Mitochondrial Dysfunction during Brain Aging: Role of Oxidative Stress and Modulation by Antioxidant Supplementation

Mitochondria and ROS Production

Although ROS can be generated from different enzymatic reactions such as xanthine oxidase, monoamine oxidase, peroxisomal fatty acyl CoA dehydrogenase, NADPH oxidase, cytochrome P450 dependent enzymes etc. and non-enzymatic reactions e.g. autoxidation of hemoglobin or catecholamines, mitochondria are the major intracellular source of ROS [14,22]. The evidence that isolated mitochondria can produce ROS came as early as 1966 and following that many studies have verified the mitochondrial production of superoxide radicals and H₂O₂ in isolated preparations and in intact cells [23–25]. The general view is that the thermodynamically favourable leakage of electrons from reduced or partially reduced redox-proteins or other redox molecules of the respiratory chain to molecular oxygen results in the formation of superoxide radicals (O₂^.−) which undergo dismutation reaction spontaneously or catalyzed by mitochondrial manganese superoxide dismutase (Mn SOD) to produce H₂O₂ which can be decomposed further by transition metals (Fenton’s reaction) to give rise to highly reactive hydroxyl radicals [22,26]. The O₂^.− radicals can also react with mitochondria derived NO to generate toxic peroxynitrite radicals. The measurement of O₂^.− radicals or H₂O₂ in isolated mitochondria or submitochondrial particles has been done under different conditions and the results have often been fallaciously extrapolated by others to in vivo conditions [22,26]. Superoxide radical generation in isolated mitochondria essentially depends on available local oxygen concentration, the concentrations of reduced redox proteins and the second order rate constants of the reactions between oxygen and the redox proteins [22]. The true quantitative assessment of O₂^.− radical production by isolated mitochondria in vitro, however, is difficult because of its rapid conversion to H₂O₂ by Mn SOD, but the measurement of H₂O₂ production or rather its release in to medium under similar conditions is probably more accurate. Under in vivo conditions a true quantitative estimate of superoxide radical production by mitochondria is virtually impossible because of uncertainties in the measurement of different parameters that affect ROS production. However, mitochondrial transmembrane electrochemical gradient, NADH/NAD⁺ ratio, QH₂/Q ratio and local concentration of oxygen are important determinants of in vivo ROS production in mitochondria [22]. It is also important to realize that steady state level of ROS will depend upon the balance between the rate of ROS production and ROS removal and mitochondria in fact can scavenge H₂O₂ at a significant rate [26]. The elaborate antioxidant defence system present in mitochondria comprising of antioxidant enzymes like Mn SOD, glutathione peroxidase, glutathione reductase, phospholipid hydroperoxide glutathione peroxidase and peroxiredoxins as also non-enzyme molecules like reduced glutathione, thioredoxins etc. can effectively scavenge the generated ROS [22,26,27]. The inter-membrane space of mitochondria also contains copper- and zinc-containing superoxide dismutase (Cu/ZnSOD) which can scavenge superoxide radicals released in to this space.

The formation of superoxide radicals in respiratory chain of mitochondria has been the subject of intense research and complex I of inner membrane is considered to be an important source, but other respiratory complexes such as complex III and complex IV along with enzymes like aconitase, α-ketoglutarate dehydrogenase, pyruvate dehydrogenase complex, glycerol-3-phosphate dehydrogenase, dihydroorotate dehydrogenase etc. also contribute to the process [22,26].

