receptors, through the caspase-8mediated truncation of Bid.14,16,17 Cardiolipin is a polyunsaturated-fatty-acid-rich phospholipid that is present only in mitochondria, and is embedded primarily in the mitochondrial inner membrane. ROS-induced peroxidation of cardiolipin may alter the fluidity and other biophysical properties of mitochondrial membranes, and induce a decrease in the activity of complexes III18 and IV,19 and dissociation of cytochrome c.20 Oxidativestress-elicited DNA damage increases the stability and activity of p53 to alter the expression profiles of several apoptosis-related genes, including proapoptotic proteins such as Bax and Puma, which promote cytochrome c release and subsequent activation of the caspase cascade.21 In addition, 4-hydroxynonenal (4-HNE), a toxic end-product of lipid peroxidation, not only conjugates with other macromolecules to disrupt their biological functions, but also emerges as an important second messenger to trigger stress-induced apoptosis.22 Therefore, defects in mitochondrial energy production and a chronic increase in intracellular ROS and oxidative stress may activate the opening of mtPTPs and initiate the apoptotic cascades.23 Moreover, apoptosis is a highly organized and orchestrated type of cell death, which is involved in embryonic development, immune responses, J Formos Med Assoc | 2009 • Vol 108 • No 8

Oxidative stress and apoptosis in human diseases

and many other physiological functions in humans and animals. Dysregulation of the cell-death process is associated with aging, cancer, autoimmune disease, and several degenerative diseases.1

mtDNA is located near the inner membrane where ROS are produced as a byproduct of mitochondrial respiration, and is one of the major immediate targets of ROS that leads to oxidative damage and subsequent mutations. Furthermore, the lack of protective histones, and inefficient DNA repair systems result in a higher frequency of mutation and oxidative damage to mtDNA compared with that of nuclear DNA. These, as a whole, lead to defects in mitochondrial respiration and oxidative phosphorylation, ROS overproduction, and progressive functional decline of affected cells.8,24

It is noteworthy that the 4977-bp deletion is located in the major arc between two origins of replication (OH and OL), and removes seven genes for polypeptides and five tRNA genes. Several mechanisms have been proposed to clarify the formation of 4977-bp-deleted mtDNA, including replication error and inefficient repair of damaged mtDNA.26–29 The mutations elicited during the repair of damaged mtDNA may explain the observations that mtDNA deletions are induced in cultured human cells by singlet oxygen, UV irradiation, cigarette smoking, and ionizing irradiation.30–32 It has been established that the content of the oxidative DNA damage, indicated by the level of 8-hydroxy 2’-deoxyguanosine (8-OHdG), is correlated with the proportion of the deleted mtDNA,33 which indicates that oxidative stress and the ensuing DNA damage play an important role in large-scale deletions of mtDNA. This also supports the idea that the 4977-bp deletion of mtDNA not only is a pathogenic mutation of mitochondrial diseases, but also serves as an indicator of oxidative damage in human cells.8

Association of mtDNA mutations and mitochondrial diseases

Accumulation of mtDNA deletions in neurodegenerative disease

A wide spectrum of mtDNA mutations, which include point mutations, large-scale deletions, small insertions and tandem duplications, are associated with mitochondrial diseases, and most of them have been demonstrated to cause mitochondrial dysfunction.1,25 Neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), Leigh syndrome, and Leber’s hereditary optic neuropathy (LHON) are associated with point mutations in the protein-coding genes. Point mutations in tRNA genes are linked to mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS), and myoclonic epilepsy with ragged-red fibers (MERRF) syndrome. Largescale deletions, including the most common 4977-bp deletion, are responsible for sporadic mitochondrial myopathy and encephalomyopathy, including chronic progressive external ophthalmoplegia (CPEO), Kearns–Sayre syndrome (KSS) and Pearson’s syndrome.

