Oxidative stress in the aging substantia nigra and the etiology of Parkinson's disease

Parkinson's disease prevalence is rapidly increasing in an aging global population. With this increase comes exponentially rising social and economic costs, emphasizing the immediate need for effective disease‐modifying treatments. Motor dysfunction results from the loss of dopaminergic neurons in the substantia nigra pars compacta and depletion of dopamine in the nigrostriatal pathway. While a specific biochemical mechanism remains elusive, oxidative stress plays an undeniable role in a complex and progressive neurodegenerative cascade. This review will explore the molecular factors that contribute to the high steady‐state of oxidative stress in the healthy substantia nigra during aging, and how this chemical environment renders neurons susceptible to oxidative damage in Parkinson's disease. Contributing factors to oxidative stress during aging and as a pathogenic mechanism for Parkinson's disease will be discussed within the context of how and why therapeutic approaches targeting cellular redox activity in this disorder have, to date, yielded little therapeutic benefit. We present a contemporary perspective on the central biochemical contribution of redox imbalance to Parkinson's disease etiology and argue that improving our ability to accurately measure oxidative stress, dopaminergic neurotransmission and cell death pathways in vivo is crucial for both the development of new therapies and the identification of novel disease biomarkers.     

Proteome analysis of human substantia nigra in Parkinson's disease

Protein expression has been compared in human substantia nigra specimens from Parkinson's disease (PD) patients and from controls, and 44 proteins expressed in this midbrain region were identified by peptide mass fingerprinting. Among them, nine showed changes in their abundance. L and M neurofilament chains are less abundant in PD specimens, whereas peroxiredoxin II, mitochondrial complex III, ATP synthase D chain, complexin I, profilin, L-type calcium channel delta-subunit, and fatty-acid binding protein are significantly more present in PD samples than in controls. Besides the consolidated view of oxidative stress involvement in PD pathogenesis, suggested by overexpression of mitochondrial and reactive oxygen species (ROS)-scavenging proteins, these results indicate a possible potentiation mechanism of afferent signals to substantia nigra following degeneration of dopaminergic neurons.     

Proteomic profiling of the substantia nigra demonstrates CNDP2 overexpression in Parkinson's disease

Despite decades of intensive investigations, the precise sequence of molecular events and the specific proteins mediating the degenerative process underlying Parkinson's disease (PD) remain unraveled. Proteomic strategies may provide unbiased tools to identify novel candidates and explore original mechanisms involved in PD. Substantia nigra pars compacta (SN) tissue, whose degeneration is the hallmark of PD, was dissected from neuropathologically confirmed PD patients (n=3) and control subjects (n=3), before being submitted to a comparative 2-DE analysis. The present study revealed a subset of neuronal and/or glial proteins that appears to be deregulated in PD and likely to contribute to neurodegeneration. Observed alterations not only consolidate well accepted concepts surrounding PD pathogenesis such as oxidative stress and mitochondrial dysfunction but also point out to novel pathways. Among the latter, cytosolic non specific dipeptidase 2 (CNDP2), a relatively unknown protein not yet reported to be associated with PD pathogenesis, was shown to be increased in the SN of PD patients, as confirmed by Western blot. Immunohistochemical analyses demonstrated the presence of CNDP2 within the cytoplasm of SN dopaminergic neurons. Altogether, our findings support a key role of CNDP2 in PD neurodegeneration, by mechanisms that could involve oxidative stress, protein aggregation or inflammation. This article is part of a Special Issue entitled: Translational Proteomics.     

F2-Isoprostanes,tocopherols and normal pregnancy

This study investigates oxidative stress and antioxidants in normal human pregnancy and post-partum period. Thirty-seven healthy women with normal pregnancies were included. Both urinary and serum samples were collected throughout the pregnancy and post-partum period. Oxidative stress was estimated by measuring the reliable in vivo marker, namely 8-iso-prostaglandin F_2α (8-iso-PGF_2α, an F₂-isoprostane) and antioxidant status was evaluated by measuring α- and γ-tocopherol in serum samples. Pregnancy was associated with successively increased levels of 8-iso-PGF_2α with advancing gestational age. The median post-partum value corresponded to the values observed in early gestation and a significant decrease was observed from late pregnancy to the post-partum period. Lipid-adjusted α- and γ-tocopherol levels decreased with advancing gestational age. This longitudinal study suggests that mild oxidative stress is involved in normal human pregnancy.     