Mitochondrial Biogenesis and Reactive Oxygen Species

Mitochondrial biogenesis is a complex process which requires multiplication of mtDNA, increase in mitochondrial mass and proliferation of mitochondria [28,29]. Only 13 subunits of respiratory chain in mitochondria are coded by mtDNA, while a large number of nuclear genes codes for different proteins of the mitochondria including those required for mtDNA replication and transcription as also mitochondrial protein synthesis [28,29]. Several transcription factors like NRF 1 (nuclear respiratory factor 1), NRF 2 (nuclear respiratory factor 2), PPARα (peroxisome proliferator-activated receptor α), ERRα (estrogen receptor-related receptor α), Sp1 (specificity protein 1) and Tfam (Transcription factor A, mitochondria) take part in the process to bring about the nucleo-mitochondrial coordination necessary for biogenesis [13,28,29]. The target genes for these transcription factors especially of NRF 1, NRF 2, Sp 1 and Tfam have been identified and together they are responsible for mtDNA replication, transcription, synthesis and assembly of respiratory chain complexes and protein import within mitochondria. The integration of the functions of such diverse transcription factors is brought about by PGC 1 (PPAR-γ coactivator 1) family of co-activators (PGC 1α, PGC 1β, PGC-1-related coactivator or PRC etc.) of which PGC 1α, is considered as the master regulator of mitochondrial biogenesis [13,28,29]. Although NRF 1 and NRF 2 are considered as the main target transcription factors for PGC 1α, many other targets of the latter have now been identified including some nuclear and cytoplasmic receptors [28,29]. Other interesting facets of PGC 1α are its inducible nature and regulation by cyclic AMP (cAMP) and cyclic AMP response element-binding protein (CREB), AMP activated protein kinase, calcium/calmodulin-dependent protein kinase type IV (CAMK IV), endothelial nitric oxide synthase (eNOS) etc. and through some of these mechanisms exercise, cold exposure and fasting can cause activation of PGC 1α and regulate mitochondrial function and biogenesis according to physiological conditions [29–31].

The relationship of ROS and mitochondrial biogenesis is another area which is being investigated vigorously with several studies indicating how ROS can induce Tfam or NRF 1 to initiate mitochondrial biogenesis. The induction of Tfam by redox activation of NRF 1 through Akt dependent phosphorylation has been shown in rat hepatoma cells [32]. Likewise mitochondrial binding of CO leads to increased formation of H₂O₂ which has been linked to Akt activation and activation of NRF 1, NRF 2 and Tfam [33]. Further, homocysteine induced ROS production has been shown to activate mitochondrial biogenesis through involvement of NRF 1 and Tfam in human endothelial cells [34]. In HeLa cells depleted of mtDNA the increased endogenous ROS has been shown to activate mitochondrial biogenesis through the involvement of NRF 1 and Tfam [35]. More interestingly, another study has elucidated the molecular pathway of ROS-dependent activation and nuclear translocation of Nrf 2 (Nuclear factor erythroid-2 related factor 2) which in turn causes promoter activation of NRF 1 to start mitochondrial biogenesis programme [36]. These scattered but substantial evidence, therefore, strongly indicates that oxidative stress may enhance mitochondrial biogenesis, but the molecular scenario is not completely clear as several studies have also reported down-regulation of mitochondrial biogenesis by increased ROS [37]. It appears that when mitochondrial functional impairment is mild to moderate, ROS may try to compensate it by stimulating mitochondrial biogenesis, but this itself may lead to an overload of ROS which then can damage the cellular components including the mitochondria. The inability of our available technology to obtain a true quantitative measurement of ROS production in vivo under physiological and pathological conditions is a big hindrance in establishing how ROS can activate or deactivate mitochondrial biogenesis programme in different situations.

Oxidative Stress and Mitochondrial Functions: An Entangled Relationship (Fig. 1)