A number of groups have investigated the role of large-scale deletions of mtDNA in neurodegenerative diseases, and found that the 4977-bp-deleted mtDNA accumulates in large quantities in highenergy-demand tissues of patients with some types of these diseases. Ro et al analyzed the 4977-bpdeleted mtDNA in muscle specimens from 36 patients with sporadic amyotrophic lateral sclerosis (ALS) and 69 age-matched controls with other neuromuscular disorders, and found that the frequency of occurrence and average level of the 4977-bpdeleted mtDNA in the ALS patients were significantly higher than those in the controls.34 Several studies have demonstrated that mitochondrial function, especially the activity of cytochrome c oxidase (COX), is decreased in brain tissues of patients with Alzheimer’s disease (AD).35,36 The content of 4977-bp-deleted mtDNA in brain tissue samples taken from AD patients with an average age of 68 years was about 6.5-fold higher

Mutations of mtDNA in Mitochondrial and Neurodegenerative Diseases mtDNA is the major target of ROS produced by mitochondria

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than that in normal subjects with an average age of 66 years.37 AD patients who died prior to age 75 years had four times more abundant 4977bp-deleted mtDNA than those who died after age 75 years. It has been suggested that mitochondrial respiratory dysfunction that results from the common deletion may play a role in the progression and pathophysiology of AD.38 On the other hand, Parkinson’s disease (PD) has been reported to be associated with deficiency of the respiratory enzyme complex I. Laser microdissection has been used to provide evidence for the clonal expansion of 4977-bp-deleted mtDNA to high levels within substantia nigra neurons in patients with PD.39,40 It has been found that neurons with COX deficiency contain a high proportion of mtDNA with the 4977-bp deletion, which indicates that mitochondrial dysfunction is caused by pathogenic mtDNA mutations. Therefore, tissues with a high-energy demand are more susceptible to mtDNA-mutation-elicited mitochondrial dysfunction in the progression of neurodegenerative diseases.

ROS and Susceptibility to Apoptosis in Patients With Mitochondrial Diseases Oxidative stress in mitochondrial diseases Most of the pathogenic mtDNA mutations have been demonstrated to cause defects in the mitochondrial respiratory chain, which lead to overproduction of ROS in the mitochondria.1,25 Piccolo et al41 found increased levels of lipid peroxides and fluorescent adducts of aldehyde with plasma proteins, and decreased concentration of reduced glutathione (GSH) in the blood of patients with CPEO syndrome. Marked granular distributions of oxidative-damage markers, 8-OHdG and 4-HNE, have been observed in the COXnegative, ragged-red fibers from patients with mitochondrial diseases.42 Therefore, increased oxidative stress elicited by mitochondrial dysfunction in the affected tissues plays an important role in the pathogenesis and progression of mitochondrial diseases. 602

Apoptosis in affected tissues from patients with mitochondrial diseases The role and involvement of apoptosis in the pathogenesis of mitochondrial diseases associated with mutated mtDNA has received increasing attention in recent years. Mirabella et al investigated apoptosis in muscle biopsies of 36 patients who carried different mtDNA mutations, and found that apoptotic features (e.g. TUNELpositive staining and immunohistochemical staining of caspase-3, p75 and Fas) were localized mainly in COX-negative muscle fibers that harbored a high percentage of single mtDNA deletions (> 40%) or point mutations in tRNA genes (> 70%).43 In another study, muscles in patients with MELAS, CPEO and MERRF were found to have only very small numbers of TUNEL-positive myonuclei: 0.13 ± 0.10%, 0.15 ± 0.14% and 0.04 ± 0.09%, respectively.44 However, Bax and Apaf-1 expression and cytochrome c release from mitochondria were noted and caspase-3 activation was detected in 9.0 ± 3.7%, 12.0 ± 4.4% and 12.4 ± 3.8% of ragged-red fibers in patients with MELAS, CPEO and MERRF, respectively, but not in muscles of healthy subjects. Umaki et al42 reported that a pronounced granular appearance of 8OHdG, 4-HNE, manganese superoxide dismutase (Mn-SOD), cytochrome c and DNase I was detected in the COX-negative, ragged-red fibers of patients with mitochondrial diseases, and that TUNEL-positive signals were found not only in myonuclei, but also in subsarcolemmal regions of the muscle. Moreover, it was found that cytochrome c was increased in the cytosol of patient’s muscles and that DNase I was accumulated in the mitochondria compared with normal muscles.42 An in situ approach was employed to address the relationship between apoptosis and respiratory function defects, ragged-red fibers and mtDNA mutation load, and the results indicated that apoptosis was associated with a high proportion of mtDNA mutation in muscle with ragged-red fibers.45 These studies support the hypothesis that apoptosis plays a key role in the pathogenesis of mitochondrial diseases associated with mtDNA mutations. However, further J Formos Med Assoc | 2009 • Vol 108 • No 8

