Antioxidant capacity in postmortem brain tissues of Parkinson’s and Alzheimer’s diseases

Calabrese V., Lodi R., Tonon C., D'Agata V., Sapienza M., Scapagnini G., Mangiameli A., Pennisi G., Stella A.M., Butterfield D.A.

There is significant evidence that the pathogenesis of several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Friedreich's ataxia (FRDA), multiple sclerosis and amyotrophic lateral sclerosis, may involve the generation of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) associated with mitochondrial dysfunction. The mitochondrial genome may play an essential role in the pathogenesis of these diseases, and evidence for mitochondria being a site of damage in neurodegenerative disorders is based in part on observed decreases in the respiratory chain complex activities in Parkinson's, Alzheimer's, and Huntington's disease. Such defects in respiratory complex activities, possibly associated with oxidant/antioxidant imbalance, are thought to underlie defects in energy metabolism and induce cellular degeneration. The precise sequence of events in FRDA pathogenesis is uncertain. The impaired intramitochondrial metabolism with increased free iron levels and a defective mitochondrial respiratory chain, associated with increased free radical generation and oxidative damage, may be considered possible mechanisms that compromise cell viability. Recent evidence suggests that frataxin might detoxify ROS via activation of glutathione peroxidase and elevation of thiols, and in addition, that decreased expression of frataxin protein is associated with FRDA. Many approaches have been undertaken to understand FRDA, but the heterogeneity of the etiologic factors makes it difficult to define the clinically most important factor determining the onset and progression of the disease. However, increasing evidence indicates that factors such as oxidative stress and disturbed protein metabolism and their interaction in a vicious cycle are central to FRDA pathogenesis. Brains of FRDA patients undergo many changes, such as disruption of protein synthesis and degradation, classically associated with the heat shock response, which is one form of stress response. Heat shock proteins are proteins serving as molecular chaperones involved in the protection of cells from various forms of stress. In the central nervous system, heat shock protein (HSP) synthesis is induced not only after hyperthermia, but also following alterations in the intracellular redox environment. The major neurodegenerative diseases, Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Huntington's disease (HD) and FRDA are all associated with the presence of abnormal proteins. Among the various HSPs, HSP32, also known as heme oxygenase I (HO-1), has received considerable attention, as it has been recently demonstrated that HO-1 induction, by generating the vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, could represent a protective system potentially active against brain oxidative injury. Given the broad cytoprotective properties of the heat shock response there is now strong interest in discovering and developing pharmacological agents capable of inducing the heat shock response. This may open up new perspectives in medicine, as molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. In particular, manipulation of endogenous cellular defense mechanisms, such as the heat shock response, through nutritional antioxidants, pharmacological compounds or gene transduction, may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration.

Montine T.J., Morrow J.D.

Alzheimer's disease (AD) is the most common dementing illness of the elderly and is a mounting public health problem. Pharmacoepidemiological data, analytical data from human tissue and body fluids, and mechanistic data mostly from murine models all have implicated oxidation products of two fatty acids, arachidonic acid (AA) and docosahexaenoic acid (DHA), in the pathogenesis of neurodegeneration. Here we review the biochemistry of AA and DHA oxidation, both enzyme-catalyzed and free radical mediated, and summarize those studies that have investigated these oxidation products as effectors of neurodegeneration and biomarkers of AD. Given the evolving appreciation for toxicity associated with current pharmaceuticals used to block AA and DHA oxidation, we close by speculating on likely areas of future research directed at suppressing this facet of neurodegeneration. If successful, these interventions are unlikely to cure AD, but may check its explosive growth and hopefully reduce its incidence and prevalence in the elderly.

Valko M., Morris H., Cronin M.

Metal-induced toxicity and carcinogenicity, with an emphasis on the generation and role of reactive oxygen and nitrogen species, is reviewed. Metal-mediated formation of free radicals causes various modifications to DNA bases, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis. Lipid peroxides, formed by the attack of radicals on polyunsaturated fatty acid residues of phospholipids, can further react with redox metals finally producing mutagenic and carcinogenic malondialdehyde, 4-hydroxynonenal and other exocyclic DNA adducts (etheno and/or propano adducts). Whilst iron (Fe), copper (Cu), chromium (Cr), vanadium (V) and cobalt (Co) undergo redox-cycling reactions, for a second group of metals, mercury (Hg), cadmium (Cd) and nickel (Ni), the primary route for their toxicity is depletion of glutathione and bonding to sulfhydryl groups of proteins. Arsenic (As) is thought to bind directly to critical thiols, however, other mechanisms, involving formation of hydrogen peroxide under physiological conditions, have been proposed. The unifying factor in determining toxicity and carcinogenicity for all these metals is the generation of reactive oxygen and nitrogen species. Common mechanisms involving the Fenton reaction, generation of the superoxide radical and the hydroxyl radical appear to be involved for iron, copper, chromium, vanadium and cobalt primarily associated with mitochondria, microsomes and peroxisomes. However, a recent discovery that the upper limit of "free pools" of copper is far less than a single atom per cell casts serious doubt on the in vivo role of copper in Fenton-like generation of free radicals. Nitric oxide (NO) seems to be involved in arsenite-induced DNA damage and pyrimidine excision inhibition. Various studies have confirmed that metals activate signalling pathways and the carcinogenic effect of metals has been related to activation of mainly redox-sensitive transcription factors, involving NF-kappaB, AP-1 and p53. Antioxidants (both enzymatic and non-enzymatic) provide protection against deleterious metal-mediated free radical attacks. Vitamin E and melatonin can prevent the majority of metal-mediated (iron, copper, cadmium) damage both in vitro systems and in metal-loaded animals. Toxicity studies involving chromium have shown that the protective effect of vitamin E against lipid peroxidation may be associated rather with the level of non-enzymatic antioxidants than the activity of enzymatic antioxidants. However, a very recent epidemiological study has shown that a daily intake of vitamin E of more than 400 IU increases the risk of death and should be avoided. While previous studies have proposed a deleterious pro-oxidant effect of vitamin C (ascorbate) in the presence of iron (or copper), recent results have shown that even in the presence of redox-active iron (or copper) and hydrogen peroxide, ascorbate acts as an antioxidant that prevents lipid peroxidation and does not promote protein oxidation in humans in vitro. Experimental results have also shown a link between vanadium and oxidative stress in the etiology of diabetes. The impact of zinc (Zn) on the immune system, the ability of zinc to act as an antioxidant in order to reduce oxidative stress and the neuroprotective and neurodegenerative role of zinc (and copper) in the etiology of Alzheimer's disease is also discussed. This review summarizes recent findings in the metal-induced formation of free radicals and the role of oxidative stress in the carcinogenicity and toxicity of metals.

