6-Hydroxydopamine (6-OHDA) induces Drp1-dependent mitochondrial fragmentation in SH-SY5Y cells

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used neurotoxin to study Parkinson's disease. Herein we studied the potential effects of 6-OHDA on mitochondrial morphology in SH-SY5Y neuroblastoma cells. By immunofluorescence and time-lapse fluorescence microscopy we demonstrated that 6-OHDA induced profound mitochondrial fragmentation in SH-SY5Y cells, an event that was similar to mitochondrial fission induced by overexpression of Fis1p, a membrane adaptor for the dynamin-related protein 1 (DLP1/Drp1). 6-OHDA failed to induce any changes in peroxisome morphology. Biochemical experiments revealed that 6-OHDA-induced mitochondrial fragmentation is an early event preceding the collapse of the mitochondrial membrane potential and cytochrome c release in SH-SY5Y cells. Silencing of DLP1/Drp1, which is involved in mitochondrial and peroxisomal fission, prevented 6-OHDA-induced fragmentation of mitochondria. Furthermore, in cells silenced for Drp1, 6-OHDA-induced cell death was reduced, indicating that a block in mitochondrial fission protects SH-SY5Y cells against 6-OHDA toxicity. Experiments in mouse embryonic fibroblasts deficient in Bax or p53 revealed that both proteins are not essential for 6-OHDA-induced mitochondrial fragmentation. Our data demonstrate for the first time an involvement of mitochondrial fragmentation and Drp1 function in 6-OHDA-induced apoptosis.     

Gallic Acid Protects 6-OHDA Induced Neurotoxicity by Attenuating Oxidative Stress in Human Dopaminergic Cell Line

Gallic acid is one of the most important polyphenolic compounds, which is considered an excellent free radical scavenger. 6-Hydroxydopamine (6-OHDA) is a neurotoxin, which has been implicated in mainly Parkinson’s disease (PD). In this study, we investigated the molecular mechanism of the neuroprotective effects of gallic acid on 6-OHDA induced apoptosis in human dopaminergic cells, SH-SY5Y. Our results showed that 6-OHDA induced cytotoxicity in SH-SY5Y cells was suppressed by pre-treatment with gallic acid. The percentage of live cells (90%) was high in the pre-treatment of gallic acid when compared with 6-OHDA alone treated cell line. Moreover, gallic acid was very effective in attenuating the disruption of mitochondrial membrane potential, elevated levels of intracellular ROS and apoptotic cell death induced by 6-OHDA. Gallic acid also lowered the ratio of the pro-apoptotic Bax protein and the anti-apoptotic Bcl-2 protein in SH-SY5Y cells. 6-OHDA exposure was up-regulated caspase-3 and Keap-1 and, down-regulated Nrf2, BDNF and p-CREB, which were sufficiently reverted by gallic acid pre-treatment. These findings indicate that gallic acid is able to protect the neuronal cells against 6-OHDA induced injury and proved that gallic acid might potentially serve as an agent for prevention of several human neurodegenerative diseases caused by oxidative stress and apoptosis.     

Neuroblastoma cells expressing the noradrenaline transporter are destroyed more selectively by 6-fluorodopamine than by 6-hydroxydopamine

6-Hydroxydopamine (6-OHDA) has been used for lesioning catecholaminergic neurons and attempted purging of neuroblastoma cells from hematopoietic stem cells in autologous bone marrow transplantation (ABMT). Neurotoxicity is mediated primarily by reactive oxygen species. In ABMT, 6-OHDA, as a purging agent, has been unsuccessful. At physiological pH it autooxidizes before targeted uptake, resulting in nonspecific cytotoxicity of nontarget cells. A catecholamine analogue, similar to 6-OHDA but with a lower rate of autooxidation enabling uptake by target cells, is thus required. Electron paramagnetic resonance spectra in this study show that 6-fluorodopamine (6-FDA) hydrolyzes slowly to 6-OHDA at physiological pH. Oxygen consumption, H(2)O(2), and quinone production are found to be intermediate between those of 6-OHDA and dopamine (DA). Relative neurotoxicity of these compounds was assessed by cell viability and DNA damage in the human neuroblastoma lines SH-SY5Y and SK-N-LO, which express and lack the noradrenaline transporter, respectively. Specific uptake of DA and 6-FDA by SH-SY5Y cells was demonstrated by competitive m-[(131)I]iodobenzylguanidine uptake inhibition. The competition by 6-OHDA was low owing to rapid autooxidation during incubation with equal toxicity toward both cell types. 6-FDA toxicity was preferential for SH-SY5Y cells and reduced in the presence of desipramine, a catecholamine uptake inhibitor. We demonstrate that 6-FDA cytotoxicity is more specific for cells expressing catecholamine reuptake systems than is 6-OHDA cytotoxicity.     

