cytochrome-c-t and Parkinson-Disease

Topics: Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Caspase 9; Cytochromes c; Galectin 3; Humans; Liver Neoplasms; Membrane Proteins; NF-kappa B; Parkinson Disease; Signal Transduction

Mitochondrial dysfunction has been implicated in Parkinson's disease. Mic60 is a critical component of mitochondrial crista remodeling and participates in maintaining mitochondrial structure and function. This study investigated whether the carnosic acid (CA) of rosemary protects the mitochondria of SH-SY5Y cells against the neurotoxicity of 6-hydroxydopamine (6-OHDA) by regulating Mic60. Our results showed that CA pretreatment reversed the reduction in the Mic60 and citrate synthase proteins, as well as the protein induction of PKA caused by 6-OHDA. Moreover, Mic60 and PINK1 siRNAs blocked the ability of CA to lessen the release of mitochondrial cytochrome c by 6-OHDA. As shown by immunoprecipitation assay, in 6-OHDA-treated cells, the interaction of Mic60 with its phosphorylated threonine residue was decreased, but the interaction with its phosphorylated serine residue was increased. PINK1 siRNA and forskolin, a PKA activator, reversed these interactions. Moreover, forskolin pretreatment prevented CA from rescuing the interaction of PINK1 and Mic60 and the reduction in cytochrome c release and mitophagy impairment in 6-OHDA-treated cells. In conclusion, CA prevents 6-OHDA-induced cytochrome c release by regulating Mic60 phosphorylation by PINK1 through a downregulation of PKA. The regulation of Mic60 by CA can be considered as a protective mechanism for the prevention of Parkinson's disease.

Topics: Apoptosis; Cell Line, Tumor; Colforsin; Cytochromes c; Humans; Mitochondria; Mitochondrial Proteins; Neuroblastoma; Oxidopamine; Parkinson Disease; Protein Kinases; RNA, Small Interfering

Heterogeneous aggregates of the human protein α-synuclein (αSyn) are abundantly found in Lewy body inclusions of Parkinson's disease patients. While structural information on classical αSyn amyloid fibrils is available, little is known about the conformational properties of disease-relevant, non-canonical aggregates. Here, we analyze the structural and dynamic properties of megadalton-sized dityrosine adducts of αSyn that form in the presence of reactive oxygen species and cytochrome c, a proapoptotic peroxidase that is released from mitochondria during sustained oxidative stress. In contrast to canonical cross-β amyloids, these aggregates retain high degrees of internal dynamics, which enables their characterization by solution-state NMR spectroscopy. We find that intermolecular dityrosine crosslinks restrict αSyn motions only locally whereas large segments of concatenated molecules remain flexible and disordered. Indistinguishable aggregates form in crowded in vitro solutions and in complex environments of mammalian cell lysates, where relative amounts of free reactive oxygen species, rather than cytochrome c, are rate limiting. We further establish that dityrosine adducts inhibit classical amyloid formation by maintaining αSyn in its monomeric form and that they are non-cytotoxic despite retaining basic membrane-binding properties. Our results suggest that oxidative αSyn aggregation scavenges cytochrome c's activity into the formation of amorphous, high molecular-weight structures that may contribute to the structural diversity of Lewy body deposits.

Topics: alpha-Synuclein; Amyloid; Amyloid beta-Peptides; Cytochromes c; Humans; Magnetic Resonance Spectroscopy; Mitochondria; Neurons; Oxidative Stress; Parkinson Disease; Protein Aggregates; Protein Conformation; Reactive Oxygen Species; Tyrosine

Preclinical Research & Development Parkinson's disease (PD) is the second most common neurodegenerative disorder that affects approximately 10 million people worldwide. The risk of developing PD and similar neurodegenerative disorders increases with age and an estimated 4% people are diagnosed with the disease before reaching the age of 50. Oxidative stress, cytotoxicity, and mitochondrial dysfunction are common features exhibited in the development of PD. The 6-hyroxydopamine (6-OHDA) model of PD is one of the most well characterized and studied models of the disease. 6-OHDA, a neurotoxin, can induce most characteristic features of the disease, including mitochondrial dysfunction in-vivo and in-vitro. SH-SY5Y is a neuroblastoma cell line of human origin that has been used for dose response studies on PD in the past. Based on previous data, we have used SH-SY5Y cells as an in-vitro model of PD to analyse the phytomedicinal potential of perillyl alcohol (PA), a monoterpenoid obtained from essential oils of various plants such as sage, peppermint and lavender. We have found that pretreatment with PA (10 μM and 20 μM) mitigated 6-OHDA (150 μM) induced cytotoxicity in a dose-dependent manner. We observed marked restoration of cell viability and mitochondrial membrane potential (MMP) as well as reduced reactive oxygen species generation, Cytochrome c immunofluorescence and DNA fragmentation after treatment with PA. On the basis of on our data, we have come to the conclusion that PA demonstrates sufficient neuroprotective activity to provide new avenues in therapy of PD and its apparent target being restoration of MMP can lead to better understanding of the disease.