Moreira P.I., Siedlak S.L., Aliev G., Zhu X., Cash A.D., Smith M.A., Perry G.

Oxidative damage of biological macromolecules is a hallmark of most neurodegenerative disorders such as Alzheimer, Parkinson and diffuse Lewy body diseases. Another important phenomenon involved in these disorders is the alteration of iron and copper homeostasis. Data from the literature support the involvement of metal homeostasis in mitochondrial dysfunction, protein alterations and nucleic acid damage which are relevant in brain function and consequently, in the development of neurodegenerative disorders. Although alterations in transition metal homeostasis, redox activity, and localization are well documented, it must be determined how alterations of specific copper- and iron-containing metalloenzymes are also involved in Alzheimer disease. The clarification of these phenomena can open a new window for understanding the mechanisms underlying neurodegeneration and, consequently, for the development of new therapeutic strategies such as gene therapy and new pharmaceutical formulations with antioxidant and chelating properties.

Zecca L., Youdim M.B., Riederer P., Connor J.R., Crichton R.R.

There is increasing evidence that iron is involved in the mechanisms that underlie many neurodegenerative diseases. Conditions such as neuroferritinopathy and Friedreich ataxia are associated with mutations in genes that encode proteins that are involved in iron metabolism, and as the brain ages, iron accumulates in regions that are affected by Alzheimer's disease and Parkinson's disease. High concentrations of reactive iron can increase oxidative-stress induced neuronal vulnerability, and iron accumulation might increase the toxicity of environmental or endogenous toxins. By studying the accumulation and cellular distribution of iron during ageing, we should be able to increase our understanding of these neurodegenerative disorders and develop new therapeutic strategies.

Berg D., Youdim M.B., Riederer P.

Substantial evidence implies that redox imbalance attributable to an overproduction of reactive oxygen species or reactive nitrogen species that overwhelm the protective defense mechanism of cells contributes to all forms of Parkinson’s disease. Factors such as dopamine, neuromelanin, and transition metals may, under certain circumstances, contribute to the formation of oxygen species such as H2O2, superoxide radicals, and hydroxyl radicals and react with reactive nitrogen species such as nitric oxide or peroxinitrite. Mitochodrial dysfunction and excitotoxicity may be a cause and a result of oxidative stress. Consequences of this redox imbalance are lipid peroxidation, oxidation of proteins, DNA damage, and interference of reactive oxygen species with signal transduction pathways. These consequences become even more harmful when genetic variations impair the normal degradation of altered proteins. Therefore, therapeutic strategies must aim at reducing free-radical formation and scavenging free-radicals.

Mazzio E.A., Soliman K.F.

The pathology of Parkinson's disease involves oxidative damage to dopaminergic neurons of the substantia nigra. Oxidation of the dopamine (DA) neurotransmitter itself may contribute to the generation of a reactive oxygen species (ROS) and subsequent neurodegeneration. Glia cells can either exacerbate injury or exert protective properties on local neurons in the brain. We investigate glial antioxidant enzyme systems relative to ROS generated during cytokine activation, monoamine oxidase (MAO) activity and autoxidation of DA in glioma cells. Rat C6 glioma cells stimulated with lipopolysaccharide Escherichia coli 0111:B4 and interferon gamma (LPS/IFN-g) produced high levels of nitric oxide (241 nmol mg−1 protein 24 h−1) but not superoxide (O−2) or hydrogen peroxide (H2O2). Basal C6 cells exhibited a rapid and robust capacity to remove exogenous H2O2 within minutes. Preincubation with sodium azide but not buthionine-[S, R]-sulfoximine attenuated this response, indicating catalase as the primary enzyme responsible for this effect. The glioma catalase reaction rate was slightly attenuated by exposure to LPS/IFN-g for 24 h. However, the reduction in catalase activity was not due to nitric oxide, because both the supernatant and sodium nitroprusside had no effect on isolated catalase enzyme activity. Hydrogen peroxide was produced only through substrate-driven MAO activity in prepared lysate. However, the quantity of H2O2 produced per unit time (0.46 nmol mg−1 protein min−1) was negligible compared with the enormous capacity for its removal by catalase (213.9 nmol mg−1 protein min−1) (≥462× greater). Similarly, H2O2 generated by DA autoxidation per unit time (0.28 nmol mg−1 protein equiv. min−1), was rapidly dissolved by glioma cells at high capacity (≥750× greater). In conclusion, C6 cells produce nitric oxide under cytokine/endotoxin-stimulated conditions. Moreover, C6 cells exhibit a dynamic H2O2 scavenging capacity, with ample facility to dispose of the peroxide generated by both MAO activity and spontaneous DA autoxidation. Copyright © 2004 John Wiley & Sons, Ltd.