Oxidative stress and apoptosis in human diseases

In addition to the accumulation of ROS-elicited deleted mtDNA described above, many studies have found increased levels of lipid peroxidation markers, including malondialdehyde and 4-HNE, in the brain tissues of AD and PD patients and in the cerebrospinal fluid of ALS patients. Moreover, protein carbonylation and nitration were also elevated in the Lewy body of patients with PD, the hippocampus and neocortex of AD patients, and motor neurons of patients with ALS.46 Recent studies on PD have revealed that a number of PD-associated proteins, including α-synuclein, Parkin, DJ-1, PINK1, LRRK2 and HTR2A are localized in the mitochondria under certain conditions, and that pathogenic mutations in some of these proteins lead to increased sensitivity to oxidative stress and apoptotic inducers.47,48

apoptosis plays a key role in the pathophysiology of AD. The role of apoptosis in PD has been substantiated by the observation of an increase in the number of TUNEL-positive neurons in the brains of PD patients.53 Bax has been shown to accumulate in the melanized substantia nigra pars compacta (SNpc) neurons that contain ubiquitin-positive Lewy bodies in PD patients, although there is no significant difference in Bax activation of postmortem brains between PD patients and healthy subjects. Furthermore, all melanized SNpc neurons in PD patients with activated caspase-3 are also immunoreactive for Bax.54 Another group has observed that the amount of Bax and activity of caspase-3 are increased greatly in melanized neurons of the substantia nigra of PD patients compared with those of healthy subjects.55 Moreover, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment causes apoptosis of nigral dopaminergic neurons through cytochrome c release and caspase activation.56,57 It has been reported that Bax is highly expressed in the substantia nigra of mice after MPTP administration and that its ablation attenuates MPTP-induced apoptosis, which indicate that Bax plays a critical role in MPTP neurotoxicity and pathogenesis of PD.58

Apoptosis in the brains of patients with neurodegenerative diseases

Response of Cybrids to Apoptotic Stimuli

Besides mitochondrial dysfunction and oxidative stress, it has been established that apoptosis is associated with the pathogenesis of neurodegenerative diseases. The brains of AD patients exhibit elevated levels of DNA fragmentation, Bax and caspase-3 activation.49,50 Research has shown that activation of caspases and caspase-mediated cleavage of the amyloid precursor protein and tau protein facilitates the production of β-amyloid (Aβ) and the formation of neurofibrillary tangles.51 Moreover, deficiency of cyclophilin D, an integral part of the mtPTPs, not only protected neurons from Aβ- and oxidative-stress-induced cell death, but also ameliorated learning and memory defects in an AD mouse model.52 These observations suggest that mitochondria-mediated

In order to study the relationship between mtDNA genotype and phenotype, a number of investigators have established, by cytoplasmic fusion, cybrids that harbor different mtDNA mutations that are related to mitochondrial and neurodegenerative diseases.59,60 We have conducted a series of studies on UV- or staurosporine (STS)-induced apoptosis of cybrids that harbor mtDNA with A3243G transition, A8344G transition, or 4977bp or 4366-bp deletions, and found that mtDNA mutation enhances the sensitivity of human cells to apoptosis.61–63 These findings are consistent with the results obtained from treatment of cybrids that harbor 4977-bp-deleted mtDNA with apoptotic stimuli such as H2O2 and TRAIL.64,65

studies are needed to provide more solid evidence.

ROS and Susceptibility to Apoptosis in Patients With Neurodegenerative Diseases Oxidative stress in neurodegenerative diseases

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A caspase-3 activity assay has revealed that cybrids that contain 4977-bp-deleted mtDNA are more sensitive to STS or UV irradiation compared with those with point mutations in the tRNA gene of mtDNA.61 This is consistent with clinical observations that a lower proportion of mtDNA with large-scale deletion is sufficient to cause the onset of mitochondrial diseases, but it requires a much higher level of mtDNA with a point mutation.66 We have also demonstrated that the activation of caspase-8 is an upstream event that amplifies the apoptotic signals by proteolytic cleavage of Bid, which in turn trigger cytochrome c release and lead the mutant cybrids to apoptosis.63 Moreover, the 4977-bp deletion of mtDNA increases the sensitivity of human cells to UV-induced apoptosis in a quantitative manner, through cytochrome c release from mitochondria, and caspase-3 activation.63 Under metabolic stress induced by culturing cells in galactose medium, LHON cybrids that harbor mtDNA mutations that affect different subunits of complex I (G3460A/ND1, G11778A/ ND4, T14484C/ND6) are triggered to undergo cell death characterized by several apoptotic hallmarks, such as chromatin condensation and nuclear DNA laddering.67 The increased release of cytochrome c into the cytosol in LHON cybrids compared with control cybrids suggests that mitochondria are involved in the activation of the apoptotic cascade.67 Similarly, cybrids that contain LHON mutations also display increased susceptibility to apoptosis induced by Fas or rotenone.68,69 In addition, cybrids that harbor the T8993G and T9176G mutations in the ATPase 6 gene associated with maternally inherited Leigh syndrome are highly susceptible to apoptosis induced by tumor necrosis factor-α or STS.70 These studies on cybrids have provided solid evidence to support the notion that point mutation and deletion of mtDNA render human cells more susceptible to apoptosis induced by different apoptotic stimuli (Table). A similar approach of cytoplasmic fusion has also been used to establish cybrids of neurodegenerative diseases, which utilize SH-SY5Y, NT2 and A549 cells as the host.71,72 PD and AD cybrids 604