Proteomic analysis of human substantia nigra identifies novel candidates involved in Parkinson's disease pathogenesis

Parkinson's disease (PD) pathology spreads throughout the brain following a region‐specific process predominantly affecting the substantia nigra (SN) pars compacta. SN exhibits a progressive loss of dopaminergic neurons responsible for the major cardinal motor symptoms, along with the occurrence of Lewy bodies in the surviving neurons. To gain new insights into the underlying pathogenic mechanisms in PD, we studied postmortem nigral tissues dissected from pathologically confirmed PD cases (n = 5) and neurologically intact controls (n = 8). Using a high‐throughput shotgun proteomic strategy, we simultaneously identified 1795 proteins with concomitant quantitative data. To date, this represents the most extensive catalog of nigral proteins. Of them, 204 proteins displayed significant expression level changes in PD patients versus controls. These were involved in novel or known pathogenic processes including mitochondrial dysfunction, oxidative stress, or cytoskeleton impairment. We further characterized four candidates that might be relevant to PD pathogenesis. We confirmed the differential expression of ferritin‐L and seipin by Western blot and demonstrated the neuronal localization of gamma glutamyl hydrolase and nebulette by immunohistochemistry. Our preliminary findings suggest a role for nebulette overexpression in PD neurodegeneration, through mechanisms that may involve cytoskeleton dynamics disruption. All MS data have been deposited in the ProteomeXchange with identifier PXD000427 ( http://proteomecentral.proteomexchange.org/dataset/PXD000427 ).     

Higher plasma levels of F2-isoprostanes are associated with slower psychomotor speed in healthy older adults

Oxidative stress has been identified as a process which is detrimental to brain health, and associated with age-related cognitive declines. Few studies to-date have examined the relationship between in vivo oxidative stress biomarkers and cognitive performance within healthy elderly populations. The current study investigated the relationship between reaction time and oxidative stress, as measured by blood plasma concentrations of F₂-isoprostanes using a sample of 251 healthy, non-demented, elderly volunteers (Male; 111: Female 140) aged 60–75 years from the Australian Research Council Longevity Intervention (ARCLI) study cohort. A Jensen Box was used in conjunction with the Hick paradigm in order to differentiate simple from choice reaction time (two, four and eight-choice conditions) as well as movement (MT) and decision times (DT). MT, but not DT, was found to be significantly slower for participants in the high F₂-isoprostane group compared to the low F₂-isoprostane group, across all stimulus choices. F₂-isoprostanes, age and Wechsler Abbreviated Scale of Intelligence (WASI) full scale intelligence quotient (IQ) were found to be significant predictors of average MT in the sample as a whole. These findings provide preliminary evidence to suggest that higher levels of oxidative stress may be associated with impaired psychomotor speed in the healthy elderly population.     

Biomarkers of oxidative stress in overweight men are not influenced by a combination of antioxidants

Abstract

The effect of antioxidant supplementation on biomarkers of oxidative stress was investigated in a 6-week intervention study in 60 overweight men. The supplement contained a combination of antioxidants aiming to correspond to the antioxidant content found in a diet rich in fruit and vegetables. Placebo, single or double dose of antioxidants was provided to the subjects. Metabolic variables, plasma antioxidants and biomarkers of oxidative stress (lipid peroxidation and DNA damage) were measured. No effect of supplementation on biomarkers of oxidative stress was observed. Both intervention groups showed substantial increases of plasma antioxidants. This study demonstrated that supplementation with a combination of antioxidants did not affect lipid peroxidation and DNA damage in overweight men, despite increased concentrations of plasma antioxidants. The absence of antioxidant supplement effect might possibly be explained by the chosen study group having a normal level of oxidative stress, duration of the intervention and/or doses of antioxidants.     

