production in mitochondria and their consequences

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Figure 1.19 Overview of ROS production in mitochondria and their consequences. ROS cause oxidative damage to mitochondrial proteins, membranes and DNA which in turn impair the ability of mitochondria to perform their metabolic functions which include ATP synthesis, tricarboxylic acid cycle (TCA), fatty acid oxidation, the urea cycle, amino acid metabolism, heme synthesis and assembly of FeS centres. ROS also activate apoptosis processes by releasing proteins located in the intermembrane space such as cytochrome C (cyt c) into the cytosol. The mitochondrial permeability transition pore (MPTP) is induced by ROS thus leading to permeability of the inner mitochondrial membrane to small molecules. This is the case in ischaemia/reperfusion injury. In addition, ROS also modulate the redox signal, affecting the activity of mitochondria, cytosol and nucleus (Brand & Murphy, 1987). Defense mechanisms against ROS

Cells have evolved a series of defense mechanisms to counteract the deleterious effects of ROS. These mechanisms include non-enzymatic, as well as enzymatic antioxidant systems (Sies, 1991) Antioxidant is defined as 'any substance that when present at low concentrations compared to that of an oxidizable substrate significantly delays or inhibits oxidation of the substrate' (Halliwell, 1989). Enzymatic systems include superoxide dismutases (SOD) and hydroperoxidases (glutathione peroxidase, catalase and other hemoprotein peroxidases) (Sies, 1991). Non-enzymatic systems include lipophilic and hydrophilic antioxidants such as tocopherols (vitamin E) which is important to break up the free radical (Burton et al., 1983). Table 1.11 shows antioxidants defense mechanisms in biological system (Sies, 1993).

Table 1.11 Antioxidant defense mechanisms in biological system (Sies, 1993)



Non enzymatic

α-tocopherol (vitamin E)




Ascorbate (vitamin C)

Glutathione (GSH)




Plasma proteins


Enzymic (direct)

Superoxide dismutases

GSH peroxidases



Ancillary enzymes

Conjugation enzymes

NADPH-quinone oxidoreductase

GSSG reductase

NADPH supply

Transport system

Repair system

radical chain breaking

singlet oxygen quencher

singlet oxygen quencher

radical scavenger

diverse antioxidant functions

diverse antioxidant functions

radical scavenger

plasma antioxidants

plant antioxidants

metal binding (e.g. coeruloplasmin)

food additives, drugs

CuZn enzyme, Mn enzyme, Fe enzyme

GPx, PHGPx, ebselen as enzyme mimic

heme protein, peroxisomes

glutathione S-transferase, UDP-glucuronosyl-transferases

two electron reduction

maintaining GSH levels

NADPH for GSSG reductase

GSSG export, thioether (S-conjugate) export

DNA repair systems, oxidized protein turnover, oxidized lipid turnover

The defense mechanisms against ROS through antioxidants and enzymes involve prevention, interception and repair processes (Sies, 1993). Prevention is the first step in the protection against ROS. These mechanisms involve metal chelation agents and enzymes. For example, cytochrome oxidase and ribonucleotide reductase prevent the spreading of the oxidants to the environment (Sies, 1993). Metal chelation agents are important to control lipid peroxidation and DNA fragmentation. These include metal-binding proteins, ferritin, transferrin, coeruloplasmin and metallothionein. Modification of the potential target site eg: stable modification of low-density lipoprotein by dehydroascorbate is another strategy to increase resistance to metal-ion oxidation (Retsky et al., 1993). Protection against radiation is achieved through specialized pigments that include melanins for UV and carotenoids for electronically excited states such as singlet oxygen (Sies, 1993). Gluthatione S-transferases are enzymes that catalyze the reaction of gluthatione (low molecular mass thiol) with reactive electrophiles species to produce thioesters, S-conjugates (Mannervik et al., 1985). The preventive strategy using antioxidants aims to channel the attack of reactive species into less harmful products, therefore reducing further damage by ROS.

The second approach for minimizing the effect of ROS is by interception or deactivation. Similarly, this can be achieved through non-enzymatic and enzymatic reactions (Sies, 1993). In non-enzymatic systems, the first objective is to stop the ROS from further activity (Sies, 1993). The second objective is to transfer the ROS to less sensitive sites in the cells, hence reducing their deleterious effect (Sies, 1993). Examples of these are the transfer of oxidants from hydrophobic phases into the aqueous phases, from membranes to the cytosol, or from lipoproteins present in the blood plasma to the aqueous phase of the plasma. Efficient intercepting of antioxidants can perform both objectives where they can react with free radicals and are also able to interact with water-soluble compounds for their own regeneration. For every 1000 potential target molecules, 1-3 antioxidants molecules are needed in any biological membranes (Sies, 1993). The enzymatic systems in eukaryotic cells include powerful antioxidants such as SOD, catalase and glutathione (GSH) peroxidases, all of which are accompanied by numerous specialized antioxidant enzymes to complete the detoxification (Table 1.12) (Sies, 1993, Chance et al., 1979). Indirect enzymes functions include i) a back up function (eg: to replenish the GSH from glutathione disulfide (GSSG) by the flavoprotein GSSG reductase) or ii) a transport or elimination of the ROS (eg: glutathione S-transferases and the transport system for the glutathione S-conjugates) (Sies, 1993).