Since ROS mediated damage is too well-known and given the fact that mitochondria form the major source of intracellular ROS, it is expected that a simple cause and effect relationship would explain mitochondrial functional alterations during ROS overload under pathological conditions. The scenario, however, is much more complex, because of the involvement of ROS in different cell signalling pathways. Redox signalling leading to altered expressions of genes has caused a paradigm shift in our understanding of the role of ROS in cell physiology and pathophysiology and this aspect needs to be emphasized while analysing the effects of oxidative stress on mitochondrial functions [38]. Oxidative stress is generally considered as a somewhat uncontrolled process mediated by reactive free radicals including ROS attacking indiscriminately various biomolecules and damaging cellular organelles, but some of the members of ROS and RNS (reactive nitrogen species) may also, under basal conditions or during a low level of oxidative stress, perform as signalling molecules to induce expression of genes coding for antioxidant enzymes and proteins, phase II detoxification enzymes, amino-acid transporters and stress-response proteins [38,39]. These genes generally contain ARE (antioxidant responsive element) sequences in the promoter regions and are activated by several redox-sensitive transcription factors like AP 1 (activator protein 1), Nrf 1, Nrf 2 etc. [38–40]. These transcription factors remain in an inactive form in cytosol usually in combination with inhibitory proteins such as Nrf 2 remaining inactive in association with KEAP 1 (Kelch-like ECH-associating protein 1) which also promotes the degradation of Nrf 2 via ubiquitin-proteasome system [38–40]. Mitochondrial or extramitochondrial ROS, lipid oxidation products, electrophilic molecules or redox-state of the cell may lead to the dissociation of the transcription factor from the clutches of the inhibitory protein and translocation to the nucleus followed by the enhanced expressions of ARE (antioxidant response element) dependent genes [38–41]. For example, the oxidation of several critical cysteine residues in KEAP 1 by oxidants causes release of Nrf 2 from KEAP 1 and the former translocates and binds to ARE containing DNA sequences (Fig. 2) for which another specific cysteine (cys 506) residue of Nrf 2 in reduced form is needed [42]. It is plausible that the redox state of the cell may lead to oxidation of cys 506 with consequent diminished binding of Nrf 2 to DNA and down regulation of the genes [38,42]. The important proteins coded by ROS responsive genes include catalase, superoxide dismutase (SOD), glutathione peroxidase, glutathione reductase, glutathione-S-transferase, heme oxygenase, γ-glutamyl cysteinyl synthetase, thioredoxin etc., but it is not clear whether such redox-dependent gene expression occurs for mitochondrial proteins in general during oxidative stress [38,39,42]. Scattered reports have, however, indicated that oxidative stress can lead to elevated levels of mitochondrial transcription factors or other proteins of oxidative-phosphorylation machinery [43,44]. Further, mitochondrial biogenesis, which requires expression changes in many nuclear and mitochondrial genes, is modulated by ROS as discussed above, but it is still not established whether ROS signalling mediated by AP 1, Nrf 1, Nrf 2 etc. are also involved in the biogenesis process.

As far as the direct damaging effects of ROS are concerned, the mitochondrial lipid, protein and DNA are like sitting ducks for attack by free radicals because of close proximity with the site of ROS generation and mtDNA is additionally vulnerable to damage because of limited ability of mitochondrial DNA repair system [14,45]. In aging as well as other pathological conditions with associated oxidative stress in brain and other tissues, various oxidative damage markers of lipid, protein and DNA accumulate within mitochondria [14,19,46]. These include common markers of lipid oxidation like malondialdehyde (MDA), 4-hydroxy nonenal (4-HNE), fluorescent lipid peroxidation products (FLPP) etc., markers of protein damage like protein carbonyls, nitrotyrosine, HNE-protein adducts and DNA damage markers like 8-oxo-deoxyguanosine. Cardiolipin, which is exclusively present in mitochondrial inner membrane, is highly vulnerable to oxidative damage and respiratory chain inhibitors can increase mitochondrial ROS production and concomitant cardiolipin oxidation [47]. The direct damaging effects of ROS on mitochondrial protein and lipid could inhibit mitochondrial bioenergetic functions, while damage to mtDNA may lead to promoter inactivation and downregulation of mitochondrial gene expression and thus may aggravate the functional impairment of the organelle [47–49]. Although it is easier to assume that enhanced ROS generation within mitochondria are primarily responsible for mitochondrial dysfunction associated with aging or other pathological states, it is entirely possible that reactive species with longer half-lives such as H₂O₂, lipid alkoxyl radicals, lipid hydroperoxides or active aldehydes like MDA, 4-HNE, acrolein etc. generated elsewhere can also reach mitochondria to inflict damage especially in the presence of heme and non-heme iron present within the mitochondria.