LC/MS analysis of cardiolipins in substantia nigra and plasma of rotenone-treated rats: Implication for mitochondrial dysfunction in Parkinson's disease

Abstract

Exposure to rotenone in vivo results in selective degeneration of dopaminergic neurons and development of neuropathologic features of Parkinson's disease (PD). As rotenone acts as an inhibitor of mitochondrial respiratory complex I, we employed oxidative lipidomics to assess oxidative metabolism of a mitochondria-specific phospholipid, cardiolipin (CL), in substantia nigra (SN) of exposed animals. We found a significant reduction in oxidizable polyunsaturated fatty acid (PUFA)-containing CL molecular species. We further revealed increased contents of mono-oxygenated CL species at late stages of the exposure. Notably, linoleic acid in sn-1 position was the major oxidation substrate yielding its mono-hydroxy- and epoxy-derivatives whereas more readily “oxidizable” fatty acid residues (arachidonic and docosahexaenoic acids) remained non-oxidized. Elevated levels of PUFA CLs were detected in plasma of rats exposed to rotenone. Characterization of oxidatively modified CL molecular species in SN and detection of PUFA-containing CL species in plasma may contribute to better understanding of the PD pathogenesis and lead to the development of new biomarkers of mitochondrial dysfunction associated with this disease.     

Neuromelanin associated redox-active iron is increased in the substantia nigra of patients with Parkinson's disease

Degeneration of dopaminergic neurones during Parkinson's disease is most extensive in the subpopulation of melanized-neurones located in the substantia nigra pars compacta. Neuromelanin is a dark pigment produced in the dopaminergic neurones of the human substantia nigra and has the ability to bind a variety of metal ions, especially iron. Post-mortem analyses of the human brain have established that oxidative stress and iron content are enhanced in association with neuronal death. As redox-active iron (free Fe2+ form) and other transition metals have the ability to generate highly reactive hydroxyl radicals by a catalytic process, we investigated the redox activity of neuromelanin (NM)-aggregates in a group of parkinsonian patients, who presented a statistically significant reduction (- 70%) in the number of melanized-neurones and an increased non-heme (Fe3+) iron content as compared with a group of matched-control subjects. The level of redox activity detected in neuromelanin-aggregates was significantly increased (+ 69%) in parkinsonian patients and was highest in patients with the most severe neuronal loss. This change was not observed in tissue in the immediate vicinity of melanized-neurones. A possible consequence of an overloading of neuromelanin with redox-active elements is an increased contribution to oxidative stress and intraneuronal damage in patients with Parkinson's disease.     

The steady-state levels of oxidative DNA damage and of lipid peroxidation (F2-isoprostanes) are not correlated in healthy human subjects

Oxidative damage to DNA in human tissues can be determined by measuring multiple products of oxidative damage to the purine and pyrimidine bases using gas chromatography-mass spectrometry (GC-MS). Oxidative damage to lipids (lipid peroxidation) can be quantitated by the mass spectrometry-based determination of F₂-isoprostanes, specific end-products of the peroxidation of arachidonic acid residues in lipids. For both DNA base damage products and 8-epi prostaglandin F_2α (PGF_2α), there is a wide variation in levels between different healthy human subjects. We measured multiple products of oxidative damage to DNA bases in white cells, and 8-epi PGF_2α in plasma, from blood samples obtained from healthy human subjects in the UK and in Portugal. No correlation of 8-epi PGF_2α levels with levels of any modified DNA base (including 8-hydroxyguanine) was observed. We conclude that no single parameter can be measured as an index of “oxidative stress” or “oxidative damage” in vivo.     

Using chemiluminescence to determine whole blood antioxidant capacity in rheumatoid arthritis and Parkinson's disease patients

