Nutlin is a chemical that selectively binds to Mdm2 in the p53 binding pocket which in turn releases and stabilizes p53 (Thompson et al., 2004). The effect of nutlin was also investigated to determine whether they could restore DNA damage induced apoptosis in AOA3 cells. Both control and AOA3 cells treated with nutlin showed activated p53 transcription indicated by increased expression of p21, Mdm2 and Puma proteins. However, incubation of AOA3 cells with nutlins failed to induce apoptosis as determined by PARP-1 cleavage. PARP-1 cleavage was readily detected in control cells indicating a normal apoptotic response after DNA damage (Gueven et al., 2007). Non-DNA damaging agent, cycloheximide, a de novo protein synthesis inhibitor also failed to induce apoptosis in AOA3 cells suggesting that these cells are generally resistant to apoptosis (Gueven et al., 2007).
126.96.36.199 DNA repair defect investigation in AOA3
A common feature of ARCA is a defective response to DNA damage and DNA repair as reported in A-T, AOA1 and AOA2 (Palau & Espinos, 2006, Gueven et al., 2004, Suraweera et al., 2007). To determine whether AOA3 possesses a defect in DNA repair, DNA single strand break (SSB) repair was investigated. The alkaline elution assay was performed to determine whether the AOA3 cells were deficient in DNA SSB repair since AOA3 was shown to be sensitive to SSB-inducing DNA damaging agents. AOA3 and control cells were exposed to different doses of H2O2. A similar time dependent reduction in the number of DNA breaks was observed in both control and AOA3 cells indicating normal kinetics of DNA SSB repair (Gueven et al., 2007).
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An additional readout for DNA SSB repair is PARP-1 activity. PARP-1 activity was determined by measuring the poly-ADP-ribose (PAR) synthesis levels in AOA3 cells. High levels of PAR were already observed in untreated AOA3 cells suggesting the constitutive hyperactivation of PARP-1 (Gueven et al., 2007). There was no PARP-1 hyperactivation observed in an untreated control cell line. As expected, H2O2 exposure increased the PARP-1 activity transiently in control cells, as has been reported elsewhere. PAR levels were significantly elevated in AOA3 cells when compared to other cell lines and controls. To confirm that high basal levels of PAR was due to hyperactive PARP, cells were treated with 3-aminobenzamide (3-AB), an inhibitor of PARP-1 (Gueven et al., 2007).
188.8.131.52 Oxidative stress status in AOA3
Oxidative stress status was measured in AOA3 by using various methods that include redox sensitive dye and immunostaining for various ROS-induced damage markers. The 5-(and 6-) chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2-DCFDA) staining was used to probe the levels of oxidative/nitrosative reactive species (ROS/RNS) in AOA3 primary fibroblast and lymphoblastoid cells. High levels of ROS/RNS in AOA3 cells were indicated by right -shifted profile when compared with control cells. Furthermore, immunostaining for oxidative DNA damage (8-oxo-dG), nitrosination of protein (nitrotyrosine), and lipid peroxidation (4HNE-Michael adducts) confirmed higher levels of oxidative stress in AOA3 (Gueven et al., 2007).
184.108.40.206 Assessment of mitochondrial dysfunction in AOA3
Mitochondria are important for apoptosis (Eisenberg-Lerner et al., 2009). Indeed, mitochondrial membrane depolarization plays an important role in the release of apoptotic-inducing factors to ensure the coordination of the apoptotic signaling (Trushina & McMurray, 2007). p53 is also involved in apoptosis by inducing several genes important for the cell cycle regulation and apoptosis (Wade et al., 2010, Farnebo et al., 2010). Transcription-dependent apoptosis via p53 is regulated by proteins such as PUMA or NOXA and BCL-2 family proteins such as BAK, BAX and BH-3 family proteins that include BID and BIM involved in the transcription-independent apoptosis through the mitochondria (Ghosh et al., 2009, Yu et al., 2009). As mentioned previously, AOA3 cells were defective in p53 stabilization (Gueven et al., 2007). Regulation of apoptosis by mitochondria is through the mitochondrial membrane potential, which is maintained in the inner mitochondrial membrane (Crompton, 1999). During apoptosis, the pro-apoptotic Bcl-2 family of proteins (BAK and BAX) are activated and lead to an increased in mitochondrial membrane permeability (Dussmann et al., 2003). Subsequently, cytochrome C and apoptosis inducing factor (AIF) are released from the mitochondria and finally lead to caspases activation and DNA degradation (Dussmann et al., 2003). Mitochondrial membrane permeability potential (MMP) was measured by fluorescence emission of a cyanine dye (JC-1). Loss of mitochondrial membrane potential was determined by loss of the aggregated (red-fluorescent) form of JC-1. This loss was observed in control after a 30 minute treatment with H2O2 In AOA3, no such loss was observed even at 60 minute post-treatment. Cytochorome C was released in control after 4 hours after etoposide treatment and continued to increase up to 8 hours (Gueven et al., 2007). However, AOA3 cells showed low levels of cytochrome C release after a 4 hours and only a slight increased after 8 hours. In order to confirm the defect in mitochondrial membrane depolarization, mitochondrial integrity in response to H2O2 was measured by determinatining of the translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus. In controls, a time-dependant nuclear accumulation was observed, however, in AOA3 cells, only a weak response was observed. Using fibroblasts cells, AIF is translocated into the nucleus within 30 minutes of treatment with H2O2 in control but no translocation was observed in AOA3 up to 2 hours post-treatment (Gueven et al., 2007). Finally, dysfunction of the mitochondria was further confirmed by reduced ATP levels (50% less) in AOA3 compared to controls.