Mitochondrial Dysfunction and Oxidative Stress in Aged Brain (table 1)

The brain is highly vulnerable to oxidative damage because of a relative lack of anti-oxidant enzymes, an abundance of oxidizable substrates like polyunsaturated fatty acids, catecholamines etc., a high content of catalytic transition metals in certain brain regions and a high rate of oxygen utilization per gram weight of tissue [14,50]. There are numerous studies which have demonstrated that aging of brain is associated with accumulation of oxidative damage markers of protein (protein carbonyls, protein 3-nitrotyrosine etc.), lipid (MDA, 4-HNE, fluorescent lipid peroxidation products etc.) and DNA (e.g. 8-hydroxy-deoxyguanosine or 8-OH-dG) [14,16,51–56]. However, it is not convincingly established whether the increased oxidative stress of aging brain results from the accumulation of pro-oxidant factors (e.g. transition metals) or increased production of ROS from different sources or a fall in antioxidant enzyme activities or a combination of all these factors [57–61]. In the context of mitochondrial protein and lipid oxidation products, increased levels of protein carbonyls and protein 3-nitrotyrosine, thiobarbituric acid reactive substances or TBARS and fluorescent lipid peroxidation products have been observed in aged rat brain along with diminished content of cardiolipin and protein thiols [61–64]. Oxidative damage to mtDNA has been reported in aging brain with the formation of 8-hydroxy-deoxyguanosine (8-OH-dG) which is the most common marker of oxidative DNA damage [65]. In human (42–97 years) brain tissue a progressive increase in 8-OH-dG has been reported both in nuclear DNA and mtDNA with aging but the extent of increase of 8-OH-dG is ten fold more in mtDNA compared to nuclear DNA [65]. Similar observations with increased 8-OH-dG levels in mtDNA compared to that in nuclear DNA in aged brains of six different mammalian species have been reported [66]. Oxidative damage to mtDNA can lead to various forms of point mutations and in the frontal cortex as well as the substantia nigra of elderly individuals a high burden of such lesions have been identified in mtDNA using sensitive cloning and sequencing methods [67]. In cortex and putamen, but not in cerebellum, a common deletion has been found to accumulate in mtDNA (Δ4977 nucleotide pair) in aged persons [68]. The cause of this deletion mutation in aged brain mtDNA has been linked to oxidative damage based on the fact that the highest accumulation of this deletion mutation is observed in those regions of brain involved in oxidative dopamine catabolism [69]. Age-dependent decrease in several DNA glycosylases involved in mtDNA repair has been reported in several brain regions in normal as well as senescence accelerated mice and this may contribute to the accumulation of somatic mutations in mtDNA in aged brain [70,71]. It is generally implied that direct oxidative damage to mitochondrial lipid and protein components brings about impairments of mitochondrial bioenergetic functions in aged brain, but alterations of mtDNA as well as nuclear DNA through oxidative damage can also contribute to the latter phenomenon by down regulating the expressions of proteins involved in oxidative phosphorylation and mitochondrial transport systems.

The most important functional deficits in aged brain mitochondria from different species like rodents, non-human primates and human beings include a loss of mitochondrial membrane potential and phosphorylation capacity, decreased respiration and ATP synthesis and activation of mitochondrial permeability transition pore [61,62,64,72–76]. Although most studies have reported a moderate decrease in the activities of respiratory complexes in aged brain mitochondria, the pattern and the extent of such decrease are quite variable [74–78]. Further, some studies have even indicated an increase in complex IV activity in aged rodent brain [79,80]. In a recent study, however, no significant change has been noticed in brain mitochondrial respiration, ATP synthesis or respiratory chain activities in aged Fischer 344 rats [63]. The mRNA and protein expression levels of different subunits of respiratory chain or F₀F₁-ATP synthase or other mitochondrial proteins in aged brain have been examined in a limited number of studies, but variabilities are quite apparent among the published results. Reduced protein expression levels of several subunits of complex I, complex IV, F₀F₁-ATP synthase and ANT-1 (adenine nucleotide translocase isoform 1) have been reported in aged rat brain, while another study has failed to demonstrate any significant alteration in the protein expression levels of subunits of cytochrome oxidase (subunit I coded by mtDNA and subunit IV coded by nuclear DNA) in aged rat cerebellar cortex [81,82]. In a large study, the mRNA expression levels of complex I, complex III, complex IV and complex V have been measured in mice brain at different ages and a significant up regulation of expression of mtDNA genes coding for subunits of respiratory complexes has been observed at 12 and 18 months compared to that present in 2 months old mice, but at 24 months the same genes are strongly down regulated [83]. On the other hand, a significant decrease in the mRNA expression of complex I (39 kDa subunit) but a clear up regulation of a gene for complex IV (subunit IV) have been reported in 28 months old rat hippocampus but not in other brain regions when compared to those in adult 6 months old animals [84].