display reduced activity of complexes I and IV, respectively, decreased ATP content, increased oxidative stress and damage, and enhanced susceptibility to MPTP or Aβ, and are characterized by the accumulation of apoptotic markers.71–74 Taking these cybrid studies together, we suggest that mitochondrial dysfunction and mtDNA-mutationinduced apoptosis are involved in the manifestation of conditions such as muscle wasting and neurological disorders in patients with mitochondrial and neurodegenerative diseases, respectively.

Animal Models for Diseases Caused by mtDNA Mutations Several laboratories have developed animal models to study the roles and mechanisms of nuclear DNA and mtDNA mutations in the pathogenesis of degenerative diseases. D257A mice with a mutant form of mtDNA polymerase γ display striking premature aging phenotypes, including reduced life span, hair loss, graying hair, kyphosis, loss of bone mass, loss of intestinal crypts, progressive decrease in circulating red blood cells, and weight loss.75,76 These mice show accelerated accumulation of mtDNA mutations in most somatic tissues, and the well-correlated expression of apoptotic markers, such as caspase-3 activation and TUNELpositive staining, particularly in tissues characterized by rapid cell turnover.76,77 These studies suggest that mtDNA mutations indeed accelerate the occurrence of apoptotic behavior in mammals. On the other hand, Inoue et al generated “mitomice” that harbor pathogenic 4696-bp-deleted mtDNA, which spans from tRNALeu to ND5, similar to the 4977-bp deletion of human mtDNA, by direct introduction of mitochondria with deletedmtDNA into mouse zygotes.78 Mito-mice manifest several symptoms of mitochondrial diseases, such as low body weight, lactic acidosis, system ischemia, hearing loss, renal failure, and male infertility,79 and the deleted mtDNA accumulates with age in somatic tissues.80 Focusing on male infertility, the group found that deleted-mtDNAelicited mitochondrial respiration defects can J Formos Med Assoc | 2009 • Vol 108 • No 8

Oxidative stress and apoptosis in human diseases

Table. Human osteosarcoma 143B-derived cybrids for study of mtDNA-mutation-induced susceptibility to apoptotic stimuli and antioxidants Type of mtDNA mutation

mtDNA gene (position)

Disease

Apoptotic inducer

Antioxidant

Apoptotic marker

A3243G transition A8344G transition 4366 bp deletion

tRNALeu(UUR)

MELAS

STS, UV

–

Caspase-3 activation

61

tRNALys

MERRF

STS, UV

–

Caspase-3 activation

61

np 9579–13944

CPEO, KSS

UV

CoQ10

62

np 8469–13447

CPEO, KSS

STS

–

UV

CoQ10, NAC

H2O2

Melatonin, CoQ10, vitamin E –

Viability, caspase-3 activation, cytochrome c release, DNA fragmentation Viability, caspase-3 activation, D4-GDI cleavage, DNA fragmentation Viability, activation of caspase-3, -8 or -9, D4-GDI cleavage, Bid cleavage, cytochrome c release, DNA fragmentation Viability, cytochrome c release Viability, caspase-3 activation Viability, annexin V stain, caspase-3 activation, DNA fragmentation Viability, chromatin condensation, DNA fragmentation, cytochrome c release Viability Annexin V stain, chromatin condensation