Topics: Cell Line; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Humans; Membrane Potential, Mitochondrial; Monoterpenes; Neuroprotective Agents; Oxidopamine; Parkinson Disease; Phytochemicals; Reactive Oxygen Species

Mitochondrial dysfunction and oxidative stress are the main toxic events leading to dopaminergic neuronal death in Parkinson's disease (PD) and identified as vital objective for therapeutic intercession. This study investigated the neuro-protective effects of the demethoxycurcumin (DMC), a derivative of curcumin against rotenone induced neurotoxicity.. SH-SY5Y neuroblastoma cells are divided into four experimental groups: untreated cells, cells incubated with rotenone (100 nM), cells treated with DMC (50 nM) + rotenone (100 nM) and DMC alone treated. 24 h after treatment with rotenone and 28 h after treatment with DMC, cell viability was assessed using the MTT assay, and levels of ROS and MMP, plus expression of apoptotic protein were analysed.. Rotenone induced cell death in SH-SY5Y cells was significantly reduced by DMC pretreatment in a dose-dependent manner, indicating the potent neuroprotective effects of DMC. Rotenone treatment significantly increases the levels of ROS, loss of MMP, release of Cyt-c and expression of pro-apoptotic markers and decreases the expression of anti-apoptotic markers.. Even though the results of the present study indicated that the DMC may serve as a potent therapeutic agent particularly for the treatment of neurodegenerative diseases like PD, further pre-clinical and clinical studies are required.

Topics: Cell Death; Cell Line, Tumor; Cell Survival; Curcuma; Curcumin; Cytochromes c; Diarylheptanoids; Dopaminergic Neurons; Humans; Insecticides; Membrane Potential, Mitochondrial; Neuroprotective Agents; Neurotoxicity Syndromes; Oxidative Stress; Parkinson Disease; Phytotherapy; Plant Extracts; Reactive Oxygen Species; Rotenone

Mutations in CHCHD2 have been identified in some Parkinson's disease (PD) cases. To understand the physiological and pathological roles of CHCHD2, we manipulated the expression of CHCHD2 in Drosophila and mammalian cells. The loss of CHCHD2 in Drosophila causes abnormal matrix structures and impaired oxygen respiration in mitochondria, leading to oxidative stress, dopaminergic neuron loss and motor dysfunction with age. These PD-associated phenotypes are rescued by the overexpression of the translation inhibitor 4E-BP and by the introduction of human CHCHD2 but not its PD-associated mutants. CHCHD2 is upregulated by various mitochondrial stresses, including the destabilization of mitochondrial genomes and unfolded protein stress, in Drosophila. CHCHD2 binds to cytochrome c along with a member of the Bax inhibitor-1 superfamily, MICS1, and modulated cell death signalling, suggesting that CHCHD2 dynamically regulates the functions of cytochrome c in both oxidative phosphorylation and cell death in response to mitochondrial stress.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Survival; Cytochromes c; DNA-Binding Proteins; Dopaminergic Neurons; Drosophila melanogaster; Drosophila Proteins; Electron Transport; Flight, Animal; Humans; Male; Mice; Mitochondria; Mitochondrial Proteins; Models, Biological; Muscles; Mutation; Nerve Degeneration; Oxidative Phosphorylation; Oxidative Stress; Parkinson Disease; Phenotype; Protein Binding; Protein Stability; Signal Transduction; Stress, Physiological; Transcription Factors; Ubiquitin-Protein Ligases; Up-Regulation

A key molecular event in the pathogenesis of Parkinson's disease is mitochondrial damage caused by α-synuclein (α-syn). Mitochondria mediates both necrosis and apoptosis, which are associated with morphological changes. However, the mechanism by which α-syn alters mitochondrial morphology remains unclear. To address this issue, we investigated mitochondrial permeability transition pore (mPTP) opening and changes in cardiolipin (CL) levels in mitochondria isolated from the brain of Thy1α-syn mice. Cytoplasmic cytochrome C and cleaved caspase-3 protein levels were upregulated in the brain of transgenic mice. Morphological analysis by atomic force microscopy (AFM) suggested a correlation between mitochondrial morphology and function in these animals. Incubation of isolated mitochondria with recombinant human α-synuclein N terminus (α-syn/N) decreased mitochondrial CL content. An AFM analysis showed that α-syn/N induced mitochondrial swelling and the formation of pore-like structures, which was associated with decreased mitochondrial transmembrane potential and complex I activity. The observed mitochondrial dysfunction was abrogated by treatment with the mPTP inhibitor cyclosporin A, although there was no recovery of CL content. These results provide insight into the mechanism by which α-syn/N directly undermines mitochondrial structure and function via modulation of mPTP opening and CL levels, and suggests that morphological analysis of isolated mitochondria by AFM is a useful approach for evaluating mitochondrial injury.

Topics: alpha-Synuclein; Animals; Apoptosis; Brain; Cytochromes c; Electron Transport Complex I; Humans; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Atomic Force; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Parkinson Disease

Parkinson's disease (PD) is the second most common neurodegenerative disease in humans. The hormone α-melanocyte-stimulating hormone (α-MSH) has been reported to be neuroprotective in previous studies. The aim of this study is to investigate the neuroprotective effects of α-MSH against the neurotoxicity of 1-methyl-4-phenylpyridinium (MPP+). Our results indicated that treatment with α-MSH in M17 cells attenuated MPP+-induced oxidative stress, embodied by exacerbated reactive oxygen species and protein carbonyls. In addition, we found that α-MSH could improve mitochondrial function in M17 cells through increasing the level of adenosine triphosphate and mitochondrial membrane potential. Furthermore, treatment with α-MSH restored the reduction of cell viability and the induction of lactate dehydrogenase release induced by α-MSH. Importantly, Hoechst staining results indicated that α-MSH treatment significantly reduces the number of apoptotic cells after treatment with MPP+. Mechanically, we found that α-MSH prevented apoptosis signals through reducing the level of cleaved caspase-3 and attenuating cytochrome c release. All these data imply that α-MSH produces a protective effect in PD. © 2015 IUBMB Life, 69(5):315-320, 2017.