Several studies have demonstrated that exposure of isolated mitochondria to in vitro oxidative stress also leads to impairment of mitochondrial functions which are in general agreement with those observed in aged brain mitochondria and thus such studies have strengthened the general assumption that oxidative stress is primarily responsible for age related decline of mitochondrial functions [47,48]. Using a redox-proteomic approach, it has been shown recently that there appears an abundance of oxidatively modified mitochondrial proteins in aged brain especially in cortex and hippocampus which are related to energy metabolism such as pyruvate kinase, ATP synthase, α-enolase, aldolase, creatine kinase etc. [85]. Finally, aging related alterations of supramolecular assembly of respiratory complexes have been reported in rat brain cortex [86]. It is probably worthwhile to remember, despite variable results reported in the literature, that the activities of oxidative-phosphorylation complexes in aged brain finally depend upon a combination of processes e.g. oxidative inactivation of the protein subunits, changes in lipid microenvironment of inner membrane by oxidative loss of cardiolipin or other phospholipids, compensatory up regulation of nuclear or mitochondrial genes coding for respiratory complexes or ATP synthase and later stage inactivation of the same genes by oxidative damage.

Despite the fact that mitochondrial biogenesis could be an important contributor to the overall health of mitochondria during brain aging, not many studies have been conducted to examine the status of PGC 1α, NRF 1, NRF 2 or Tfam in the latter condition. Likewise isolated reports have indicated enhanced ROS production from aged brain mitochondria, but not many systematic studies have been made to analyse how ARE-dependent genes are activated in aged brain by increased mitochondrial production of ROS [61].

Mitochondrial Dysfunction, Apoptosis, Cognitive Decline and the Aging Brain

Since increased oxidative stress and several forms of mitochondrial dysfunctions appear to co-exist in aged brain, the activation of apoptotic pathway in the latter is a likely phenomenon. However, the existence of apoptosis in normal brain aging remains controversial, and most recent studies have demonstrated that neuronal loss in normal brain aging is minimal and localized in certain brain areas only, which clearly rules out apoptosis as a key element of brain aging [87,88]. Cognitive decline during brain aging, on the other hand, is well established from a large number of experimental and clinical studies and in certain cases of human beings the cognitive deficit progresses to an irreversible and a devastating failure of memory and higher functions of brain as seen in Alzheimer’s disease. Although some cognitive domains remain unaffected during brain aging, others like declarative memory, working memory, attention and spatial learning show a considerable decline [89]. Motor learning is considerably slower in aged population, but the motor memory is largely spared [90]. In aged animals, a significant decline of Morris Water Task and Transverse Patterning Discrimination Task has been observed indicating an impairment of hippocampal function [91]. Both in animals and human beings, the age-related decline of cognition involves hippocampus, medial temporal lobe and pre-frontal cortex [91,92]. In hippocampal neurons from aged animals, decreased membrane excitability, diminished inhibitory post-synaptic potential, decreased Long Term Potentiation (LTP) and reduced sensitivity to carbachol have been reported [93]. The molecular details of age-related cognitive failure is not known, but altered gene expression profile, oxidative damage, mitochondrial dysfunction, metabolic deficits, changes in neuro-transmitter turnover or receptor population may all contribute to the process. In a large number of studies, however, mitochondrial dysfunction in brain and various learning and memory deficits have been examined together in experimental animals and very often both have been prevented significantly by treatment with synthetic or plant-derived antioxidants [94–96]. Such results, however, do not necessarily prove that brain mitochondrial dysfunction during aging is causally related to cognitive decline, as various other age-related alterations are possibly also modified by the same drug treatment. However, both point mutations and large-scale deletions in mtDNA are present in aged brain and also in different tissues in classical mitochondrial disorders like mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), myoclonic epilepsy and ragged red fibers (MERRF) syndrome, Kearns-Sayre Syndrome (KSS), chronic progressive external ophthalmoplegia (CPEO) etc. where dementia is often an accompanying feature implying that mitochondrial respiratory impairment may be causally linked with cognitive decline. An interesting paper has described the development of heteroplasmic trans-mitochondrial mice carrying a deletion mutation in mtDNA (Δ4696 nucleotide pair) extending from the gene for tRNA^Lys to ND5 gene and presenting with characteristic deficit of spatial remote memory [97]. Such mito-mice could be used as good models to study the relation between mitochondrial respiratory defects and cognitive decline.