Bcl-x L blocks mitochondrial multiple conductance channel activation and inhibits 6-OHDA-induced death in SH-SY5Y cells

Apoptosis is an active process that is regulated by different signalling pathways. One of the more important organelles involved in apoptosis regulation is the mitochondrion. Electron chain transport disruption increases free radical production leading to multiple conductance channel opening, release of cytochrome c and caspase activation. This death pathway can be blocked by anti-apoptotic members of the Bcl-2 protein family that might shift redox potential to a more reduced state, preventing free radical-mediated damage. 6-Hydroxydopamine (6-OHDA) has been widely used to generate Parkinson's disease-like models. It is able to generate free radicals and to induce catecholaminergic cell death. In this paper we have used the human neuroblastoma cell line SH-SY5Y overexpressing Bcl-x(L) as a model to gain insights into the mechanisms through which Bcl-x(L) blocks 6-OHDA-induced cell death and to identify the molecular targets for this action. Herein, we present evidence supporting that the Bcl-x(L)-anti-apoptotic signal pathway seems to prevent mitochondrial multiple conductance channel opening, cytochrome c release and caspase-3 like activity following 6-OHDA treatment in the human neuroblastoma cell line SH-SY5Y.     

6-Hydroxydopamine activates the mitochondrial apoptosis pathway through p38 MAPK-mediated, p53-independent activation of Bax and PUMA

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used toxin to study Parkinson's disease. In previous work, we have demonstrated that 6-OHDA increases mitochondrial membrane permeability leading to cytochrome c release, but the precise mechanisms involved in this process remain unknown. Herein we studied the mechanism of increased mitochondrial permeability of SH-SY5Y neuroblastoma cells in response to 6-OHDA. Cytochrome c release induced by 6-OHDA occurred, in both SH-SY5Y cells and primary cultures, in the absence of mitochondrial swelling or a decrease in mitochondrial calcein fluorescence, suggesting little involvement of the mitochondrial permeability transition pore in this process. In contrast, 6-OHDA-induced cell death was associated with a significant translocation of the pro-apoptotic Bax protein from the cytosol to mitochondria and with a significant induction of the BH3-only protein PUMA. Experiments in mouse embryonic fibroblasts deficient in Bax or PUMA demonstrated a role for both proteins in 6-OHDA-induced apoptosis. Although 6-OHDA elevated both total and nuclear p53 protein levels, activation of p53 was not essential for subsequent cell death. In contrast, we found that p38 mitogen-activated protein kinase (MAPK) was activated early during 6-OHDA-induced apoptosis, and that treatment with the p38 MAPK inhibitor SKF86002 potently inhibited PUMA induction, green fluorescent protein-Bax redistribution and apoptosis in response to 6-OHDA. These data demonstrate a critical involvement of p38 MAPK, PUMA, and Bax in 6-OHDA-induced apoptosis.     

Protective effect of (+/-) isoborneol against 6-OHDA-induced apoptosis in SH-SY5Y cells.