4977 bp deletion

G3460A transition G11778A transition T14484C transition

ND1

T8993G transversion T9176G transversion

ATPase 6

LHON

H2O2, TRAIL Fas

–

Galactose

Mn-SOD

Rotenone TNF-α, STS

GSH NAC, CoQ10, L-acetyl-carnitine

ND4 ND6

MILS

Reference

61

61–63

64 65 68

67, 88

69 70

mtDNA = mitochondrial DNA; MELAS = mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes; MERRF = myoclonic epilepsy with ragged-red fibers; CPEO = chronic progressive external ophthalmoplegia; KSS = Kearns–Sayre syndrome; LHON = Leber’s hereditary optic neuropathy; MILS = maternally inherited Leigh syndrome; CoQ10 = coenzyme Q10; NAC = N-acetylcysteine; Mn-SOD = manganese superoxide dismutase; GSH = glutathione; STS = staurosporine; TNF = tumor necrosis factor; UV = ultraviolet light.

induce oligospermia, asthenozoospermia, and meiotic arrest at the zygotene stage, as well as enhanced apoptosis.81 Additionally, other mice models with deficiencies in adenine nucleotide translocator 1, Mn-SOD or mitochondrial transcription factor A show several mitochondrial-disease-like J Formos Med Assoc | 2009 • Vol 108 • No 8

phenotypes.82 These studies on animal models have demonstrated that mtDNA mutations and mitochondrial dysfunction can sensitize the animals to apoptotic markers, symptoms of premature aging, and degenerative and mitochondrial diseases. 605

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Antioxidant Therapy for Mitochondrial Diseases and Neurodegenerative Diseases In previous studies on cybrids that harbor 4977bp-deleted mtDNA, we have observed that mitochondrial respiratory function is decreased, and the levels of ROS and oxidative damage to DNA and lipids are increased, in relation to the level of 4977-bp-deleted mtDNA.83,84 These results are consistent with the findings that the intracellular levels of ROS and the 8-OHdG in skin fibroblasts of CPEO patients are higher than those of controls.85 As described above, distinct apoptotic features have been observed in the affected muscle fibers that carry pathogenic mtDNA mutations,42–45 and in mutant cybrids treated with different apoptotic stimuli.61–63 Therefore, we contend that mtDNA-elicited ROS overproduction plays a key role in the susceptibility to apoptosis of human cells that harbor a pathogenic mutation of mtDNA triggered by various stimuli. We and our collaborators have demonstrated that coenzyme Q10 (CoQ10) and melatonin are effective in attenuating apoptosis of human cells that harbor the 4977-bp deletion of mtDNA.62,64 Recently, we have observed that N-acetylcysteine (NAC) treatment effectively decreases H2O2 production in a dose-dependent manner, and attenuates sensitivity to UV-triggered apoptosis of cybrids that harbor 4977-bp-deleted mtDNA (unpublished data). In similar studies, enhanced ROS production and decreased antioxidant defense capacities have been observed in LHON cybrids that harbor mtDNA mutations associated with LHON.86,87 GSH has been found to protect cybrids from cell death induced by treatment with tert-butyl hydroperoxide or rotenone.69 Overexpression of Mn-SOD in LHON cybrids also reduces the accumulation of ROS and attenuates metabolic-stress-induced apoptosis.88 Moreover, it has been shown that CoQ10 attenuates oxidative stress and damage, increases complex I activity, and restores ATP to control levels in PD cybrids.89 NAC treatment attenuates the accumulation of oxidative-stress markers and enhanced apoptosis of AD cybrids.90 606

Therefore, inhibition of ROS overproduction by antioxidants and free-radical-scavenging enzymes may protect mutant cybrids from apoptosis triggered by different stimuli (Table). To date, there is no effective drug to prevent the progression of mitochondrial and neurodegenerative diseases. Consequently, it is a rational approach to develop therapeutic agents that target mitochondria and ROS, to protect cells from apoptosis and slow the progression of these diseases. Based on the observations that CoQ10 and NAC decrease intracellular ROS and alleviate oxidativestress-induced apoptosis in human cells that harbor pathogenic mtDNA mutations, we suggest that antioxidant therapy is full of potential to become a good therapeutic strategy. Indeed, several antioxidants including CoQ10 have been undergoing clinical trials for patients with mitochondrial or neurodegenerative diseases. In addition to scavenging free radicals, CoQ10 may enhance mitochondrial function by stabilizing the mitochondrial inner membrane and raising the efficiency of ATP synthesis. The serum CoQ10 levels of some patients with mitochondrial diseases are significantly lower before CoQ10 treatment than those of normal subjects and increase significantly after administration of CoQ10.91 It has been reported that CoQ10 attenuates the fatigability of daily activities, and increases endurance to exercise and global muscle strength in some patients with mitochondrial diseases,91 and improves the visual acuity in some LHON patients.92 Moreover, other clinical studies have demonstrated that CoQ10 therapy improves the pancreatic β-cell function and attenuates the symptoms of patients with MELAS, such as lactic acidosis and stroke-like episodes.93,94 Currently, CoQ10 is on the participant-recruited stage of a phase III clinical trail for patients with mitochondrial diseases.95 A randomized, double-blind, placebo-controlled study in patients with early PD has revealed that CoQ10 is safe and welltolerated at dosages of up to 1200 mg/day, and that it slows the progressive deterioration of function.96 However, another study has indicated that CoQ10 does not display symptomatic effects in J Formos Med Assoc | 2009 • Vol 108 • No 8