Antioxidant Supplementation and Mitochondrial Dysfunction in Aged Brain (table 2)

Since oxidative stress has an overwhelming influence on biochemical and physiological alterations associated with brain aging, numerous attempts have been made to halt the age-related deterioration of brain functions in normal animals as well as in senescence accelerated mice by antioxidant supplementation either as a single antioxidant agent or a combination of agents using synthetic compounds or plant derived compounds or even crude preparations from plants or fruits [98–102]. The antioxidants are administered for a prolonged period to aging animals along with the diet or sometimes by systemic methods and the animals are then examined for various learning and memory tasks and subsequently sacrificed for the biochemical analysis of oxidative damage parameters or other alterations in the brain. This review will, however, restrict itself to those studies where antioxidant supplementations have been provided to aged animals for preventing the age-related mitochondrial dysfunctions in the brain. It has been shown that a very high dose of α-tocopherol improves survival, neurological deficits and brain mitochondrial respiratory chain activities in aging mice [96]. Several compounds like α-lipoic acid, coenzyme Q and acetyl-L-carnitine have antioxidant properties and can also act as substrates for various mitochondria associated reactions. Both α- lipoic acid and acetyl-L-carnitine when fed to rats have been proved to be highly effective in preventing age-related oxidative dysfunctions of mitochondria and decline of cognition and ambulatory activity, and coenzyme Q may also be useful in this regard as the brain mitochondrial level of Q₁₀ can be increased after oral administration [94,95,103,104]. Using a combination of N-acetylcysteine, α-tocopherol and α-lipoic acid as dietary supplements for over 4 months, a significant protection has been observed on brain mitochondrial dysfunction associated with aging [72]. This combination of drugs also raises the brain levels of SOD and catalase which may potentiate the antioxidant effects of these drugs [72]. Most of the antioxidants used in different studies to prevent age-related oxidative damage and mitochondrial dysfunctions work either as scavengers of ROS or lipid and protein derived radicals or reduce the disulphide linkages in oxidized proteins [14,105,106]. Some like N-acetylcysteine can increase the intracellular level of reduced glutathione which is an important compound to maintain the redox-state of the brain [107]. It must be appreciated that antioxidants in vivo can keep the critical cysteine residues in reduced condition to facilitate the binding of AP 1 or Nrf 2 to ARE and to activate the expressions of genes coding for antioxidant enzymes or phase II detoxification enzymes which may potentiate the protective actions of the former [38,42]. On the other hand, as discussed earlier, the initial activation of AP 1 or Nrf 2 is triggered by ROS and other oxidants in the cytosol and antioxidants by scavenging the latter may prevent the activation of these transcription factors and downregulate the ARE dependent genes [42]. Thus the in vivo action of many antioxidants may be determined by its direct radical scavenging action as also through the opposing actions on the activation and DNA binding of transcription factors like AP 1, Nrf 2 etc., not to mention the bioavailability of these antioxidants in the brain or other tissues or specific sites within the tissue such as mitochondria.