Finally, antioxidants and enzymes may also repair the damage since the prevention and interception processes are not completely efficient to remove the ROS. Endogenous ROS and environmental insults such as cigarette smoking, UV light can cause replication errors during DNA synthesis mechanism (Hegde et al., 2008). Oxidative DNA damage which include base damages, single strand or double strand breaks are repaired by DNA repair enzymes that include exonucleases, endonucleases and specific glycosylases that remove these lesions (Frei, 1994). DNA single strand breaks are repaired predominantly by base excision repair (BER) where DNA glycosylases remove the damage base and then AP endonuclease cuts the phosphodiester bond at the damage site (Hegde et al., 2008, Krokan et al., 2000). Subsequently, a new base is synthesized by a DNA polymerase and finally DNA ligase nick-seal the DNA strand (Hegde et al., 2008, Krokan et al., 2000). DNA double strand breaks (DBSs) are repaired by non-homologous end joining (NHEJ) and homologous recombinant (HR) (Mao et al., 2008). HR is a highly accurate process whereas NHEJ is an error-prone process where it joins the two ends of the DNA break together (Mao et al., 2008). In NHEJ, the Ku heterodimer 70/80 recognizes the DNA double strand break and forms a complex with DNA-dependent protein kinase catalytic subunit (DNA PKcs) (Czornak et al., 2008, Feldmann et al., 2000). Subsequently, ligation is performed by a ligation complex that includes DNA ligase IV, a cofactor XRCC4 and XLF (Cernunnos) (Czornak et al., 2008, Feldmann et al., 2000). In HR, nucleases consisting of the MRN complex process the DSBs to form ssDNA (Czornak et al., 2008). These ssDNA are then coated with replication protein A (RPA). Subsequently, RAD51 replaces RPA and form a nucleoprotein filament which consists of RAD52, RAD54, BRCA1 and BRCA2 (Czornak et al., 2008). The nucleoprotein filament then invades into the D-loop and the DNA polymerase synthesizes the complementary strand. Finally, DNA ligases seal the nick between the two strands (Czornak et al., 2008).

1.8.4 Reactive oxygen specieas (ROS) and neurodegenerative diseases

Oxidative stress and mitochondrial dysfunction have been described as the major factors contributing to neuronal loss (Trushina & McMurray, 2007). However whether oxidative stress is the initiator of primary events associated with neurodegeneration or secondary effects is still largely unknown. Examples of neurodegenerative diseases associated with oxidative damage include Alzheimer's, Parkinson's, Mild cognitive impairment (MCI), Hungtinton's and Amyotrophic lateral sclerosis (ALS) (Trushina & McMurray, 2007). I will just briefly illustrate the role of oxidative stress in neurodegeneration by presenting briefly the role of ROS in ALS. Role of ROS in Amyotrophic lateral sclerosis (ALS)

ALS is characterized by degeneration of motor neurons in the cortex, brainstem and spinal cord (Kamat et al., 2008). Approximately about 10% of all ALS cases are familial (FALS) and the rest are sporadic (SALS) (Lin & Beal, 2006, Ferrante et al., 1997). While, the cause of neurodegeneration in SALS is unclear, 20% of all FALS is associated with mutations in CuZnSOD1 (SOD1) and consequently reduced protein activity (Lin & Beal, 2006). SOD1 plays an important role in converting superoxide anion into hydrogen peroxide (H2O2), a less reactive substance (Boillee & Cleveland, 2008). The pathogenesis of ALS has been associated with increased cellular oxidative stress (Smith et al., 1998). The hypothesis that mutations in SOD1 are the cause of ALS is supported by the identification of free radical damage and abnormal free radical metabolism in CSF and post-mortem tissue samples from an ALS patient (Tohgi et al., 1999, Smith et al., 1998, Ferrante et al., 1997). ALS fibroblasts also displayed increased sensitivity to oxidative damage (Aguirre et al., 1998). Ferrente and colleagues provided further evidence for oxidative damage in both FALS and SALS patients (Ferrante et al., 1997). Several markers were used to identify the oxidative damage in these patients. These markers include protein carbonyl groups (protein oxidation), DNA-8-hydroxy-2'-deoxyguanine (OH8dG) (oxidative DNA damage) and MDA (lipid peroxidation). Protein carbonyl and nuclear OH8dG were increased in SALS motor cortex but not in FALS patients. Immunohistochemical studies demonstrated increased neuronal staining for hemeoxygenase-1, malondialaldehyde-modified protein and OH8dG in the spinal cord of both FALS and SALS patients (Ferrante et al., 1997). Furthermore, the oxidative protein marker (3-nitrotyrosine) was increased in CSF of SALS patients (Tohgi et al., 1999). This was supported by several other studies that showed increased 3-nitrotyrosine in the spinal cord and anterior horn cells of the FALS and SALS patients (Beal, 1996). Smith and colleagues reported elevated levels of lipid peroxidation (4-HNE) in CSF of SALS patients compared to patients with other neurological diseases (Smith et al., 1998).