220.127.116.11 Restoration of DNA damage-induced apoptosis by antioxidants
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To determine whether PARP-1 hyperactivation and the defect in apoptosis in AOA3 are linked to high levels of ROS, cells were treated with antioxidants and DNA damage-induced apoptosis was monitored. Cells were pretreated with the antioxidant isoindoline nitroxide 5-carboxy-1,1,3,3-tetramethylisoindolin2-yloxyl (CTMIO) for 1 hour before DNA damage was induced by etoposide. CTMIO, is a nitroxide antioxidant which belongs to the class of catalytic antioxidants as it oxidizes metals and prevent them from entering the Fenton-reaction, thus reducing formation of free radicals (Damiani et al., 2000, Hosokawa et al., 2004). It can repeatedly inactivate ROS and is more potent than scavengers such as vitamins (Damiani et al., 2000, Hosokawa et al., 2004). While etoposide-induced apoptosis was observed in control cells after a treatment with CTMIO as shown by PARP-1 cleavage, both an hour and overnight treatment with CTMIO did not restore the etoposide-induced cleavage in these cells. N-Acetyl Cysteine (NAC) is a metabolite of the sulfur-containing amino acid, Cysteine, which acts as an antioxidant by increasing intracellular glutathione at the cellular level. NAC is produced in the human body and plays a role in the sulfation cycle, acting as a sulfur donor in phase II detoxification and as a methyl donor to convert homocysteine to methionine (Kerksick & Willoughby, 2005). NAC has been used as a dietary supplement in clinical practice for many decades (Reliene et al., 2008). Reliene and collegues showed that NAC suppressed genome rearrangements linked to cancer in a short term study (Reliene et al., 2008). A long-term study demonstrated that NAC reduced the incidence and multiplicity of lymphoma and improved some aspects of motor performance (Reliene et al., 2008). In another study, treatment with NAC revealed significant protective effects against HNE-induced neuronal death in cerebellar granule neurons via preservation of the mitochondrial membrane potential and intracellular GSH levels (Arakawa et al., 2006). Desferal or desferrioxamine mesylate is a metal chelators and has been used for treatment of iron-overload diseases such thalassemia and spontaneous intracerebral hemorrhage (ICH) (Porter, 2009, Selim, 2009). Desferal chelates Fe3+ and hemosiderin to form a stable complex, thus preventing iron from entering the Haber-Weiss reaction which subsequently reduces the formation of hydroxyl radicals (Selim, 2009). AOA3 cells were treated with 200nM Desferal and 500Î¼M NAC for 1 day (short term effect) and 9 days (long term effect) and the etoposide-induced apoptosis as well as the ability to reduced oxidative stress were determined. After 9 days treatment with 500Î¼M NAC and 200nM desferal, etoposide-induced apoptosis was restored in AOA3 cells. A single treatment with NAC or desferal did not restore capacity for apoptosis in AOA3. Antioxidants significantly reduced PAR levels in AOA3 cells after 9 days treatment, supporting that PARP-1 hyperactivation is not upstream of apoptosis inhibition, but is a consequence of high levels of oxidative stress. After 1 day of treatment with both antioxidants, a significant reduction in ROS/RNS was indicated by lower proportion of the cells in the high fluorescent region for both antioxidants. However, 9 days treatment did not reduce the ROS/RNS level any further. Although 1 day treatment with NAC and desferal reduced ROS/RNS levels, 4-HNE and nitrotyrosine immunoreactivity were still observed in AOA3 cells. However, levels of 4HNE and nitrotyrosine were reduced after 9 days treatment with NAC. Desferal reduced 4HNE immunoreactivity within 1 day only at high dose (Gueven et al., 2007). Thus, treating AOA3 cells with antioxidants reduced levels of ROS and restored apoptosis.