Prior R.L., Hoang H., Gu L., Wu X., Bacchiocca M., Howard L., Hampsch-Woodill M., Huang D., Ou B., Jacob R.

Methods are described for the extraction and analysis of hydrophilic and lipophilic antioxidants, using modifications of the oxygen radical absorbing capacity (ORAC(FL)) procedure. These methods provide, for the first time, the ability to obtain a measure of "total antioxidant capacity" in the protein free plasma, using the same peroxyl radical generator for both lipophilic and hydrophilic antioxidants. Separation of the lipophilic and hydrophilic antioxidant fractions from plasma was accomplished by extracting with hexane after adding water and ethanol to the plasma (hexane/plasma/ethanol/water, 4:1:2:1, v/v). Lipophilic and hydrophilic antioxidants were efficiently partitioned between hexane and aqueous solvents. Conditions for controlling temperature effects and decreasing assay variability using fluorescein as the fluorescent probe were validated in different laboratories. Incubation (37 degrees C for at least 30 min) of the buffer to which AAPH was dissolved was critical in decreasing assay variability. Lipophilic antioxidants represented 33.1 +/- 1.5 and 38.2 +/- 1.9% of the total antioxidant capacity of the protein free plasma in two independent studies of 6 and 10 subjects, respectively. Methods are described for application of the assay techniques to other types of biological and food samples.

Gsell W., Conrad R., Hickethier M., Sofic E., Frölich L., Wichart I., Jellinger K., Moll G., Ransmayr G., Beckmann H., Riederer P.

"Oxidative stress" may be of significance in the etiopathogenesis of dementia of Alzheimer type (DAT). Therefore, we measured activities of the enzymes superoxide dismutase (SOD) and catalase (CAT), which detoxicate reactive oxygen species. Enzyme activities were measured postmortem in basal ganglia, cortical, and limbic brain regions of patients with DAT and age-matched controls. SOD activity increased with age in basal nucleus of Meynert. However, there was no significant difference in SOD activity between DAT and controls. CAT activity was independent of age and postmortem time. There were significant reductions in CAT activity in parietotemporal cortex, basal ganglia, and amygdala in DAT compared with controls (p < 0.05 to 0.01). Our findings are in line with the assumption that reactive oxygen species could contribute to the pathogenesis of DAT. Absence of these changes in basal nucleus of Meynert might reflect retrograde degeneration of cholinergic fibers.

Sofic E., Rustembegovic A., Kroyer G., Cao G.

The role of free radicals (FR) in the pathogenesis and in the progression of many diseases has been often discussed, but not widely investigated. However, the total antioxidant capacity in the serum seems to be of great evidence. Total antioxidant capacity was determined using oxygen absorbance capacity assay (ORAC) in serum of patients suffering from depression, schizophrenia, Alzheimer's disease (AD), anorexia nervosa, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Aids-encephalopathy, diabetic polyneuropathy (PNP), cardiomyopathy (CM), renal disease, and healthy individuals as controls (C). The results showed that the total antioxidant capacity in serum decreased significantly (p < 0.01) by 24, 20, 13, and 17% for anorexia nervosa, Aids-encephalopathy, PNP and CM respectively. In serum of patients with renal disease significantly elevated antioxidant capacity was found. The data indicated that increased oxidative stress can be involved in the pathogenesis or in the progression of PNP and CM. Decrease of serum antioxidant capacity in patients with anorexia nervosa and Aids-encephalopathy are probably due primarily to malnutrition and secondly to insufficient antioxidant and immune system. In renal disease, the accumulation of urea in serum seems to be responsible for high antioxidant capacity. In contrast, there were no changes in PD, AD, depression syndrome and schizophrenia.

Serra J.A., Domínguez R.O., de Lustig E.S., Guareschi E.M., Famulari A.L., Bartolomé E.L., Marschoff E.R.

Antioxidant profiles in Parkinson's disease (PD; n = 15), dementias of Alzheimer's type (DAT; 18) and Vascular (VD; 15), and control subjects (C; 14) were studied. Cu-Zn superoxide dismutase (SOD), catalase (CAT), glutathione system (GLU) and thiobarbituric acid reactive substances (TBARS) were measured in erythrocytes; antioxidant capacity (TRAP) in plasma. Biochemical variables were analyzed simultaneously using multivariate and non-parametric methods. Clinical diagnostic resulted associated with the main source of variability in antioxidant variables (Kruskal-Wallis: H = 32.58, p = 0.000001). Comparison of PD and C resulted highly significant (z = 4.47, p = 0.000047), demonstrating an association between oxidative stress and PD. SOD and TBARS were significantly higher in pathological groups against C (p = 0.0000001, p = 0.051); TRAP resulted lower (p = 0.00015). Discriminant functions constructed using biochemical variables separated pathological groups (93% success) from C, and DAT (88.9%) from VD (73.3%); but not PD from DAT or VD. Antioxidant profiles of PD patients showed characteristics overlapping with DAT (60%) and with VD (40%), suggesting biochemical similarities between them.