Topics: 1-Methyl-4-phenylpyridinium; Adenosine Triphosphate; alpha-MSH; Cell Line, Tumor; Cell Survival; Cytochromes c; Humans; Membrane Potential, Mitochondrial; Neuroblastoma; Neuroprotective Agents; Neurotoxicity Syndromes; Parkinson Disease; Reactive Oxygen Species

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 1-Methyl-4-phenylpyridinium; Animals; Apoptosis; Calcium-Binding Proteins; Caspase 3; Cells, Cultured; Cytochromes c; Disease Models, Animal; DNA-Binding Proteins; Dopaminergic Neurons; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nerve Tissue Proteins; Nucleobindins; Parkinson Disease; Proto-Oncogene Proteins c-raf

Parkinson's Disease (PD) is one of the most common neurodegenerative diseases. α-Synuclein (α-Syn)-encoded by SNCA, the first-identified PD-related gene-is the main component of Lewy bodies, which are a pathological hallmark of PD. We previously reported that α-Syn accumulates in mitochondria in PD, causing mitochondrial abnormalities and disrupting mitochondrial membrane potential (Δψm) and mitochondrial potential transition pore (mPTP) opening by interacting with the voltage-dependent anion channel (VDAC) and adenine nucleotide translocator. However, the mechanistic basis of mitochondrial impairment caused by α-Syn has yet to be elucidated. It has been suggested that the amino acid residues Q62, V63, and N65 of α-Syn are important for the interaction of the protein with membranes. To investigate whether this underlies the mitochondrial dysfunction induced by α-Syn overexpression, we mutated these residues to alanine and transfected HEK293T and MN9D cells with the mutated forms of α-Syn protein. The V63A and N65A mutations prevented mitochondrial Ca(2+) overload and Δψm dysregulation as well as complex I inactivation and reactive oxygen species production while blocking mPTP opening and caspase 9 activation, possibly by reducing α-Syn accumulation in mitochondria. These results indicate that V63 and N65 are critical residues mediating mitochondrial inactivation. These findings provide novel insight into the molecular events contributing to PD pathogenesis.

Topics: alpha-Synuclein; Amino Acids; Animals; Apoptosis; Calcium; Caspase 9; Cell Survival; Cytochromes c; Electron Transport Complex I; HEK293 Cells; Humans; Membrane Potential, Mitochondrial; Mice; Mitochondria; Parkinson Disease; Reactive Oxygen Species

Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting 2% of the population aged over 65 years old. Mitochondrial defects and oxidative stress actively participate in degeneration of dopaminergic (DA) neurons in PD. Paeonolum, a main component isolated from Moutan cortex, has potent antioxidant ability. Here, we have examined the effects of paeonolum against MPP(+)-induced neurotoxicity in zebrafish and PC12 cells.. The overall viability and neurodegeneration of DA neurons was assessed in ETvmat2:green fluorescent protein (GFP) transgenic zebrafish, in which most monoaminergic neurons are labeled by GFP. Damage to PC12 cells was measured using a cell viability assay and assessment of nuclear morphology. Intracellular reactive oxygen species (ROS) and the level of total GSH were assessed. The mitochondrial cell death pathway including mitochondrial membrane potential, cytochrome C release and caspase-3 activity were also examined in PC12 cells.. Paeonolum protected against MPP(+)-induced DA neurodegeneration and locomotor dysfunction in zebrafish in a concentration-dependent manner. Similar neuroprotection was replicated in the PC12 cellular model of MPP(+) toxicity. Paeonolum attenuated MPP(+)-induced intracellular ROS accumulation and restored the level of total GSH in PC12 cells. Furthermore, paeonolum significantly inhibited the mitochondrial cell death pathway induced by MPP(+).. Collectively, the present study demonstrates that paeonolum protects zebrafish and PC12 cells against MPP(+)-induced neurotoxicity.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Antioxidants; Caspase 3; Cell Death; Cytochromes c; Dopamine; Dopaminergic Neurons; Humans; Membrane Potential, Mitochondrial; Mitochondria; MPTP Poisoning; Neuroprotective Agents; Neurotoxins; Oxidative Stress; Paeonia; Parkinson Disease; PC12 Cells; Phytotherapy; Plant Extracts; Rats; Reactive Oxygen Species; Zebrafish

Parkinson's disease (PD) is the second most common neurodegenerative disorder that mainly affects the movement of the aged populations. Lycopene is a carotenoid with unique pharmacological properties and its efficacy on experimental Hunginton's disease and brain ischemia has shown intense neuroprotective effects. The present study was aimed to explore the neuroprotective effect of lycopene against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced PD mice. Administration of lycopene (5, 10 and 20 mg/kg/day orally) protected MPTP induced depletion of striatal dopamine (DA) and its metabolites in a dose dependent manner. It also attenuated MPTP-induced oxidative stress and motor abnormalities seen in PD mice. Our western blot studies showed that treatment with lycopene reversed MPTP induced apoptosis may be due to its antioxidant and antiapoptotic properties. As to conclude, lycopene reverses neurochemical deficts, oxidative stress, apoptosis and physiological abnormalities in PD mice and offer promise strategy in the treatment of this neurodegenerative disease.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Apoptosis; Carotenoids; Caspase 3; Caspase 8; Caspase 9; Corpus Striatum; Cytochromes c; Dopamine; Hand Strength; Lycopene; Male; Mice, Inbred C57BL; Neuroprotective Agents; Oxidative Stress; Parkinson Disease; Proto-Oncogene Proteins c-bcl-2; Substantia Nigra