The consequences of oxidative stress and inflammation are implicated in a wide range of diseases, including rheumatoid arthritis and Parkinson's disease. The status of antioxidant capacity in rheumatoid arthritis and Parkinson's disease remains unclear, in part due to common practice of assaying erythrocytes separately to plasma. This method removes any synergistic interactions between plasma and erythrocyte‐based antioxidants. The experiments in this report tested antioxidant capacity in whole blood, erythrocytes and plasma by group and disease stage. Medically diagnosed patients were recruited along with appropriate control group participants. Fasting venous blood was assayed using chemiluminescence methods for: time to maximum light emitted, maximum light emitted, and plasma antioxidant capacity in vitamin E analogue units. Here we demonstrate that whole blood exhibits higher antioxidant capacity than either plasma or erythrocytes assayed separately. We report increased oxidative stress in the blood of rheumatoid arthritis patients by group (p = 0.018, p = 0.049). We show increased antioxidant capacity in Parkinson's disease patients by group (p < 0.001). For later stage Parkinson's disease patients, we report reduced oxidative stress (p = 0.025), and increased antioxidant capacity and for erythrocytes (p < 0.001, p = 0.004) and whole blood (p < 0.001, p = 0.003). Early stage Parkinson's disease showed higher antioxidant capacity on only one measure (p = 0.008). Whole blood chemiluminescence is a useful technique for determining redox status in disease and might help clarify the role of oxidative stress in rheumatoid arthritis and Parkinson's disease.     

Neuroprotective effects of ginkgetin against neuroinjury in Parkinson's disease model induced by MPTP via chelating iron

Abstract

Disruption of neuronal iron homeostasis and oxidative stress are closely related to the pathogenesis of Parkinson's disease (PD). Ginkgetin, a natural biflavonoid isolated from leaves of Ginkgo biloba L, has many known effects, including anti-inflammatory, anti-influenza virus, and anti-fungal activities, but its underlying mechanism of the neuroprotective effects in PD remains unclear. The present study utilized PD models induced by 1-methyl-4-phenylpyridinium (MPP⁺) and 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to explore the neuroprotective ability of ginkgetin in vivo and in vitro. Our results showed that ginkgetin could provide significant protection from MPP⁺-induced cell damage in vitro by decreasing the levels of intracellular reactive oxygen species and maintaining mitochondrial membrane potential. Meanwhile, ginkgetin dramatically inhibited cell apoptosis induced by MPP+ through the caspase-3 and Bcl2/Bax pathway. Moreover, ginkgetin significantly improved sensorimotor coordination in a mouse PD model induced by MPTP by dramatically inhibiting the decrease of tyrosine hydroxylase expression in the substantia nigra and superoxide dismutase activity in the striatum. Interestingly, ginkgetin could strongly chelate ferrous ion and thereby inhibit the increase of the intracellular labile iron pool through downregulating L-ferritin and upregulating transferrin receptor 1. These results indicate that the neuroprotective mechanism of ginkgetin against neurological injury induced by MPTP occurs via regulating iron homeostasis. Therefore, ginkgetin may provide neuroprotective therapy for PD and iron metabolism disorder related diseases.     

Identification and quantification of dolichol and dolichoic acid in neuromelanin from substantia nigra of the human brain

Neuromelanin (NM) isolated from the substantia nigra of the human brain is found to contain a series of dolichoic acids (dol-CA) containing 14-20 isoprene units. This is the first observation of dol-CA in a natural system. Using internally spiked nor-dolichol and nor-dolichoic acid standards, the concentrations of dolichol (dol) and dol-CA present in NM were determined. Remarkably, dol was only four times as abundant as dol-CA in NM. The distribution of dol-CA chains lengths in NM also differed from that of dol, suggesting that the enzyme(s) responsible for the conversion of dol to dol-CA prefer a dolichol substrate containing 19 isoprene units.     

Effects of cypermethrin on monoamine transporters,xenobiotic metabolizing enzymes and lipid peroxidation in the rat nigrostriatal system

Abstract

Long-term exposure to cypermethrin induces the nigrostriatal dopaminergic neurodegeneration in adult rats and its pre-exposure in the critical periods of brain development enhances the susceptibility during adulthood. Monoamine transporters, xenobiotic metabolizing enzymes and oxidative stress play critical roles in the nigrostriatal dopaminergic neurodegeneration. The study was undertaken to investigate the effects of cypermethrin on DAT, VMAT 2, CYP2E1, GST Ya, GST Yc and GSTA4-4 expressions, CYP2E1 and GST activities and lipid peroxidation in the nigrostriatal system of adult rats with/without post-natal exposure to cypermethrin. Cypermethrin reduced VMAT 2 and increased CYP2E1 expressions without causing significant change in DAT. Although GSTA4-4 mRNA expression and lipid peroxidation were increased, no significant changes were observed in GST Ya and GST Yc expressions and total GST activity. The results obtained demonstrate that long-term exposure to cypermethrin modulates VMAT 2, CYP2E1, GSTA4-4 expressions and lipid peroxidation, which could contribute to the nigrostriatal dopaminergic neurodegeneration.     