Marcus D.L., Thomas C., Rodriguez C., Simberkoff K., Tsai J.S., Strafaci J.A., Freedman M.L.

The overall peroxidation activity in brain tissue by region from patients with Alzheimer's disease (AD) and age-matched controls was determined employing the thiobarbituric acid-reactive substances (TBARS) assay, a measure of lipid peroxidation, followed by a determination the activities of the antioxidant enzymes Cu/Zn superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), in the frontal, temporal, and cerebellar cortex of 10 AD and 9 control brains. The level of TBARS was elevated in all regions, with particular statistical significance in the temporal cortex when compared to age-matched controls. SOD activity was significantly decreased in AD frontal and AD temporal cortex, while catalase activity was significantly decreased in AD temporal cortex. There was no significant difference in GSH-Px activity found in any of the regions examined. This study supports the theory that in AD the brain is affected by increased oxidative stress which, when combined with a decrease in SOD activity, produces oxidative alterations, seen most significantly in temporal cortex in AD, where the pathophysiologic changes are most severe.

Jenner P., Olanow C.W.

Current concepts of the pathogenesis of Parkinson9s disease (PD) center on the formation of reactive oxygen species and the onset of oxidative stress leading to oxidative damage to substantia nigra pars compacta.Extensive postmortem studies have provided evidence to support the involvement of oxidative stress in the pathogenesis of PD; in particular, these include alterations in brain iron content, impaired mitochondrial function, alterations in the antioxidant protective systems (most notably superoxide dismutase [SOD] and reduced glutathione [GSH]), and evidence of oxidative damage to lipids, proteins, and DNA. Iron can induce oxidative stress, and intranigral injections have been shown to induce a model of progressive parkinsonism. A loss of GSH is associated with incidental Lewy body disease and may represent the earliest biochemical marker of nigral cell loss. GSH depletion alone may not result in damage to nigral neurons but may increase susceptibility to subsequent toxic or free radical exposure. The nature of the free radical species responsible for cell death in PD remains unknown, but there is evidence of involvement of hydroxyl radical (OH sup [bullet]), peroxynitrite, and nitric oxide. Indeed, OH sup [bullet] and peroxynitrite formation may be critically dependent on nitric oxide formation. Central to many of the processes involved in oxidative stress and oxidative damage in PD are the actions of monoamine oxidase-B (MAO-B). MAO-B is essential for the activation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to 1-methyl-4-phenylpyridinium ion, for a component of the enzymatic conversion of dopamine to hydrogen peroxide (H2 O2), and for the activation of other potential toxins such as isoquinolines and beta-carbolines. Thus, the inhibition of MAO-B by drugs such as selegiline may protect against activation of some toxins and free radicals formed from the MAO-B oxidation of dopamine. In addition, selegiline may act through a mechanism unrelated to MAO-B to increase neurotrophic factor activity and upregulate molecules such as glutathione, SOD, catalase, and BCL-2 protein, which protect against oxidant stress and apoptosis. Consequently, selegiline may be advantageous in the long-term treatment of PD. NEUROLOGY 1996;47(Suppl 3): S161-S170

Cao G., Giovanoni M., Prior R.L.

The objective of this study was to evaluate the effects of growth and aging on the antioxidant capacities of rat serum and brain, by using two different reactive oxygen species (ROS) generators and two different methods of expressing the results. The antioxidant capacity was determined by using the oxygen radical absorbance capacity (ORAL) assay. The protein content of serum increased significantly during the growth period of day 1 to 6 months of age. The protein content in brain (cortex and cerebellum) extract also tended to increase with the growth. The antioxidant capacity of serum decreased during growth, if the ORAC values were expressed on the basis of serum protein concentration. The antioxidant capacity in brain cortex and cerebellum declined significantly during growth, regardless of which ROS generator was used in the ORAC assay or how the results were expressed (units/ml or units/mg protein). Therefore, the rat pups had higher antioxidant capacity than the adult and old rats. The low plasma antioxidant capacity reported in premature neonates may simply result from a low protein concentration of the plasma, which occurs in neonates. No aging effect was observed on the antioxidant capacity of serum and brain in the adult and old rats.

Prior R.L., Wang H., Wu A.H., Verdon C.P., Cao G.

Abstract Reactive oxygen species are of interest in biology and medicine because of evidence relating them to aging and disease processes. A relatively simple but sensitive and reliable method for quantifying the oxygen radical absorbance capacity (ORAC) of antioxidants in biological tissues has been automated for use with the COBAS FARA II centrifugal analyzer with a fluorescence-measuring attachment. In this assay, beta-phycoerythrin (beta-PE) is used as an indicator protein, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) as a peroxyl radical generator, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) as a calibrator for antioxidant activity. This assay is unique because the reaction goes to completion so that both inhibition time and inhibition degree are considered in quantifying ORAC (micromoles of Trolox equivalent per liter or per gram of tissue). This method can be used not only for serum but also other tissue and food samples and is suitable for application to a range of nutritional and clinical conditions.

Armstrong O.J., Neal E.S., Vidovic D., Xu W., Borges K.