18.104.22.168 Model for the defect in AOA3
A summary comparing the cellular and molecular characteristics of AOA3 with several ARCA is shown in Table 1.6. AOA3 showed normal ATM protein expression and kinase activity, defective p53 stabilization and hyperactivation of PARP-1. The hyperactivation of PARP-1 indicates the presence of unrepaired DNA damage in the AOA3 cells. The ATM protein was absent in A-T as mutation in the ATM gene results in truncated protein. In addition, A-T also showed absence of kinase activity, defective in p53 stabilization and normal PARP-1 activity. A-TLD patient's cells showed normal ATM protein with reduced kinase activity and normal p53 stabilization. AOA1 appear to have normal ATM protein and PARP-1 activity. AOA1, AOA2 and AOA3 had normal, whereas A-T cells showed increased basal level of apoptosis. AOA3 cells have defective in DNA damage-induced apoptosis as well as in A-T cells. Oxidative stress which has been implicated in many neurodegenerative diseases was increased in cells from AOA and A-T diseases. All diseases showed hypersensitivity to a variety of DNA damaging agents as indicates in Table 1.6.
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A model taking into account all the molecular and cellular data on AOA3 is described in Figure 1.4. An unknown "X" factor(s) triggers the formation of reactive molecules (ROS) which can react and cause oxidative damage to DNA, proteins and lipid. Subsequently, this results in oxidative stress in the cells. Oxidative stress is defined as the imbalance between the production of ROS and their antioxidants defense mechanisms. Oxidative damage of the macromolecules leads to hyperactivation of PARP-1 in AOA3 cells. Oxidative stress also inhibits the mitochondrial membrane depolarization and causes low mitochondrial membrane potential. The mitochondrial membrane potential is important in order to allow the release of the cytochrome c from the mitochondria into the cytosol and for the translocation of the AIF into the nucleus after DNA damage exposure. Cytochrome c, together with APAF-1 and ATP form the apoptosome and activate the caspase-3,7 and finally lead to DNA degradation in the nucleus. Mitochondrial membrane damage lead to failure of cytochrome c and AIF released from the mitochondria resulting in failure of caspase activation and the absence of apoptosis. In response to DNA damage, p53/p73 are stabilized, however ROS inhibits p53/p73 stabilization in AOA3 which in turn prevents the induction of effector genes involved in DNA damage repair mechanisms. Antioxidants are important substances which reduce the harmful effect of ROS. These include enzymes such as SOD and catalase and antioxidants such as vitamin. The addition of antioxidants such as NAC and iron-chelator agent, desferal into AOA3 cells reduced ROS levels and restored the apoptotic capacity of these cells. In summary, we postulate that oxidative stress in AOA3 cells may give rise to mitochondrial dysfunction and finally lead to disease state.