Oxidative stress caused by dopamine (DA) may play an important role in the pathogenesis of Parkinson's disease (PD). (+/-) Isoborneol is a monoterpenoid alcohol present in the essential oils of numerous medicinal plants and is a known antioxidant. In this study, we investigated the neuroprotective effect of isoborneol against 6-hydroxydopamine (6-OHDA)-induced cell death in human neuroblastoma SH-SY5Y cells. Pretreatment of SH-SY5Y cells with isoborneol significantly reduced 6-OHDA-induced generation of reactive oxygen species (ROS) and 6-OHDA-induced increases in intracellular calcium. Furthermore, apoptosis induced by 6-OHDA was reversed by isoborneol treatment. Isoborneol protected against 6-OHDA-induced increases in caspase-3 activity and cytochrome C translocation into the cytosol from mitochondria. Isoborneol prevented 6-OHDA from decreasing the Bax/Bcl-2 ratio. We also observed that isoborneol decreased the activation of c-Jun N-terminal kinase and induced activation of protein kinase C (PKC) which had been suppressed by 6-OHDA. Our results indicate that the protective function of isoborneol is dependent upon its antioxidant potential and strongly suggest that isoborneol may be an effective treatment for neurodegenerative diseases associated with oxidative stress.     

Pyrroloquinoline Quinone is a Potent Neuroprotective Nutrient Against 6-Hydroxydopamine-Induced Neurotoxicity

Pyrroloquinoline quinone (PQQ), which is an essential nutrient, has been shown to act as an antioxidant. Reactive oxygen species (ROS) are thought to be responsible for neurotoxicity caused by the neurotoxin 6-hydroxydopamine (6-OHDA). In this study, we investigated the ability of PQQ to protect against 6-OHDA-induced neurotoxicity using human neuroblastoma SH-SY5Y. When SH-SY5Y cells were exposed to 6-OHDA in the presence of PQQ, PQQ prevented 6-OHDA-induced cell death and DNA fragmentation. Flow cytometry analysis using the ROS-sensitive fluorescence probe, dihydroethidium, revealed that PQQ reduced elevation of 6-OHDA-induced intracellular ROS. In contrast to PQQ, antioxidant vitamins, ascorbic acid and α-tocopherol, had no protective effect. Moreover, we showed that PQQ effectively scavenged superoxide, compared to the antioxidant vitamins. Therefore, our results suggest the protective effect of PQQ on 6-OHDA-induced neurotoxicity is involved, at least in part, in its function as a scavenger of ROS, especially superoxide.     

Rescue from death but not from functional impairment: caspase inhibition protects dopaminergic cells against 6-hydroxydopamine-induced apoptosis but not against the loss of their terminals

Despite the identification of several mutations in familial Parkinson's disease (PD), the underlying mechanisms of dopaminergic neuronal loss in idiopathic PD are still unknown. To study whether caspase-dependent apoptosis may play a role in the pathogenesis of PD, we examined 6-hydroxydopamine (6-OHDA) toxicity in dopaminergic SH-SY5Y cells and in embryonic dopaminergic mesencephalic cultures. 6-OHDA induced activation of caspases 3, 6 and 9, chromatin condensation and cell death in SH-SY5Y cells. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethylketone (zVAD-fmk) or adenovirally mediated ectopic expression of the X-chromosomal inhibitor of apoptosis protein (XIAP) blocked caspase activation and prevented death of SH-SY5Y cells. Similarly, zVAD-fmk provided protection from 6-OHDA-induced loss of tyrosine hydroxylase-positive neurones in mesencephalic cultures. In contrast, zVAD-fmk failed to protect mesencephalic dopaminergic neurones from 6-OHDA-induced loss of neurites and reduction of [(3)H]dopamine uptake. These data suggest that, although caspase inhibition provides protection from 6-OHDA-induced death of dopaminergic neurones, the neurones may remain functionally impaired.     

Protective effects of the thioredoxin and glutaredoxin systems in dopamine-induced cell death

Although the etiology of sporadic Parkinson disease (PD) is unknown, it is well established that oxidative stress plays an important role in the pathogenic mechanism. The thioredoxin (Trx) and glutaredoxin (Grx) systems are two central systems upholding the sulfhydryl homeostasis by reducing disulfides and mixed disulfides within the cell and thereby protecting against oxidative stress. By examining the expression of redox proteins in human postmortem PD brains, we found the levels of Trx1 and thioredoxin reductase 1 (TrxR1) to be significantly decreased. The human neuroblastoma cell line SH-SY5Y and the nematode Caenorhabditis elegans were used as model systems to explore the potential protective effects of the redox proteins against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. 6-OHDA is highly prone to oxidation, resulting in the formation of the quinone of 6-OHDA, a highly reactive species and powerful neurotoxin. Treatment of human cells with 6-OHDA resulted in an increased expression of Trx1, TrxR1, Grx1, and Grx2, and small interfering RNA for these genes significantly increased the cytotoxic effects exerted by the 6-OHDA neurotoxin. Evaluation of the dopaminergic neurons in C. elegans revealed that nematodes lacking trxr-1 were significantly more sensitive to 6-OHDA, with significantly increased neuronal degradation. Importantly, both the Trx and the Grx systems were also found to directly mediate reduction of the 6-OHDA-quinone in vitro and thus render its cytotoxic effects. In conclusion, our results suggest that the two redox systems are important for neuronal survival in dopamine-induced cell death.     