IntroductionIntermittent fasting enhances neural bioenergetics, is neuroprotective, and elicits antioxidant effects in various animal models. There are conflicting findings on seizure protection, where intermittent fasting regimens often cause severe weight loss resembling starvation which is unsustainable long-term. Therefore, we tested whether a less intensive intermittent fasting regimen such as time-restricted feeding (TRF) may confer seizure protection.MethodsMale CD1 mice were assigned to either ad libitum-fed control, continuous 8 h TRF, or 8 h TRF with weekend ad libitum food access (2:5 TRF) for one month. Body weight, food intake, and blood glucose levels were measured. Seizure thresholds were determined at various time points using 6-Hz and maximal electroshock seizure threshold (MEST) tests. Protein levels and mRNA expression of genes, enzyme activity related to glucose metabolism, as well as mitochondrial dynamics were assessed in the cortex and hippocampus. Markers of antioxidant defence were evaluated in the plasma, cortex, and liver.ResultsBody weight gain was similar in the ad libitum-fed and TRF mouse groups. In both TRF regimens, blood glucose levels did not change between the fed and fasted state and were higher during fasting than in the ad libitum-fed groups. Mice in the TRF group had increased seizure thresholds in the 6-Hz test on day 15 and on day 19 in a second cohort of 2:5 TRF mice, but similar seizure thresholds at other time points compared to ad libitum-fed mice. Continuous TRF did not alter MEST seizure thresholds on day 28. Mice in the TRF group showed increased maximal activity of pyruvate dehydrogenase in the cortex, which was accompanied by increased protein levels of mitochondrial pyruvate carrier 1 in the cortex and hippocampus. There were no other major changes in protein or mRNA levels associated with energy metabolism and mitochondrial dynamics in the brain, nor markers of antioxidant defence in the brain, liver, or plasma.ConclusionsBoth continuous and 2:5 TRF regimens transiently increased seizure thresholds in the 6-Hz model at around 2 weeks, which coincided with stability of blood glucose levels during the fed and fasted periods. Our findings suggest that the lack of prolonged anticonvulsant effects in the acute electrical seizure models employed may be attributed to only modest metabolic and antioxidant adaptations found in the brain and liver. Our findings underscore the potential therapeutic value of TRF in managing seizure-related conditions.

Haque S., Ahmad F., Ramamorthy S., Areeshi M.Y., Ashraf G.M.

Abstract: Mitochondria regulate multiple aspects of neuronal development, physiology, plasticity, and pathology through their regulatory roles in bioenergetic, calcium, redox, and cell survival/death signalling. While several reviews have addressed these different aspects, a comprehensive discussion focussing on the relevance of isolated brain mitochondria and their utilities in neuroscience research has been lacking. This is relevant because the employment of isolated mitochondria rather than their in situ functional evaluation, offers definitive evidence of organelle-specificity, negating the interference from extra mitochondrial cellular factors/signals. This mini-review was designed primarily to explore the commonly employed in organello analytical assays for the assessment of mitochondrial physiology and its dysfunction, with a particular focus on neuroscience research. The authors briefly discuss the methodologies for biochemical isolation of mitochondria, their quality assessment, and cryopres- ervation. Further, the review attempts to accumulate the key biochemical protocols for in organello assessment of a multitude of mitochondrial functions critical for neurophysiology, including assays for bioenergetic activity, calcium and redox homeostasis, and mitochondrial protein translation. The purpose of this review is not to examine each and every method or study related to the functional assessment of isolated brain mitochondria, but rather to assemble the commonly used protocols of in organello mitochondrial research in a single publication. The hope is that this review will provide a suitable platform aiding neuroscientists to choose and apply the required protocols and tools to address their particular mechanistic, diagnostic, or therapeutic question dealing within the confines of the research area of mitochondrial patho-physiology in the neuronal perspective.

Lin C., Wei P., Chen C., Huang Y., Lin J., Lo Y., Lin J., Lin C., Wu Y., Chang K., Lee-Chen G.

Parkinson’s disease (PD) is a common neurodegenerative disease characterized by progressive loss of dopaminergic (DAergic) neurons in ventral brain. A disaccharide trehalose has demonstrated the potential to mitigate the DAergic loss in disease models for PD. However, trehalose is rapidly hydrolyzed into glucose by trehalase in the intestine, limiting its potential for clinical practice. Here we investigated the neuroprotective potentials of two trehalase-indigestible analogs, lactulose and melibiose, in sub-chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. Treatment with MPTP generated significant motor deficits, inhibited dopamine levels and down-regulated dopamine transporter (DAT) in striatum. Expression levels of genes involved in anti-oxidative stress pathways, including superoxide dismutase 2 (SOD2), nuclear factor erythroid 2-related factor 2 (NRF2) and NAD(P)H dehydrogenase (NQO1) were also down-regulated, while expressions of oxidative stress marker 4-hydroxynonenal (4-HNE), microglial activation marker ionized calcium-binding adapter molecule 1 (IBA1) and astrocyte activation marker glial fibrillary acidic protein (GFAP) in ventral midbrain were up-regulated following MPTP treatment. MPTP also reduced the activity of autophagy, evaluated by autophagosomal marker microtubule-associated protein 1 light chain 3 (LC3)-II. Lactulose and melibiose significantly rescued motor deficits, increased dopamine in striatum, reduced levels of 4-HNE, IBA1 and GFAP, up-regulated SOD2, NRF2 and NQO1 levels, as well as LC3-II/LC3-I ratio in ventral midbrain with MPTP treatment. Our findings indicate the potential of lactulose and melibiose to protect DAergic neurons in PD.