Parkinson's disease (PD) is a common disabling movement disorder owing to progressive depletion of dopamine in nigrostriatal region, and can be experimentally accelerated by the neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). MPTP-treated mice are a representative animal model for searching for the therapeutic agents for PD without adverse effect. In this study we investigated the effect of geraniol (GE) on chronic MPTP/probenecid (MPTP/p) induced apoptotic changes in nigrostriatal region. We observed that chronic exposure to MPTP/p led to increased expression of apoptotic markers, results in neurodegeneration and motor behavioral impairments in mice. Pretreatment with GE to MPTP/p significantly improved motor functions and ameliorated striatal antioxidant balance. In addition, GE attenuated the expression of apoptotic markers evident by the normalized Bcl-2/Bax ratio and decreased expression of cytochrome-C and caspase-9 in the substantia nigra and striatum of MPTP/p induced mice model of PD. The findings of the present study suggested that GE, a new therapeutic potential avenue may have beneficial effects in slowing or preventing the progression of PD and other neurodegenerative disorders.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Acyclic Monoterpenes; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Catalase; Cytochromes c; Glutathione Peroxidase; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Motor Activity; Parkinson Disease; Probenecid; Proto-Oncogene Proteins c-bcl-2; Random Allocation; Real-Time Polymerase Chain Reaction; RNA, Messenger; Superoxide Dismutase; Terpenes

Apoptotic stimuli induce the release of cytochrome c from the mitochondria to the cytosol, and this released cytochrome c promotes the formation of the apoptosome, which contains cytochrome c, Apaf-1 and caspase-9, resulting in the activation of caspase-9 and the promotion of apoptotic cell death. To investigate the role of the apoptosome in patients with Parkinson׳s disease (PD), we performed immunohistochemical studies on apoptosome-related proteins in formalin-fixed, paraffin-embedded sections from 8 normal subjects, 10 patients with PD and 5 patients with dementia with Lewy bodies (DLB). Furthermore, we performed double-labeling immunohistochemistry for cleaved caspase-9 and CD68 in some sections from 8 normal subjects and 10 patients with PD. In the substantia nigra and locus ceruleus from both control and PD cases, the somata and processes of melanin-containing neurons were immunostained for cytochrome c, Apaf-1 and caspase-9. In the same areas from the PD cases, brainstem-type Lewy bodies were also immunoreactive for cytochrome c, Apaf-1 and caspase-9, and cleaved caspase-9 immunoreactivity was detected in brainstem-type Lewy bodies and CD68-immunopositive microglia. In addition to brainstem-type Lewy bodies, cortical Lewy bodies were also immunoreactive for these apoptosome-related proteins in the frontal and temporal cortices from the DLB cases. Our results suggest that apoptosome formation accompanied by caspase-9 activation may occur in the substantia nigra and locus ceruleus in brains affected by PD, and that a mitochondria-dependent apoptotic pathway may be partially associated with the pathogenesis of PD.

Topics: Aged; Aged, 80 and over; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Apoptosomes; Apoptotic Protease-Activating Factor 1; Brain; Caspase 3; Caspase 9; Cytochromes c; Female; Humans; Lewy Bodies; Lewy Body Disease; Male; Middle Aged; Parkinson Disease

The aim of the present study was to explore the neuroprotective effects of Gynostemma pentaphyllum polysaccharides (GP) in a 1-methyl-4-phenylpyridiniumion (MPP(+))-induced cellular model of Parkinson's disease (PD) and the underlying mechanisms. Our results indicated that exposure of PC12 cells to 1mM MPP(+) significantly decreased the cell viability when examined by MTT assay, LDH assay, and annexin-V-FITC and propidium iodide (PI) apoptosis detection assays. MPP(+)-induced apoptosis in PC12 cells was accompanied by an increased Bax/Bcl-2 ratio, release of mitochondrial cytochrome c into the cytosol, activation of caspase-3/9 and cleavage of poly (ADP-ribose) polymerase (PARP). However, pretreatment of PC12 cells with 50μg/ml GP prior to MPP(+) exposure effectively attenuated the cytotoxicity and improved cell viability via inhibiting elevated Bax/Bcl-2 ratio, as well as the release of cytosolic cytochrome c. Furthermore, GP was effective in attenuating caspase-3/9 activation and cleavage of PARP in MPP(+)-exposed PC12 cells. These results suggest that the GP has protective effects against MPP(+)-induced neuronal apoptosis in PC12 cells by suppressing apoptosis-related protein, and therefore, might likely be a promising candidate for the treatment of Parkinson's disease (PD).