Thus, in summary, ALS is caused by mutations in the SOD1 gene which acts as an antioxidant to reduce the harmful effect of ROS. Mutations in SOD1 lead to decreased activity of SOD1 resulting in increased oxidative damage and motor neuron degeneration in ALS patients (Tohgi et al., 1999).

1.9 General conclusion and project aims

Although cells from the AOA3 patient have been extensively characterized, the cause of the disease still remains unknown. Recently, a novel form of Autosomal Recessive Cerebellar Ataxia (ARCA) known as ARCA2 has been identified. ARCA2 demonstrates similar clinical characteristics to AOA3. Mutations in the ADCK3 gene have been identified as the cause of ARCA2. Given the role of ADCK3 in p53-dependant apoptosis and its role in the ETC, the mitochondrial dysfunction observed in AOA3 indicates that ADCK3 may be a genuine candidate for the defect observed in AOA3. Besides the characterization of the defect in AOA3, characterization of cells derived from ARCA2 patients will also be performed to compare the molecular and cellular characteristics with those of AOA3. Given that antioxidant correct the molecular features of AOA3 to some extent, a similar approach will also be investigated for ARCA2 cells. Thus, the overall aim of this project is to identify the molecular/genetic nature of the defect in AOA3 and to demonstrate the efficacy of antioxidants as potential therapeutics to treat AOA3 and other related neurodegenerative disorders.

The specific aims of this project are:

To characterize AOA3 cells and identify the mitochondrial dysfunction

This chapter focuses on the characterization of AOA3 cells and the identification of the mitochondrial dysfunctions in these cells. To study the mitochondrial function, several assays were performed including mitochondrial morphology and mass, mitochondrial functions (quantification of mitochondrial membrane potential, DNA damage-induced apoptosis, p53 stabilization, cell metabolic activity and mitochondrial superoxide production, mitochondrial superoxide formation and OXPHOS activity). Given that mitochondria are the major producer of ROS, identification of oxidative stress and antioxidant defense mechanisms in AOA3 cells havebeen investigated. Sequencing of mtDNA genome of AOA3 was also performed to identify any potential mutations which may contribute to the disease.

To compare the cellular and molecular characteristics of AOA3 and ARCA2

While the cause of ARCA2 has been attributed to mutations in the ADCK3 gene, nothing is really known about the cellular and molecular characteristics of these cells. Thus, this chapter aimed to characterize ARCA2 cells using the assays that are described in the first aim. These assays were selected because ADCK3 gene is a potential mitochondrial kinase and is believed to play a role in mitochondrial function. Moreover, ARCA2 shares similar clinical characteristics with AOA3 that include cerebellar degeneration, ataxia, and childhood onset. Given the similarities between AOA3 and ARCA2, the presence of oxidative stress and mitochondrial dysfunctions in AOA3 and the role of ADCK3 gene in mitochondrial function, sequencing of the ADCK3 gene in AOA3 was carried out to determine whether ADCK3 may contribute to the AOA3 phenotype. In addition, ADCK3 antibodies have also been produced to determine ADCK3 protein expression levels in AOA3 and ARCA2 cells.

To determine the pathogenicity of ADCK3 mutation (Y429C) in control cells

DNA sequencing of the ADCK3 gene was performed in AOA3 cells and revealed a paternal inheritance of the Y429C mutation in this gene. To investigate the pathogenicicty of this mutation, ADCK3 knock down was performed. Using ADCK3 siRNA knock down was carried in the patient's mother to determine whether this mutation is important for the development of AOA3. The Y429C (AOA3) and Y514C (ARCA2) mutants were made using site directed mutagenesis to determine the effects of these mutations in control cells. The Y429C (AOA3) mutant was also transfected into ARCA2 (1-33654) cells to identify the pathogenicity of this mutation in ARCA2 cells. Pathogenicity of both mutations was assessed by several assays including mitochondrial membrane potential, mitochondrial superoxide production and mitochondrial respiration.

Efficacy of antioxidants in the correction of mitochondrial dysfunction in AOA3 and ARCA2 cells

Oxidative has been associated with many neurological and mitochondrial dysfunctions. The addition of antioxidants which have beneficial effects on the mitochondrial function and oxidative stress could therefore be used as a therapeutic strategy in neurodegenerative diseases. The effectiveness of several antioxidants such as Idebenone, MitoQ and DecylQ to correct the mitochondrial dysfunction observed in AOA3 and ARCA2 cells were investigated.