Loeffler D.A., Klaver A.C., Coffey M.P., Aasly J.O., LeWitt P.A.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most frequent cause of inherited Parkinson's disease (PD). The most common PD-associated LRRK2 mutation, G2019S, induces increased production of reactive oxygen species in vitro. We therefore hypothesized that individuals with PD-associated LRRK2 mutations might have increased concentrations of oxidative stress markers and/or decreased total antioxidant capacity (TAC) in their cerebrospinal fluid (CSF). We measured two oxidative stress markers, 8-hydroxy-2’-deoxyguanosine (8-OHdG) and 8-isoprostane (8-ISO), and TAC in CSF from LRRK2 mutation-bearing PD patients (LRRK2 PD = 19), sporadic PD patients (sPD = 31), and healthy control subjects with or without these mutations (LRRK2 CTL = 30, CTL = 27). 8-OHdG and 8-ISO levels were increased in LRRK2 CTL subjects, while TAC was similar between groups. 8-ISO was negatively correlated, and TAC was positively correlated, with Montreal Cognitive Assessment (MoCA) scores in LRRK2 PD, LRRK2 CTL, and CTL subjects. Correlations in both groups of PD patients between the two oxidative stress markers and Unified Parkinson Disease Rating Scale (UPDRS) Total scores were weak, while TAC was negatively correlated with these scores. These findings suggest that oxidative stress may be increased in the CNS in healthy individuals with PD-associated LRRK2 mutations, perhaps contributing to their risk for PD. Further, TAC may decrease in the CNS with the progression of PD, and when cognitive impairment is present regardless of the presence or absence of PD.

Prasad K.N.

Parkinson disease (PD) is a slow progressive neurological disorder associated with abnormal functions of extrapyramidal system. Studies suggest that increased oxidative stress, chronic inflammation and glutamate play a dominant role in the initiation and progression of PD. Mitochondria are very sensitive to increased oxidative stress and damaged mitochondria produce more free radicals. Mutation in DJ-1, alpha-synuclein, PINK-1 or PARKIN gene associated with familial PD impairs mitochondrial functions which can increase oxidative stress. Oxidative damage initiates chronic inflammation, whereas excessive amounts of glutamate participate in neuronal death via free radicals. Thus, attenuation of oxidative stress, chronic inflammation and glutamate levels may reduce incidence, progression, and in combination with drug therapy, improve the management of PD. This review suggests that an elevation of the levels of antioxidant enzymes and phase-2-detoxifying enzymes, and dietary and endogenous antioxidants simultaneously are essential for attenuating these biochemical abnormalities optimally. The levels of dietary and endogenous antioxidants can be increased by supplementation; however, the levels of antioxidant enzymes and detoxifying enzymes requires an activation of a nuclear transcriptional factor Nrf2 and its binding with the antioxidant response elements (AREs) in the nucleus. This review discusses the regulation of Nrf2 activation, and identifies agents that activate Nrf2 by reactive oxygen species (ROS)-dependent and-independent mechanisms. This review also describes studies on individual antioxidants in PD to show that a single antioxidant cannot activate Nrf2 and enhance the levels of multiple antioxidants at the same time. It suggests a mixture of micronutrients that may accomplish the above goal.

Perfeito R., Ribeiro M., Rego A.C.

Alpha-synuclein (α-syn) is a major component of Lewy bodies found in sporadic and inherited forms of Parkinson’s disease (PD). Mutations in the gene encoding α-syn and duplications and triplications of wild-type (WT) α-syn have been associated with PD. Several mechanisms have been implicated in the degeneration of dopaminergic neurons in PD, including oxidative stress and mitochondrial dysfunction. Here we defined the occurrence of oxidative stress in SH-SY5Y cells overexpressing WT α-syn in a doxycycline (Dox) regulated manner, before and after exposure to iron (500 µM), and determined the changes in proteins involved in the intracellular antioxidant defense system. Data evidenced an increase in caspase-3 activation and diminished reducing capacity of −Dox cells, associated with decreased activity of mitochondria complex I and reduced mitochondrial transcription factor A (TFAM) levels in these cells. Furthermore, total and mitochondrial reactive oxygen species levels were higher under basal conditions in cells overexpressing α-syn (−Dox) and this increase was apparently correlated with diminished levels and activities of SOD1 and SOD2 in −Dox cells. Moreover, both reduced and oxidized glutathione levels were diminished in −Dox cells under basal conditions, concomitantly with decreased activity of GCL and reduced protein levels of GCLc. The effects caused by iron (500 µM) were mostly independent of α-syn expression and triggered different antioxidant responses to possibly counterbalance higher levels of free radicals. Overall, data suggest that overexpression of α-syn modifies the antioxidant capacity of SH-SY5Y cells due to altered activity and protein levels of SOD1 and SOD2, and decreased glutathione pool.

Çubukçu H.C., Yurtdaş M., Durak Z.E., Aytaç B., Güneş H.N., Çokal B.G., Yoldaş T.K., Durak İ.

Parkinson’s disease (PD) is one of the common neurodegenerative disorders. Oxidative stress is considered as a contributing factor to the development of PD. The present study aims to investigate serum oxidative stress status in patients with PD. Oxidative stress was assessed by measuring serum nitric oxide levels, lipid hydroperoxide concentrations, and nitric oxide synthase activity. In addition, total serum antioxidant capacity (TAC) was evaluated using the serum 2,2-Diphenyl-1-picryl-hydrazyl (DPPH) free-radical scavenging method in 32 patient with Parkinson’s disease and 32 control subjects. Our results indicated that serum nitric oxide and lipid hydroperoxide levels were significantly lower in patients with PD than controls. Moreover, nitric oxide levels were found to be negatively correlated with Unified Parkinson’s Disease Rating Scale (UPDRS). However, no statistical difference was observed in total serum antioxidant capacities and nitric oxide synthase activities between patients and controls. The present study indicates that although antioxidant capacity was not changed, lipid hydroperoxide (LPO) level was found decreased. This might show pre-oxidative process in these patients. In addition, decreased nitric oxide (NO) level and negative correlation observed between NO level and disease rating scale implicated a role for NO in the disease process.