Phosphorylation of p66shc mediates 6-hydroxydopamine cytotoxicity

6-Hydroxydopamine (6-OHDA) is a neurotoxin that has been widely used to generate Parkinson's disease (PD) models. Increased oxidative stress is suggested to play an important role in 6-OHDA-induced cell death. Given the lessened susceptibility to oxidative stress exhibited by mice lacking p66shc, this study investigated the role of p66shc in the cytotoxicity of 6-OHDA. 6-OHDA induced cell death and p66shc phosphorylation at Ser36 in SH-SY5Y cells. Pre-treatment with the protein kinase C β (PKCβ) inhibitor hispidin suppressed 6-OHDA-induced p66shc phosphorylation. Elimination of H(2)O(2) by catalase reduced cell death and p66shc phosphorylation induced by 6-OHDA. Cells deficient in p66shc were more resistant to 6-OHDA-induced cell death than wild-type cells. Furthermore, reconstitution of wild-type p66shc, but not the S36A mutant, in p66shc-deficient cells increased susceptibility to 6-OHDA. These results indicate that H(2)O(2) derived from 6-OHDA is an important mediator of cell death and p66shc phosphorylation induced by 6-OHDA and that p66shc phosphorylation at Ser36 is indispensable for the cytotoxicity of 6-OHDA.     

Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells

The role of the main intracellular energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK) in the induction of autophagic response and cell death was investigated in SH-SY5Y human neuroblastoma cells exposed to the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). The induction of autophagy in SH-SY5Y cells was demonstrated by acridine orange staining of intracellular acidic vesicles, the presence of autophagosome- and autophagolysosome-like vesicles confirmed by transmission electron microscopy, as well as by microtubule-associated protein 1 light-chain 3 (LC3) conversion and p62 degradation detected by immunoblotting. 6-OHDA induced phosphorylation of AMPK and its target Raptor, followed by the dephosphorylation of the major autophagy inhibitor mammalian target of rapamycin (mTOR) and its substrate p70S6 kinase (S6K). 6-OHDA treatment failed to suppress mTOR/S6K phosphorylation and to increase LC3 conversion, p62 degradation and cytoplasmatic acidification in neuroblastoma cells in which AMPK expression was downregulated by RNA interference. Transfection of SH-SY5Y cells with AMPK or LC3β shRNA, as well as treatment with pharmacological autophagy inhibitors suppressed, while mTOR inhibitor rapamycin potentiated 6-OHDA-induced oxidative stress and apoptotic cell death. 6-OHDA induced phosphorylation of p38 mitogen-activated protein (MAP) kinase in an AMPK-dependent manner, and pharmacological inhibition of p38 MAP kinase reduced neurotoxicity, but not AMPK activation and autophagy triggered by 6-OHDA. Finally, the antioxidant N-acetyl cysteine antagonized 6-OHDA-induced activation of AMPK, p38 and autophagy. These data suggest that oxidative stress-mediated AMPK/mTOR-dependent autophagy and AMPK/p38-dependent apoptosis could be valid therapeutic targets for neuroprotection.     