Topics: 1-Methyl-4-phenylpyridinium; Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Survival; Cytochromes c; Gene Expression Regulation; Gynostemma; L-Lactate Dehydrogenase; Neurons; Neuroprotective Agents; Parkinson Disease; PC12 Cells; Poly(ADP-ribose) Polymerases; Polysaccharides; Proteolysis; Proto-Oncogene Proteins c-bcl-2; Rats

Parkinson's disease is characterized by the progressive and selective loss of dopaminergic neurons in the substantia nigra. It has been postulated that endogenously formed CysDA (5-S-cysteinyldopamine) and its metabolites may be, in part, responsible for this selective neuronal loss, although the mechanisms by which they contribute to such neurotoxicity are not understood. Exposure of neurons in culture to CysDA caused cell injury, apparent 12-48 h post-exposure. A portion of the neuronal death induced by CysDA was preceded by a rapid uptake and intracellular oxidation of CysDA, leading to an acute and transient activation of ERK2 (extracellular-signal-regulated kinase 2) and caspase 8. The oxidation of CysDA also induced the activation of apoptosis signal-regulating kinase 1 via its de-phosphorylation at Ser967, the phosphorylation of JNK (c-Jun N-terminal kinase) and c-Jun (Ser73) as well as the activation of p38, caspase 3, caspase 8, caspase 7 and caspase 9. Concurrently, the inhibition of complex I by the dihydrobenzothiazine DHBT-1 [7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid], formed from the intracellular oxidation of CysDA, induces complex I inhibition and the subsequent release of cytochrome c which further potentiates pro-apoptotic mechanisms. Our data suggest a novel comprehensive mechanism for CysDA that may hold relevance for the selective neuronal loss observed in Parkinson's disease.

Topics: Animals; Apoptosis; Caspases; Cells, Cultured; Cytochromes c; Dopamine; Electron Transport Complex I; MAP Kinase Kinase Kinase 5; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinase 9; Nerve Tissue Proteins; Neurons; Parkinson Disease

Rotenone a widely used pesticide that inhibits mitochondrial complex I has been used to investigate the pathobiology of PD both in vitro and in vivo. Studies have shown that the neurotoxicity of rotenone may be related to its ability to generate reactive oxygen species (ROS), leading to neuronal apoptosis. The current study was carried out to investigate the neuroprotective effects of hesperidin, a citrus fruit flavanol, against rotenone-induced apoptosis in human neuroblastoma SK-N-SH cells. We assessed cell death, mitochondrial membrane potential, ROS generation, ATP levels, thiobarbituric acid reactive substances, reduced glutathione (GSH) levels, and the activity of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) using well established assays. Apoptosis was determined in normal, rotenone, and hesperidin treated cells, by measuring the protein expression of cytochrome c (cyt c), caspases 3 and 9, Bax, and Bcl-2 using the standard western blotting technique. The apoptosis in rotenone-induced SK-N-SH cells was accompanied by the loss of mitochondrial membrane potential, increased ROS generation, the depletion of GSH, enhanced activities of enzymatic antioxidants, upregulation of Bax, cyt c, and caspases 3 and 9, and downregulation of Bcl-2, which were attenuated in the presence of hesperidin. Our data suggests that hesperidin exerts its neuroprotective effect against rotenone due to its antioxidant, maintenance of mitochondrial function, and antiapoptotic properties in a neuroblastoma cell line.

Topics: Adenosine Triphosphate; Apoptosis; bcl-2-Associated X Protein; Biomarkers; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Hesperidin; Humans; Membrane Potential, Mitochondrial; Models, Biological; Neuroprotective Agents; Oxidative Stress; Parkinson Disease; Reactive Oxygen Species; Rotenone

Dangguijakyak-san protects dopamine neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity under postmenopausal conditions.. Dangguijakyak-san (DJS), a famous traditional herbal formula, has long been used to treat gynecological disorders, including postmenopausal symptoms. This study evaluated the effects and mechanism of DJS on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in a postmenopausal mouse model induced by ovariectomy.. Three weeks after ovariectomy, C57bl/6 female mice were divided randomly into (1) control, (2) MPTP (30 mg/kg/day, i.p., 5 days), (3) MPTP+estrogen (50 μg/kg/day, i.p., 5 days), and (4) MPTP+DJS (50 mg/kg/day, p.o., 5 days) groups. We investigated the behavioral recovery and dopamine neuron protection of DJS using the pole test and tyrosine hydroxylase (TH) immunohistochemistry. We also explored the mechanism by assessing the protein expression of Bax, Bcl-2, cytochrome c, and cleaved caspase-3.. DJS treatment restored the movement behavior impaired by MPTP, showing a similar or better effect than estrogen. DJS protected TH-immunoreactive cells and fibers in the nigrostriatal region from MPTP toxicity. In addition, DJS inhibited the Bcl-2 decrease and Bax increase in mitochondria, cytochrome c release to the cytosol, and caspase-3 activation induced by MPTP.. DJS showed behavior recovery and dopamine neuron protection against MPTP-induced toxicity via anti-apoptotic activities in ovariectomized female mice. These results suggest that DJS treatment is effective for postmenopausal neurodegenerative diseases.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; bcl-2-Associated X Protein; Behavior, Animal; Brain; Caspase 3; Cytochromes c; Disease Models, Animal; Dopaminergic Neurons; Drugs, Chinese Herbal; Estrogens; Female; Mice; Mice, Inbred C57BL; Mitochondria; Movement; MPTP Poisoning; Neuroprotective Agents; Ovariectomy; Parkinson Disease; Phytotherapy; Poria; Postmenopause; Proto-Oncogene Proteins c-bcl-2; Random Allocation; T-Lymphocytes, Helper-Inducer