Since both ROS and RNS are highly reactive, it will be conceptually ideal to trap these species at the sites of their origin before they can interact with other biomolecules to trigger the damage reactions. The mitochondria-targeted antioxidants could thus be more effective in scavenging the ROS and RNS from within the mitochondria and in countering mitochondrial decay in a variety of pathological conditions including brain aging and neurodegenerative disorders. Several ingenious methods have been developed to achieve this. Since mitochondrial transmembrane potential is negative inside (−150 to −180 mV) with respect to cytosol, lipophilic cationic compounds can accumulate within mitochondria in high concentrations and this has provided a tool to target antioxidants to the inside of mitochondria by conjugating them to compounds like triphenylphosphonium cation or TPP⁺ [108]. Thus coenzyme Q and vitamin E have been conjugated to TPP⁺ to produce Mito-Q and Mito-vitE respectively which can localize within mitochondria driven by the negative transmembrane potential and can scavenge ROS and prevent lipid peroxidation within mitochondria [108,109]. At very low concentration, they are capable of preventing H₂O₂ mediated apoptosis in cultured cells and when fed to animals they can reach various organs like heart, liver and the brain, although some studies using ³H labelled Mito-Q or Mito-vitE have indicated that their entry into brain is somewhat restricted [108–110]. Based on similar ideas, choline esters of glutathione and N-acetylcysteine have been synthesized to make these well known antioxidant compounds enter mitochondria in significant amounts [110].

A novel class of small mitochondria targeted peptides (SS or Szeto-Schiller peptides) have been synthesized which contain alternating aromatic and basic amino acids (aromatic cationic peptides) and these can pass through the plasma membrane and then accumulate within mitochondria in potential independent manner. A specific motif in the peptide is probably responsible for mitochondrial localization of these compounds and the tyrosine or dimethyl tyrosine residues of the peptide can effectively scavenge H₂O₂, ^.OH, ^.OONO [110–112]. The compounds can prevent mitochondrial ROS production and also Ca²⁺-dependent mitochondrial swelling [111]. In animal models of neurodegenerative disorders, the SS peptides can prevent neuronal cell loss and they also produce protective actions against ischemia-reperfusion injury with ex vivo and in vivo models [111,112]. Although extensive animal studies with mitochondria targeted antioxidants have not been completed, it is expected that these novel compounds would be extremely useful in combating mitochondrial decay associated with brain aging and neurodegenerative disorders.

Concluding Remarks

Oxidative stress and mitochondrial dysfunctions are intricately related, but more incisive studies are necessary to understand the various ramifications and nuances of this relationship, especially in the context of mitochondrial biogenesis and ROS-dependent gene expressions and the signalling mechanisms. This will help us to better understand the complex process of brain aging as also the genesis of several neurodegenerative disorders like Alzheimer’s disease and Parkinson’s disease where ROS and mitochondrial dysfunctions play critical roles. The antioxidant supplementation therapy for age-related brain mitochondrial decay and other biochemical and cognitive deficits must be critically evaluated in view of newer roles of ROS as signalling molecules and proper protocols should be developed for clinical studies with normally aged subjects or patients of neurodegenerative disorders like Alzheimer’s disease. The account provided in this review also indicates that the effects of antioxidants in vivo are dependent upon a multitude of factors other than the direct radical scavenging actions of the former and their bioavailability. These in vivo factors that regulate the antioxidant effects could be tissue-specific as well as species-dependent which probably explains the failure of many clinical studies with antioxidant supplementation despite encouraging results in cell based and animal models. These issues are to be resolved, but the identification of newer therapeutic targets and the development of novel antioxidants should continue along with the elucidation of complex pathways of cell signalling and the innovation of newer technologies for quantitation of ROS in vivo.