Sulforaphane as an inducer of glutathione prevents oxidative stress-induced cell death in a dopaminergic-like neuroblastoma cell line

The total GSH depletion observed in the substantia nigra (SN) appears to be responsible for subsequent oxidative stress (OS), mitochondrial dysfunction, and dopaminergic cell loss in patients with Parkinson's disease. A strategy to prevent the OS of dopaminergic cells in the SN may be the use of chemopreventive agents as inducers of endogenous GSH, antioxidant and phase 2 enzymes. In this study, we demonstrated that treatment of the dopaminergic-like neuroblastoma SH-SY5Y cell line with sulforaphane (SF), a cruciferous vegetables inducer, resulted in significant increases of total GSH level, NAD(P)H : quinone oxidoreductase-1, GSH-transferase and -reductase, but not GSH-peroxidase, catalase and superoxide dismutase activities. Further, the elevation of GSH levels, GSH-transferase and NAD(P)H:quinone oxidoreductase-1 activities was correlated to an increase of the resistance of SH-SY5Y cells to toxicity induced by H₂O₂ or 6-hydroxydopamine (6-OHDA). The pre-treatment of SH-SY5Y cells with SF was also shown to prevent various apoptotic events (mitochondrial depolarization, caspase 9 and 3 activation and DNA fragmentation) and necrosis elicited by 6-OHDA. Further, the impairment of antioxidant capacity and reactive oxygen species formation at intracellular level after exposure to 6-OHDA was effectively counteracted by pre-treatment with SF. Last, both the cytoprotective and antioxidant effects of SF were abolished by the addition of buthionine sulfoximine supporting the main role of GSH in the neuroprotective effects displayed by SF. These findings show that SF may play a role in preventing Parkinson's disease.     

Discovery of novel PDE9A inhibitors with antioxidant activities for treatment of Alzheimer’s disease

Phosphodiesterase-9 (PDE9) is a promising target for treatment of Alzheimer’s disease (AD). To discover multifunctional anti-AD agents with capability of PDE9 inhibition and antioxidant activity, a series of novel pyrazolopyrimidinone derivatives, coupling with the pharmacophore of antioxidants such as ferulic and lipolic acids have been designed with the assistance of molecular docking and dynamics simulations. Twelve out of 14 synthesised compounds inhibited PDE9A with IC₅₀ below 200?nM, and showed good antioxidant capacities in the ORAC assay. Compound 1h, the most promising multifunctional anti-AD agent, had IC₅₀ of 56?nM against PDE9A and good antioxidant ability (ORAC (trolox)?=?3.3). The selectivity of 1h over other PDEs was acceptable. In addition, 1h showed no cytotoxicity to human neuroblastoma SH-SY5Y cells. The analysis on structure-activity relationship (SAR) and binding modes of the compounds may provide insight into further modification.     

Phloroglucinol Attenuates Motor Functional Deficits in an Animal Model of Parkinson's Disease by Enhancing Nrf2 Activity

In this study, we investigated whether phloroglucinol (1, 3, 5 - trihydroxybenzene) has therapeutic effects in cellular and animal model of Parkinson''s disease (PD). PD is the second most common, chronic and progressive neurodegenerative disease, and is clinically characterized with motor dysfunctions such as bradykinesia, rigidity, postural instability, gait impairment, and resting tremor. In the brains of PD patients, dopaminergic neuronal loss is observed in the Substantia nigra. Although the exact mechanisms underlying PD are largely unknown, mitochondrial dysfunction and oxidative stress are thought to be critical factors that induce the onset of the disease. Here, phloroglucinol administration was shown to attenuate motor functional deficits evaluated with rota-rod and apomorphine-induced rotation tests in 6-hydroxydopamine (6-OHDA)-induced PD animal models. Moreover, phloroglucinol ameliorated the loss of synapses as assessed with protein levels and immunoreactivity against synaptophysin in the midbrain region of the 6-OHDA-lesioned rats. In addition, in SH-SY5Y cultures, the cytotoxicity of 6-OHDA was reduced by pre-treatment with phloroglucinol. The increase in the reactive oxygen species, lipid peroxidation, protein carbonyl formation and 8-hydroxyguanine caused by treatment with 6-OHDA was attenuated by phloroglucinol in SH-SY5Y cells. Furthermore, phloroglucinol treatment rescued the reduced levels of nuclear Nrf2, antioxidant enzymes, i.e., catalase and glutathione peroxidase, in 6-OHDA-treated cells. Taken together, phloroglucinol has a therapeutic potential for treatment of PD.     