Ataxia Telangiectasia Like Disorder
Ataxia Oculomotor Apraxia type 1
Ataxia Oculomotor Apraxia type 2
Ataxia oculomotor apraxia type 3
ATM protein kinase absent
Defective p53 stabilization
Normal PARP-1 activity
Normal ATM protein, reduced kinase activity, Normal p53 stabilization
Normal ATM protein
Normal PARP-1 activity
Normal PARP-1 activity
Normal ATM protein and kinase
Defective p53 stabilisation
Elevated basal apoptosis level
Normal basal level
Normal basal level
Normal basal level
Defective DNA damage induced apoptosis
Normal DNA damage induced apoptosis
Defective DNA damage induced apoptosis
Presence of oxidative stress
Hypersensitivity to :
Hydrogen Peroxide (H2O2)
Mitomycin C (MMC)
Methylmethane sulfonate (MMS)
Table 1.6 Comparison of cellular and molecular analyses of different forms of ataxia (Gueven et al., 2007)
XDAMAGE to DNA, proteins and lipids
Mitochonria membrane potential (MMP)
Figure 1.4 Model for the defect in AOA3. Increased levels of ROS result in oxidative damage to DNA, protein and lipid leading to PARP-1 hyperactivation. Oxidative stress causes a decrease in mitochondrial membrane potential (MMP), resulting in failure of cytochrome C release from the mitochondria into the cytosol and AIF translocation from the mitochondria into the nucleus. This in turn inhibits caspase activation resulting in failure to induce apoptosis. DNA damage induces p53/p73 stabilization, while ROS inhibits this process and leads to failure to induce effector genes. Antioxidant treatments reduced the levels of ROS, thus restoring apoptosis in AOA3 cells. Thus, we postulate that increased levels of ROS lead to oxidative stress and may alternatively contribute to the neurodegenerative phenotyoe observed in AOA3 [Taken from (Gueven et al., 2007)]
1.5 Mitochondrial diseases
Mitochondrial diseases are a heterogenous group of diseases caused by
mutations in mitochondrial DNA (mtDNA) or nuclear genes which codes for mitochondrial components (Shoffner & Wallace, 1992). The mitochondrion is an important organelle in cells and is involved in many cellular processes such as energy production through the oxidative phosphorylation (OXPHOS), calcium homeostasis, ROS metabolism, and also acts as a regulator of apoptosis (Wallace et al., 2005). mtDNA mutations affect areas that include brain, heart, skeletal muscle, kidney and the endocrine system and specific symptoms include blindness, deafness, movement disorders, dementias, cardiovascular disease, muscle weakness, renal dysfunction and endocrine disorders such as diabetes (DiMauro & Schon, 2003, Wallace, 1999). However, the most frequent clinical presentations of mitochondrial diseases are neurological and neuro-muscular syndromes (D'Souza et al., 2007). Neurological disorders with mitochondrial involvement are listed in Table 1.7. The effects of defective OXPHOS in neurons are due to two different mechanisms (Schapira, 2006, Di Donato, 2000). First, a decrease in energy production resulting in neuronal depolarization, followed by activation of excitatory amino acid receptors and impaired intracellular Ca2+ homeostasis that lead to protease activation and cell death (Di Donato, 2000). Second, a defect in OXPHOS leads to increased levels of reactive oxygen species (ROS) which then cause damage to the cell membranes, protein and DNA (Schapira, 2006).
The first mitochondrial disease was described in 1988 where a missense mutation was identified in Leber's hereditary optic neuropathy (LHON) (Wallace et al., 1988). LHON is caused by mutations in OXPHOS subunit I, III, IV and V. About 40-60% of LHON are caused by G to A point mutations in the ND4 gene at position 11778 (MTND4*LHON11778) (Wallace et al., 1988). Patients with LHON have acute, painless loss of vision in the central visual field with opthalmoscopic features including circumpapillary telangiectasia microangiopathy and swelling of the nerve fiber layer around the optic nerve (Newman, 1993). Mitochondrial defects commonly show a delayed onset of disease which varies between individuals depending on the metabolic requirement and the mutations involved (Crimi & Rigolio, 2008). Mitochondrial diseases due to mutations in the mtDNA genes are diverse and usually due to an age-related decline in OXPHOS activity, thus many of these diseases are late-onset and progressive (Crimi & Rigolio, 2008). The diversity and variability of the mitochondrial diseases often makes the diagnosis very challenging and difficult (Crimi & Rigolio, 2008). Mitochondrial diseases often lead to mitochondrial dysfunction which is known partly to be the cause of several syndromes that include diabetes mellitus, some forms of cardiovascular disease, Alzheimer and Parkinson diseases (DiMauro et al., 2006). mtDNA mutations can also be acquired for example in ageing and cancer (Taylor & Byrd, 2005). The mitochondrial theory of ageing suggests that accumulation of somatic mtDNA mutations lead to a decrease in mitochondrial function (Harman, 1992). The association of mtDNA mutations with cancer was revealed by a G10398A mtDNA polymorphism resulting in increased risk of breast cancer in African-American women (Canter et al., 2005). Examples of cancer associated with mtDNA mutations included renal adenocarcinoma, colon, head and neck tumors, thyroid, breast and prostate cancer (Crimi & Rigolio, 2008).