Van Laar V.S., Roy N., Liu A., Rajprohat S., Arnold B., Dukes A.A., Holbein C.D., Berman S.B.

Disruption of the dynamic properties of mitochondria (fission, fusion, transport, degradation, and biogenesis) has been implicated in the pathogenesis of neurodegenerative disorders, including Parkinson's disease (PD). Parkin, the product of gene PARK2 whose mutation causes familial PD, has been linked to mitochondrial quality control via its role in regulating mitochondrial dynamics, including mitochondrial degradation via mitophagy. Models using mitochondrial stressors in numerous cell types have elucidated a PINK1-dependent pathway whereby Parkin accumulates on damaged mitochondria and targets them for mitophagy. However, the role Parkin plays in regulating mitochondrial homeostasis specifically in neurons has been less clear. We examined whether a stressor linked to neurodegeneration, glutamate excitotoxicity, elicits Parkin-mitochondrial translocation and mitophagy in neurons. We found that brief, acute exposure to glutamate causes Parkin translocation to mitochondria in neurons, in a calcium- and N-methyl-d-aspartate (NMDA) receptor-dependent manner. In addition, we found that Parkin accumulates on endoplasmic reticulum (ER) and mitochondrial/ER junctions following excitotoxicity, supporting a role for Parkin in mitochondrial-ER crosstalk in mitochondrial homeostasis. Despite significant Parkin-mitochondria translocation, however, we did not observe mitophagy under these conditions. To further investigate, we examined the role of glutamate-induced oxidative stress in Parkin-mitochondria accumulation. Unexpectedly, we found that glutamate-induced accumulation of Parkin on mitochondria was promoted by the antioxidant N-acetyl cysteine (NAC), and that co-treatment with NAC facilitated Parkin-associated mitophagy. These results suggest the possibility that mitochondrial depolarization and oxidative damage may have distinct pathways associated with Parkin function in neurons, which may be critical in understanding the role of Parkin in neurodegeneration.

Parrott M.D., Winocur G., Bazinet R.P., Ma D.W., Greenwood C.E.

Food combinations have been associated with lower incidence of Alzheimer's disease. We hypothesized that a combination whole-food diet containing freeze-dried fish, vegetables, and fruits would improve cognitive function in TgCRND8 mice by modulating brain insulin signaling and neuroinflammation. Cognitive function was assessed by a comprehensive battery of tasks adapted to the Morris water maze. Unexpectedly, a "Diet × Transgene" interaction was observed in which transgenic animals fed the whole-food diet exhibited even worse cognitive function than their transgenic counterparts fed the control diet on tests of spatial memory (p < 0.01) and strategic rule learning (p = 0.034). These behavioral deficits coincided with higher hippocampal gene expression of tumor necrosis factor-α (p = 0.013). There were no differences in cortical amyloid-β peptide species according to diet. These results indicate that a dietary profile identified from epidemiologic studies exacerbated cognitive dysfunction and neuroinflammation in a mouse model of familial Alzheimer's disease. We suggest that normally adaptive cellular responses to dietary phytochemicals were impaired by amyloid-beta deposition leading to increased oxidative stress, neuroinflammation, and behavioral deficits.

Sofic E., Salkovic-Petrisic M., Tahirovic I., Sapcanin A., Mandel S., Youdim M., Riederer P.

Low intracerebroventricular (icv) doses of streptozotocin (STZ) produce regionally specific brain neurochemical changes in rats that are similar to those found in the brain of patients with sporadic Alzheimer’s disease (sAD). Since oxidative stress is thought to be one of the major pathologic processes in sAD, catalase (CAT) activity was estimated in the regional brain tissue of animals treated intracerebroventricularly with STZ and the multitarget iron chelator, antioxidant and MAO-inhibitor M30 [5-(N-methyl-N-propargylaminomethyl)-8-hydroxyquinoline]. Five-day oral pre-treatment of adult male Wistar rats with 10 mg/kg/day M30 dose was followed by a single injection of STZ (1 mg/kg, icv). CAT activity was measured colorimetrically in the hippocampus (HPC), brain stem (BS) and cerebellum (CB) of the control, STZ-, M30- and STZ + M30-treated rats, respectively, 4 weeks after the STZ treatment. STZ-treated rats demonstrated significantly lower CAT activity in all three brain regions in comparison to the controls (p < 0.05 for BS and CB, p < 0.01 for HPC). M30 pre-treatment of the control rats did not influence the CAT activity in HPC and CB, but significantly increased it in BS (p < 0.05). M30 pre-treatment of STZ-treated rats significantly increased CAT activity in the HPC in comparison to the STZ treatment alone (p < 0.05) and normalized to the control values. These findings are in line with the assumption that reactive oxygen species contribute to the pathogenesis of STZ in a rat model of sAD and indicate that multifunctional iron chelators such as M30 might also have beneficial effects in this non-transgenic sAD model.

Lim J.L., Wilhelmus M.M., de Vries H.E., Drukarch B., Hoozemans J.J., van Horssen J.