Toxicity of 6-hydroxydopamine: live cell imaging of cytoplasmic redox flux

Oxidative stress contributes to several debilitating neurodegenerative diseases. To facilitate direct monitoring of the cytoplasmic oxidation state in neuronal cells, we have developed roTurbo by including several mutations: F223R, A206K, and six of the mutations for superfolder green fluorescent protein. Thus we have generated an improved redox sensor that is much brighter in cells and oxidizes more readily than roGFP2. Cytoplasmic expression of the sensor demonstrated the temporal pattern of 6-hydroxydopamine (6-OHDA) induced oxidative stress in a neuroblastoma cell line (SH-SY5Y). Two distinct oxidation responses were identified in SH-SY5Y cells but a single response observed in cells lacking monoamine transporters (HEK293). While both cell lines exhibited a rapid transient oxidation in response to 6-OHDA, a second oxidative response coincident with cell death was observed only in SH-SY5Y cells, indicating an intracellular metabolism of 6-OHDA, and or its metabolites are involved. In contrast, exogenously applied hydrogen peroxide induced a cellular oxidative response similar to the first oxidation peak, and cell loss was minimal. Glucose deprivation enhanced the oxidative stress induced by 6-OHDA, confirming the pivotal role played by glucose in maintaining a reduced cytoplasmic environment. While these studies support previous findings that catecholamine auto-oxidation products cause oxidative stress, our findings also support studies indicating 6-OHDA induces lethal oxidative stress responses unrelated to production of hydrogen peroxide. Finally, temporal imaging revealed the sporadic nature of the toxicity induced by 6-OHDA in neuroblastoma cells.     

Neuroprotective effects of SCM198 on 6-hydroxydopamine-induced behavioral deficit in rats and cytotoxicity in neuronal SH-SY5Y cells

6-Hydroxydopamine (6-OHDA), one of the most investigated Parkinson's disease neurotoxins, is widely used to study mechanisms of cell death in dopaminergic neurons. In the present study, we demonstrated that SCM198, a new compound based on the active component of Herba leonuri, significantly reduced 6-OHDA-induced cell death in dopaminergic SH-SY5Y cells and attenuated apomorphine-elicited rotational behavior in 6-OHDA-lesioned rats. Pretreatment with SCM198 (0.001, 0.01, 0.1, 1, and 10 μM) concentration-dependently increased the cell viability as measured in MTT and LDH leakage assays compared with 6-OHDA-injured cells. Tocopherol, an antioxidant used as positive control, had similar effect at 10 μM to SCM198 1 μM. Furthermore, we assessed oxidative stress and subsequent apoptosis, the critical players in dopaminergic neurodegeneration, with 0.1, 1, and 10 μM of SCM198 in SH-SY5Y cells exposed to 6-OHDA. Pretreatment with SCM198 significantly increased antioxidant enzyme superoxide dismutase activity, ameliorated intracellular reactive oxygen species generation, prevented the dissipation of mitochondrial membrane potential, decreased apoptotic cell death in Hoechst 33258 staining, as well as down-regulated Bax and up-regulated Bcl-2 in both mRNA and protein levels compared with 6-OHDA damaged cells. Moreover, intragastrical administration of SCM198 (18 or 60 mg kg?1 day?1) for 4 weeks significantly ameliorates apomorphine-induced contralateral rotations in 6-OHDA-lesioned rats. These results support the neuroprotective effects of SCM198 against 6-OHDA-induced toxicity in vivo and in vitro with the underlying mechanisms of inhibiting oxidative stress and apoptosis. Therefore we suggest that SCM198 might provide a useful therapeutic strategy for neurodegenerative diseases such as Parkinson's disease.     