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. The pathogenesis of PD remains incompletely understood, but it appears to involve both genetic susceptibility and environmental factors. Treatment for PD that prevents neuronal death in the dopaminergic system and abnormal protein deposition in the brain is not yet available. Evidence from human and animal studies has suggested that oxidative damage critically contributes to neuronal loss in PD. Here we test whether curcumin, a potent antioxidant compound, derived from the curry spice turmeric, can protect against mutant A53T α-synuclein-induced cell death. We used PC12 cells that inducibly express A53T α-synuclein. We found that curcumin protected against A53T α-synuclein-induced cell death in a dose-dependent manner. We further found that curcumin can reduce mutant α- synuclein-induced intracellular reactive oxygen species (ROS) levels, mitochondrial depolarization, cytochrome c release, and caspase-9 and caspase-3 activation. This study demonstrate that curcumin protected against A53T mutant α-synuclein-induced cell death via inhibition of oxidative stress and the mitochondrial cell death pathway, suggesting that curcumin may be a candidate neuroprotective agent for A53T α-synuclein-linked Parkinsonism, and possibly for other genetic or sporadic forms of PD.

Topics: alpha-Synuclein; Animals; Antioxidants; Caspase 3; Caspase 9; Cell Death; Curcuma; Curcumin; Cytochromes c; Humans; Mitochondria; Mutation; Neuroprotective Agents; Parkinson Disease; PC12 Cells; Rats; Reactive Oxygen Species

The present study investigates the protective effects of water extract of ginseng (Panax ginseng C.A. Meyer) against 1-methyl-4-phenylpyridinium ion (MPP(+))-induced cytotoxicity in SH-SY5Y human neuroblastoma cells and explores the underlying mechanisms. The approach may be used for screening therapeutic agents for degenerative disorders such as Parkinson's disease.. SH-SY5Y human neuroblastoma cells were used to analyze the protective effects of water extract of ginseng (WEG) against multiple parameters such as MPP(+)-induced viability, oxidative injury, expression of Bax, Bcl-2, cytochrome c and cleaved caspase-3.. WEG exerted inhibitory effect on cell death, overproduction of ROS, elevated Bax/Bcl-2 ratio, release of cytochrome c and activation of caspase-3 expression in MPP(+)-treated SH-SY5Y cells.. WEG exhibited significant protective effects against MPP(+)-induced cytotoxicity in SH-SY5Y cells possibly through the suppression of ROS generation and the inhibition of mitochondria-dependent apoptotic pathway.

Topics: 1-Methyl-4-phenylpyridinium; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cytochromes c; Drug Evaluation, Preclinical; Enzyme Inhibitors; Herbicides; Humans; Mitochondria; Neuroblastoma; Neurotoxins; Panax; Parkinson Disease; Phytotherapy; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

Except for its redox properties, cytochrome c is an inert protein. However, dissociation of the bond between methionine-80 and the heme iron converts the cytochrome into a peroxidase. Dissociation is accomplished by subjecting the cytochrome to various conditions, including proteolysis and hydrogen peroxide (H(2)O(2))-mediated oxidation. In affected cells of various neurological diseases, including Parkinson's disease, cytochrome c is released from the mitochondrial membrane and enters the cytosol. In the cytosol cytochrome c is exposed to cellular proteases and to H(2)O(2) produced by dysfunctional mitochondria and activated microglial cells. These could promote the formation of the peroxidase form of cytochrome c. In this study we investigated the catalytic and cytolytic properties of the peroxidase form of cytochrome c. These properties are qualitatively similar to those of other heme-containing peroxidases. Dopamine as well as sulfhydryl group-containing metabolites, including reduced glutathione and coenzyme A, are readily oxidized in the presence of H(2)O(2). This peroxidase also has cytolytic properties similar to myeloperoxidase, lactoperoxidase, and horseradish peroxidase. Cytolysis is inhibited by various reducing agents, including dopamine. Our data show that the peroxidase form of cytochrome c has catalytic and cytolytic properties that could account for at least some of the damage that leads to neuronal death in the parkinsonian brain.

Topics: Antioxidants; Cytochromes c; Dopamine; Humans; Parkinson Disease; Peroxidases

Bax is translocated into the mitochondrial membrane and oligomerized therein to initiate mitochondrial apoptotic signaling. Our previous study indicated that reactive oxygen species (ROS)-mediated activation of mitogen-activated protein kinase (MAPK) and caspase is critically involved in 6-hydroxydopamine (6-OHDA)-mediated neurodegeneration. Here, we specifically attempted to examine whether and how these death signaling pathways may be linked to Bax translocation and oligomerization. We found that 6-OHDA treatment triggered translocation and oligomerization of Bax onto the mitochondria in MN9D dopaminergic neuronal cells. These events preceded cytochrome c release into the cytosol. Cross-linking assay revealed that co-treatment with a ROS scavenger or a pan-caspase inhibitor inhibited 6-OHDA-induced Bax oligomerization. Among several candidates of ROS-activated MAPKs and caspases, we found that co-treatment with PD169316 or VDVAD specifically inhibited 6-OHDA-induced Bax oligomerization, suggesting critical involvement of p38 MAPK and caspase-2. Consequently, overexpression of a dominant negative form of p38 MAPK or a shRNA-mediated knockdown of caspase-2 indeed inhibited 6-OHDA-induced Bax oligomerization. However, activation of p38 MAPK and caspase-2 was independently linked to oligomerization of Bax. This specificity was largely confirmed with a Bax 6A7 antibody known to detect activated forms of Bax on the mitochondria. Taken together, our data suggest that there is an independent amplification loop of Bax translocation and oligomerization via caspase-2 and p38 MAPK during ROS-mediated dopaminergic neurodegeneration.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 2; Caspase Inhibitors; Cell Line; Cytochromes c; Dopaminergic Neurons; Enzyme Activation; Enzyme Inhibitors; Imidazoles; Mice; Mitochondria; Oxidopamine; p38 Mitogen-Activated Protein Kinases; Parkinson Disease; Phosphorylation; Polymerization; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction

Parkinson disease (PD), a prevalent neurodegenerative motor disorder, is characterized by the rather selective loss of dopaminergic neurons and the presence of α-synuclein-enriched Lewy body inclusions in the substantia nigra of the midbrain. Although the etiology of PD remains incompletely understood, emerging evidence suggests that dysregulated iron homeostasis may be involved. Notably, nigral dopaminergic neurons are enriched in iron, the uptake of which is facilitated by the divalent metal ion transporter DMT1. To clarify the role of iron in PD, we generated SH-SY5Y cells stably expressing DMT1 either singly or in combination with wild type or mutant α-synuclein. We found that DMT1 overexpression dramatically enhances Fe(2+) uptake, which concomitantly promotes cell death. This Fe(2+)-mediated toxicity is aggravated by the presence of mutant α-synuclein expression, resulting in increased oxidative stress and DNA damage. Curiously, Fe(2+)-mediated cell death does not appear to involve apoptosis. Instead, the phenomenon seems to occur as a result of excessive autophagic activity. Accordingly, pharmacological inhibition of autophagy reverses cell death mediated by Fe(2+) overloading. Taken together, our results suggest a role for iron in PD pathogenesis and provide a mechanism underlying Fe(2+)-mediated cell death.

Topics: alpha-Synuclein; Apoptosis; Autophagy; Cation Transport Proteins; Cell Line; Cytochromes c; Humans; Iron; Mutant Proteins; Neurons; Oxidative Stress; Parkinson Disease; Phagosomes; Proteasome Endopeptidase Complex; Ubiquitin

To study the relationship between oxidative DNA damage and mitochondrial apoptosis relative proteins in rat striatum induced by 6-hydroxydopamine (6-OHDA) during the pathogenesis of Parkinson's disease (PD).. 6-OHDA was stereotactically injected into the bilateral right striatum of rats to produce PD models. Assays for 8-oxo-dG immunohistochemistry and Western blot for MTH1, Cytochrome c, Cl-caspase 9 and Cl-caspase 3 in right striatum was separately conducted.. In 10 successful PD rats, compared with either sham or normal group, there were obvious more 8-oxo-dG positive cells in lesioned striatum while there was a lower expression of MTH1. Furthermore, the expressions of such intrinsic apoptotic pathway factors as cytoplasmic Cytochrome c, Cl-caspase 9 and Cl-caspase 3 were highly up-regulated in lesioned striatum.. Oxidative DNA damage plays a key role in the pathogenesis of PD. Furthermore Cytochrome c, caspase 9 and caspase 3 are involved in the regulation of apoptosis under oxidative DNA damage induced by 6-OHDA.

Topics: Animals; Apoptosis; Caspase 3; Caspase 9; Corpus Striatum; Cytochromes c; DNA Damage; Male; Mitochondrial Proteins; Oxidopamine; Parkinson Disease; Rats; Rats, Sprague-Dawley

Previously, we suggested that tetrahydrobiopterin (BH4), an obligatory cofactor for dopamine synthesis, as an intrinsic contributor to dopaminergic neuron vulnerability. The BH4 toxicity is observed in dopamine-producing cells, including Cath.a cells, but not in non-dopaminergic cells. Furthermore, the dopaminergic cell death induced by BH4 is apoptotic in nature and involves oxidative stress, similar to that observed in Parkinson's disease. Accordingly, various antioxidants have been found to protect dopaminergic cells from BH4. This study was undertaken to evaluate protective effects of the dopamine receptor agonist bromocriptine on BH4-induced Cath.a cell death, because bromocriptine has been reported to be an antioxidant with a neuroprotective activity. In the presence of bromocriptine, the increase in LDH activity and mitochondrial cytochrome c release induced by BH4 were significantly abolished. This cytoprotective effect was phosphatidylinositol 3-kinase (PI3K)/Akt pathway-dependent. In addition, bromocriptine was found to up-regulate the expressions of nuclear factor-E2-related factor-2 and antioxidant enzymes including NAD(P)H quinone oxidoreductase 1. Our findings show that bromocriptine stimulates antioxidant defense mechanisms in Cath.a cells and suggest a potential use of bromocriptine as a neuroprotectant.

Topics: Animals; Antioxidants; Biopterins; Bromocriptine; Cell Death; Cell Line; Cytochromes c; Dopamine; Dopamine Agonists; Enzymes; L-Lactate Dehydrogenase; Mice; Neurons; Neuroprotective Agents; NF-E2-Related Factor 2; Oxidative Stress; Parkinson Disease; Phosphatidylinositol 3-Kinases; Receptors, Dopamine; Signal Transduction; Up-Regulation