Activation of microglial cells and impaired mitochondrial function are common pathological characteristics of many neurological diseases and contribute to increased generation of reactive oxygen species (ROS). It is nowadays accepted that oxidative damage and mitochondrial dysfunction are key hallmarks of classical neuroinflammatory and neurodegenerative diseases, such as multiple sclerosis, Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. To counteract the detrimental effects of ROS and restore the delicate redox balance in the central nervous system (CNS), cells are equipped with an endogenous antioxidant defense mechanism consisting of several antioxidant enzymes. The production of many antioxidant enzymes is regulated at the transcriptional level by the transcription factor nuclear factor E2-related factor 2 (Nrf2). Although evidence is accumulating that activation of the Nrf2 pathway represents a promising therapeutic approach to restore the CNS redox balance by reducing ROS-mediated neuronal damage in experimental models of neurodegenerative disorders, only a few Nrf2-activating compounds have been tested in a clinical setting. We here provide a comprehensive synopsis on the role of ROS in common neurodegenerative disorders and discuss the therapeutic potential of the Nrf2 pathway.

Jansen E.H., Beekhof P.K., Cremers J.W., Viezeliene D., Muzakova V., Skalicky J.

The antioxidant status of serum or plasma can be determined using several commercially available assays. Here, four different assays, total antioxidant status (TAS), its second-generation assay (TAS2), biological antioxidant potential (BAP), and enzymatic assay using horseradish peroxidase (EAOC), were applied on human serum samples to test the temperature stability of antioxidants, upon storage of serum for 12 months. The two or three most commonly used temperatures for storage, that is, - 20, - 70 (or - 80), and - 196°C, were selected. The general conclusion is that all assays were stable at the temperatures tested. In addition, there were almost no statistically significant differences between the samples stored at different temperatures. Only the rank order of the EAOC assay was not very good in samples stored at - 20°C. Also three components contributing to the total antioxidant capacity, uric acid, creatinine and bilirubin, showed no statistically significant differences between the temperatures. Therefore, storage at - 20°C is sufficient to maintain a proper assay outcome of most of the total antioxidant assays, although storage at - 70/80°C is to be preferred for longer storage times.

Chen Y., Wu Y., Wu Y., Lee-Chen G., Chen C.

Abstract Oxidative stress has been reported as one of the pathogeneses of Parkinson's disease (PD). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates the expression of the target genes involved in antioxidant pathway. The promoter polymorphisms of the Nrf2-encoding gene, NFE2L2, have been shown to affect the promoter activity. Inconsistent findings of the associations between NFE2L2 promoter polymorphisms and PD have been reported in a multiple candidate gene study, a whole-genome association study, and a case–control study in multiple ethnicity groups. This study enrolled a total of 1006 individuals composed of 480 PD patients and 526 controls to evaluate if there was an association of the NFE2L2 promoter polymorphisms with PD susceptibility in the Taiwan population. Three promoter single nucleotide polymorphisms (SNPs) rs35652124, rs6706649, and rs6721961 were examined using polymerase chain reaction and restriction analysis. The associations of each of the SNPs and the haplotypes constructed by the variations with PD susceptibility were examined. In spite of adequate statistic power, we observe no association of the three SNPs and their haplotypes with PD in a Taiwanese population. One of the reasons for the association disparity may include the genomic differences in ethnicities and environmental factors in different geographical regions.

Pienaar I.S., Lu B., Schallert T.

Animal models that make use of chemical toxins to adversely affect the nigrostriatal dopaminergic pathway of rodents and primates have contributed significantly towards the development of symptomatic therapies for Parkinson's disease (PD) patients. Although their use in developing neuro-therapeutic and -regenerative compounds remains to be ascertained, toxin-based mammalian and a range of non-mammalian models of PD are important tools in the identification and validation of candidate biomarkers for earlier diagnosis, as well as in the development of novel treatments that are currently working their way into the clinic. Toxin models of PD have and continue to be important models to use for understanding the consequences of nigrostriatal dopamine cell loss. Functional assessment of these models is also a critical component for eventual translational success. Sensitive behavioural testing regimens for assessing the extent of dysfunction exhibited in the toxin models, the degree of protection or improvement afforded by potential treatment modalities, and the correlation of these findings with what is observed clinically in PD patients, ultimately determines whether a potential treatment moves to clinical trials. Here, we review existing published work that describes the use of such behavioural outcome measures associated with toxin models of parkinsonism. In particular, we focus on tests assessing sensorimotor and cognitive function, both of which are significantly and progressively impaired in PD.

Binolfi A., Quintanar L., Bertoncini C.W., Griesinger C., Fernández C.O.

Alpha-synuclein (AS) aggregation is associated with neurodegeneration in Parkinson's disease (PD). At the same time, alterations in metal ion homeostasis may play a pivotal role in the progression of AS amyloid assembly and the onset of PD. Elucidation of the structural basis directing AS–metal interactions and their effect on AS aggregation constitutes a key step toward understanding the role of metal ions in AS amyloid formation and neurodegeneration. This work provides a comprehensive review of recent advances attained in the bioinorganic chemistry of AS amyloid diseases. A hierarchy in AS–metal ion interactions has been established: while the physiologically relevant divalent metal ions iron and manganese interact at a non-specific, low-affinity binding interface in the C-terminus of AS, copper binds with high affinity at the N-terminal region and it is the most effective metal ion in accelerating AS filament assembly. The strong link between metal binding specificity and its impact on aggregation is discussed here on a mechanistic basis. A detailed description of the structural features and coordination environments of copper to AS is presented and discussed in the context of oxidative cellular events that might lead to the development of PD. Overall, the research observations presented here support the notion that perturbations in copper metabolism may be a common upstream event in the pathogenesis of neurodegenerative processes.