SOD3 Ameliorates Aβ<Subscript>25–35</Subscript>-Induced Oxidative Damage in SH-SY5Y Cells by Inhibiting the Mitochondrial Pathway

This study was designed to investigate the protective effects of extracellular superoxide dismutase (SOD3) against amyloid beta (Aβ_25–35)-induced damage in human neuroblastoma SH-SY5Y cells and to elucidate the mechanisms responsible for this beneficial effect. SH-SY5Y cells overexpressing SOD3 were generated by adenoviral vector-mediated infection and Aβ_25–35 was then added to the cell culture system to establish an in vitro model of oxidative stress. Cell viability, the generation of intracellular reactive oxygen species (ROS), the expression and activity of antioxidant enzymes, the levels of lipid peroxidation malondialdehyde (MDA), the expression of mitochondrial apoptosis-related genes and calcium images were examined. Following Aβ_25–35 exposure, SOD3 overexpression promoted the survival of SH-SY5Y cells, decreased the production of ROS, decreased MDA and calcium levels, and decreased cytochrome c, caspase-3, caspase-9 and Bax gene expression. Furthermore, SOD3 overexpression increased the expression and activity of antioxidant enzyme genes and Bcl-2 expression. Together, our data demonstrate that SOD3 ameliorates Aβ_25–35-induced oxidative damage in neuroblastoma SH-SY5Y cells by inhibiting the mitochondrial pathway. These data provide new insights into the functional actions of SOD3 on oxidative stress-induced cell damage.     

The mitochondria-targeted anti-oxidant MitoQ reduces aspects of mitochondrial fission in the 6-OHDA cell model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder for which available treatments provide symptom relief but do not stop disease progression. Mitochondria, and in particular mitochondrial dynamics, have been postulated as plausible pharmacological targets. Mitochondria-targeted antioxidants have been developed to prevent mitochondrial oxidative damage, and to alter the involvement of reactive oxygen species (ROS) in signaling pathways. In this study, we have dissected the effect of MitoQ, which is produced by covalent attachment of ubiquinone to a triphenylphosphonium lipophilic cation by a ten carbon alkyl chain. MitoQ was tested in an in vitro PD model which involves addition of 6-hydroxydopamine (6-OHDA) to SH-SY5Y cell cultures. At sublethal concentrations of 50 μM, 6-OHDA did not induce increases in protein carbonyl, mitochondrial lipid peroxidation or mitochondrial DNA damage. However, after 3 h of treatment, 6-OHDA disrupts the mitochondrial morphology and activates the machinery of mitochondrial fission, but not fusion. Addition of 6-OHDA did not increase the levels of fission 1, mitofusins 1 and 2 or optic atrophy 1 proteins, but does lead to the translocation of dynamin related protein 1 from the cytosol to the mitochondria. Pre-treatment with MitoQ (50 nM, 30 min) results in the inhibition of the mitochondrial translocation of Drp1. Furthermore, MitoQ also inhibited the translocation of the pro-apoptotic protein Bax to the mitochondria. These findings provide mechanistic evidence for a role for redox events contributing to mitochondrial fission and suggest the potential of mitochondria-targeted therapeutics in diseases that involve mitochondrial fragmentation due to oxidative stress.     

Neuronal transfer of the human Cu/Zn superoxide dismutase gene increases the resistance of dopaminergic neurons to 6-hydroxydopamine

Several mechanisms are thought to be involved in the progressive decline in neurons of the substantia nigra pars compacta (SNpc) that leads to Parkinson's disease (PD). Neurotoxin 6-hydroxydopamine (6-OHDA), which induces parkinsonian symptoms in experimental animals, is thought to be formed endogenously in patients with PD through dopamine (DA) oxidation and may cause dopaminergic cell death via a free radical mechanism. We therefore investigated protection against 6-OHDA by inhibiting oxidative stress using a gene transfer strategy. We overexpressed the antioxidative Cu/Zn-superoxide dismutase (SOD1) enzyme in primary culture dopaminergic cells by infection with an adenovirus carrying the human SOD1 gene (Ad-hSOD1). Survival of the dopaminergic cells exposed to 6-OHDA was 50% higher among the SOD1-producing cells than the cells infected with control adenoviruses. In contrast, no significant increased survival of (6-OHDA)-treated dopaminergic cells was observed when they were infected with an adenovirus expressing the H(2) O(2) -scavenging glutathione peroxidase (GPx) enzyme. These results underline the major contribution of superoxide in the dopaminergic cell death process induced by 6-OHDA in primary cultures. Overall, this study demonstrates that the survival of the dopaminergic neurons can be highly increased by the adenoviral gene transfer of SOD1. An antioxidant gene transfer strategy using viral vectors expressing SOD1 is therefore potentially beneficial for protecting dopaminergic neurons in PD.