Membrane phospholipid peroxidation promotes loss of dopaminergic neurons in psychological stress-induced Parkinson's disease susceptibility

Parkinson's disease (PD) is a neurodegenerative disorder associated with α-synuclein aggregation and dopaminergic neuron loss in the midbrain. There is evidence that psychological stress promotes PD progression by enhancing glucocorticoids-related oxidative damage, however, the mechanisms involved are unknown. The present study demonstrated that plasma membrane phospholipid peroxides, as determined by phospholipidomics, triggered ferroptosis in dopaminergic neurons, which in turn contributed to stress exacerbated PD-like motor disorder in mice overexpressing mutant human α-synuclein. Using hormonomics, we identified that stress stimulated corticosteroid release and promoted 15-lipoxygenase-1 (ALOX15)-mediated phospholipid peroxidation. ALOX15 was upregulated by α-synuclein overexpression and acted as a fundamental risk factor in the development of chronic stress-induced parkinsonism and neurodegeneration. Further, we demonstrated the mechanism by which corticosteroids activated the PKC pathway and induced phosphatidylethanolamine-binding protein-1 (PEBP1) to form a complex with ALOX15, thereby facilitating ALOX15 to locate on the plasma membrane phospholipids. A natural product isolated from herbs, leonurine, was screened with activities of inhibiting the ALOX15/PEBP1 interaction and thereby attenuating membrane phospholipid peroxidation. Collectively, our findings demonstrate that stress increases the susceptibility of PD by driving membrane lipid peroxidation of dopaminergic neurons and suggest the ALOX15/PEBP1 complex as a potential intervention target.     

End products of lipid peroxidation in erythrocyte membranes in Alzheimer's disease

Alzheimer's disease (AD) is accompanied by oxidative stress in the brain. Because the brain tissue is rich in polyunsaturated fatty acids, it is prone to the free radical attack resulting in lipid peroxidation. Intermediates of lipid peroxidation may diffuse from the primary site, cross the blood-brain barrier and modify erythrocyte membranes in the bloodstream. We exposed isolated erythrocyte membranes from patients with AD and the control group to in vitro free radical damage and monitored the accumulation of the end products of lipid peroxidation, lipofuscin-like pigments (LFPs), by fluorescence spectroscopy. LFPs were analyzed by means of tridimensional and synchronous fluorescence spectroscopy. The levels of LFP formed during in vitro peroxidation were significantly higher in erythrocyte membranes from patients with AD compared with the control group. Furthermore, the chemical composition of LFP in AD was different from the control group. The analysis of the specific modifications of erythrocyte membranes in AD is of great medical importance regarding the need of a diagnostic blood biomarker.     

JNK2 and JNK3 combined are essential for apoptosis in dopamine neurons of the substantia nigra,but are not required for axon degeneration

Activation of c‐jun N‐terminal kinase (JNK) by the mitogen‐activated protein kinase cascade has been shown to play an important role in the death of dopamine neurons of the substantia nigra, one of the principal neuronal populations affected in Parkinson’s disease. However, it has remained unknown whether the JNK2 and JNK3 isoforms, either singly or in combination, are essential for apoptotic death, and, if so, the mechanisms involved. In addition, it has been unclear whether they play a role in axonal degeneration of these neurons in disease models. To address these issues we have examined the effect of single and double jnk2 and jnk3 null mutations on apoptosis in a highly destructive neurotoxin model, that induced by intrastriatal 6‐hydroxydopamine. We find that homozygous jnk2/3 double null mutations result in a complete abrogation of apoptosis and a prolonged survival of the entire population of dopamine neurons. In spite of this complete protection at the cell soma level, there was no protection of axons. These studies provide a striking demonstration of the distinctiveness of the mechanisms that mediate cell soma and axon degeneration, and they illustrate the need to identify and target pathways of axon degeneration in the development of neuroprotective therapeutics.