There are 5 unique characteristics of mitochondrial genetics based on a detailed analysis of mtDNA diseases pedigree (Shoffner & Wallace, 1990, Wallace et al., 1992). First, pathogenic mtDNA mutations are maternally inherited, presumably because sperm contributes almost no cytoplasm to the zygote (Case & Wallace, 1981). Second, mutations arise in mtDNA genome due to several factors. These factors include lack of histone protection, insufficient DNA repair mechanisms and increased levels of ROS through OXPHOS in the mitochondria (Turnbull et al., 2010). Pathogenic mutations in mtDNA can be divided into 3 categories which are rearrangement mutations, point mutations in RNA genes affecting the mitochondrial protein translation and point mutations resulting in a specific OXPHOS defects (DiMauro et al., 2006). Examples of mtDNA diseases caused by rearrangement mutations in the mtDNA genome are Kearns-Sayre syndrome, Progressive external opthalmoplegia (CPEO) and Pearson syndrome (Crimi & Rigolio, 2008). Mutations in mtDNA genome associated with neurological syndrome are mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and myoclonic epilepsy with ragged-red fibres (MERRF) (Crimi & Rigolio, 2008). mtDNA rearrangement mutations also have been identified in age-related degenerative diseases such as AD and ALS (Wallace et al., 1992). Increased incidence of mtDNA rearrangement in AD brain may be due to elevated level of mtDNA oxidative damage (Mariani et al., 2005). Diseases involved mutations in mtDNA gene which encode for polypeptides in the respiratory complexes are LHON and neuropathy, ataxia and retinitis pigmentosa (NARP) (Crimi & Rigolio, 2008).
However some of the mitochondrial diseases are caused by mutations in nuclear genes which encode for mitochondrial components (DiMauro & Schon, 2003). Mutations in the nuclear DNA encoding for mitochondrial genes may affect structural subunits of the respiratory chain, their assembly, the mtDNA replication and proteins transportation through the mitochondrial double membrane. They may also affect mitochondrial antioxidant defenses (Zeviani & Antozzi, 1997, Orth & Schapira, 2001). Mutations in structural genes lead to Leigh syndrome, which is the most common disease in this group. Mutations in assembly genes such as SURF 1, SCO1 and COX10 which code for assembly proteins of complex IV result in Leigh syndromes and hyperthropic cardiomyopathy (Jing et al., 2002). Several metabolic diseases such as hypertension, hypercholestrolaemia and hypomagnesemia (renal ductal convoluted tubule defect) have been linked to a mutation in the mtDNA tRNAIle (Jing et al., 2002). This mutation is associated with low levels of mitochondrial ATP production and secondary clinical findings of migraine, hearing loss, hypertrophic cardiomyopathy and mitochondrial myopathy (Jing et al., 2002).
Table 1.7 Neurodegenerative disorders with mitochondrial involvement (Schon & DiMauro, 2003)
Neurodegeneration due to : Mutated gene product
a) Primary mutations in mtDNA
Leigh syndrome CCOIII, ND5, tRNATrp, tRNAVal
LHON/Parkinsonism/Dystonia Complex I mtDNA-encoded subunits
Motor Neuron Disease CCO I
NARP/MILS ATPase 6
Parkinsonism 125 rRNA
b) Nuclear gene mutations in mitochondrion -targeted proteins affecting OXPHOS
Leigh syndrome with complex I deficiency Complex I nDNA-encoded subunits
Leigh syndrome with complex II deficiency SDH flavoprotein
Leigh syndrome with complex IV deficiency SURF1, SCO2
Leigh syndrome with PDH deficiency PDH E1-ï¡ subunit
c) Nuclear gene mutations in other mitochondrion-targeted proteins
Amyotrophic lateral sclerosis (ALS) Cu, Zn-SOD
Friedreich ataxia Frataxin
Hereditary spastic paraplegia Paraplegin, HSP60
Mohr-Tranebjaerg syndrome Deafness/dystoniaprotein1 (TIMM8A)
Wilson disease Cu-transporting ATPase (ATP7B)
d) Nuclear gene mutations in non-mitochondrion targeted proteins
Alzhemier's Disease (AD) ABPP, presenilin1, presenilin2
Huntington's Disease (AD) Huntingtin
Parkinson's Disease (PD) Parkin, ï¡-synuclein
Progressive Supranuclear Palsy (PSP) Tau protein
e) Putative secondary mitochondrial involvement
Sporadic AD Unknown
Sporadic ALS Unknown
Sporadic PD Unknown