Oxidative stress and apoptosis in human diseases

mid-stage PD.97 Therefore, a phase III clinical trail of 1200 mg and 2400 mg CoQ10 is now underway to verify whether it has symptomatic or neuroprotective effects. Furthermore, as a potential neuroprotective agent, CoQ10 is also undergoing clinical trials in patients with AD, ALS, and Huntington’s disease.98

Conclusion It has been established that mitochondrial dysfunction and apoptosis play an important role in the pathogenesis and progression of mitochondrial and degenerative diseases. In light of these observations, we provide a hypothetical model of mitochondrial role in these diseases (Figure). The mtDNA mutation-elicited deficiency of mitochondrial respiration and oxidative phosphorylation cause a progressive decline in ATP synthesis and increased ROS formation and then results in a vicious cycle. The overproduction

Mitochondrion Inefficient repair of damaged mtDNA Replication error mtDNA mutations Mitochondrial dysfunction

Vicious cycle

ROS overproduction

Accumulation of oxidative damage to lipids, proteins and nucleic acids

ROSresponsive signaling pathways

Alteration in gene expression profile Reduced ATP synthesis

Cell dysfunction

of ROS from dysfunctional mitochondria may directly cause oxidative damage to cellular macromolecules and in turn lead to alteration of their biochemical and physiological functions, and alter gene expression profiles to lead cells into apoptosis. We believe that the mtDNA mutationelicited energy insufficiency and enhanced oxidative damage may cause cell dysfunction, and that the mtDNA mutation-induced susceptibility to apoptosis in high energy-demand tissues is involved in the manifestation and clinical progression of mitochondrial and neurodegenerative diseases, respectively. Several clinical studies have revealed the efficacy and amount of CoQ10 needed for clinical treatment of mitochondrial and neurodegenerative diseases. The abovementioned findings support the notion that antioxidant therapy is a potential therapeutic strategy for the treatment of these diseases through attenuation of intracellular ROS and alleviation of oxidative stressinduced apoptosis in human cells that harbor pathogenic mtDNA mutations.

Figure. The roles of mitochondria in mitochondrial and neurodegenerative diseases. Pathogenic mtDNA mutations produce defective respiratory subunits, which are assembled to form an inefficient mitochondrial respiration and oxidative phosphorylation system. Mitochondrial dysfunction cause reduced ATP synthesis and ROS overproduction. Overproduction of ROS from dysfunctional mitochondria may directly cause oxidative damage to cellular macromolecules, such as lipids, proteins and nucleic acids, and in turn lead to instability of membrane structure, accumulation of unfolded and misfolded proteins, and DNA mutations. Oxidative stress and ensuing DNA damage accelerate the formation of mtDNA mutations through replication error and repair of damaged mtDNA. Moreover, ROS and 4-HNE also activate numerous ROS-responsive signaling pathways that result in alteration of gene-expression profiles, which render cells more prone to apoptosis. During the process of aging and progression of degenerative diseases, mtDNA mutations and oxidative damage accumulate and lead to progressive decline in bioenergetic function and enhanced ROS formation, which culminates in a vicious cycle. The mtDNA-mutation-elicited energy insufficiency and enhanced oxidative damage cause cell dysfunction, and increase the susceptibility to apoptosis in tissues of high energy demand of patients with mitochondrial or neurodegenerative diseases. Most importantly, mtDNA-mutation-induced apoptosis is involved in the manifestation and clinical progression of muscle wasting and neurological disorders in a number of these debilitating diseases. mtDNA = mitochondrial DNA; ROS = reactive oxygen species; 4-HNE = 4-hydroxynonenal.

Apoptosis

Mitochondrial diseases & neurodegenerative diseases

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