Damage of presynaptic mitochondria could result in release of proapoptotic factors that threaten the integrity of the entire neuron. We discovered that alpha-synuclein (Syn) forms a triple complex with anionic lipids (such as cardiolipin) and cytochrome c, which exerts a peroxidase activity. The latter catalyzes covalent hetero-oligomerization of Syn with cytochrome c into high molecular weight aggregates. Syn is a preferred substrate of this reaction and is oxidized more readily than cardiolipin, dopamine, and other phenolic substrates. Co-localization of Syn with cytochrome c was detected in aggregates formed upon proapoptotic stimulation of SH-SY5Y and HeLa cells and in dopaminergic substantia nigra neurons of rotenone-treated rats. Syn-cardiolipin exerted protection against cytochrome c-induced caspase-3 activation in a cell-free system, particularly in the presence of H(2)O(2). Direct delivery of Syn into mouse embryonic cells conferred resistance to proapoptotic caspase-3 activation. Conversely, small interfering RNA depletion of Syn in HeLa cells made them more sensitive to dopamine-induced apoptosis. In human Parkinson disease substantia nigra neurons, two-thirds of co-localized Syn-cytochrome c complexes occurred in Lewy neurites. Taken together, these results indicate that Syn may prevent execution of apoptosis in neurons through covalent hetero-oligomerization of cytochrome c. This immediate protective function of Syn is associated with the formation of the peroxidase complex representing a source of oxidative stress and postponed damage.

Topics: Animals; Apoptosis; Cardiolipins; Cell Line, Tumor; Cloning, Molecular; Cross-Linking Reagents; Cytochromes c; HeLa Cells; Humans; Lipids; Mice; Neuroblastoma; Neurons; Oxidative Stress; Parkinson Disease; Peroxidases; RNA, Small Interfering; Synucleins

Environmental exposure to neurotoxic metals through various sources including exposure to welding fumes has been linked to an increased incidence of Parkinson's disease (PD). Welding fumes contain many different metals including vanadium typically present as particulates containing vanadium pentoxide (V2O5). However, possible neurotoxic effects of this metal oxide on dopaminergic neuronal cells are not well studied. In the present study, we characterized vanadium-induced oxidative stress-dependent cellular events in cell culture models of PD. V2O5 was neurotoxic to dopaminergic neuronal cells including primary nigral dopaminergic neurons and the EC50 was determined to be 37 microM in N27 dopaminergic neuronal cell model. The neurotoxic effect was accompanied by a time-dependent uptake of vanadium and upregulation of metal transporter proteins Tf and DMT1 in N27 cells. Additionally, vanadium resulted in a threefold increase in reactive oxygen species generation, followed by release of mitochondrial cytochrome c into cytoplasm and subsequent activation of caspase-9 (>fourfold) and caspase-3 (>ninefold). Interestingly, vanadium exposure induced proteolytic cleavage of native protein kinase Cdelta (PKCdelta, 72-74 kDa) to yield a 41 kDa catalytically active fragment resulting in a persistent increase in PKCdelta kinase activity. Co-treatment with pan-caspase inhibitor Z-VAD-FMK significantly blocked vanadium-induced PKCdelta proteolytic activation, indicating that caspases mediate PKCdelta cleavage. Also, co-treatment with Z-VAD-FMK almost completely inhibited V2O5-induced DNA fragmentation. Furthermore, PKCdelta knockdown using siRNA protected N27 cells from V2O5-induced apoptotic cell death. Collectively, these results demonstrate that vanadium can exert neurotoxic effects in dopaminergic neuronal cells via caspase-3-dependent PKCdelta cleavage, suggesting that metal exposure may promote nigral dopaminergic degeneration.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspase 9; Cation Transport Proteins; Cell Line; Cell Survival; Cysteine Proteinase Inhibitors; Cytochromes c; DNA Fragmentation; Dopamine; Dose-Response Relationship, Drug; Environmental Pollutants; Inhibitory Concentration 50; Mesencephalon; Mitochondria; Neurons; Neurotoxicity Syndromes; Oxidative Stress; Parkinson Disease; Protein Kinase C-delta; Rats; Reactive Oxygen Species; RNA Interference; Signal Transduction; Time Factors; Transferrin; Vanadium Compounds

Autosomal-recessive mutations in the Parkin gene are the second most common cause of familial Parkinson's disease (PD). Parkin deficiency leads to the premature demise of the catecholaminergic neurons of the ventral midbrain in familial PD. Thus, a better understanding of parkin function may elucidate molecular aspects of their selective vulnerability in idiopathic PD. Numerous lines of evidence suggest a mitochondrial function for parkin and a protective effect of ectopic parkin expression. Since mitochondria play a critical role in cell survival/cell death through regulated cytochrome c release and control of apoptosis, we sought direct evidence of parkin function in this pathway. Mitochondria were isolated from cells expressing either excess levels of human parkin or shRNA directed against endogenous parkin and then treated with peptides corresponding to the active Bcl-2 homology 3 (BH3) domains of pro-apoptotic proteins and the threshold for cytochrome c release was analyzed. Data obtained from both rodent and human neuroblastoma cell lines showed that the expression levels of parkin were inversely correlated with cytochrome c release. Parkin was found associated with isolated mitochondria, but its binding per se was not sufficient to inhibit cytochrome c release. In addition, pathogenic parkin mutants failed to influence cytochrome c release. Furthermore, PINK1 expression had no effect on cytochrome c release, suggesting a divergent function for this autosomal recessive PD-linked gene. In summary, these data demonstrate a specific autonomous effect of parkin on mitochondrial mechanisms governing cytochrome c release and apoptosis, which may be relevant to the selective vulnerability of certain neuronal populations in PD.

Topics: Animals; Cell Line; Cytochromes c; Guinea Pigs; Humans; Mice; Mitochondria; Parkinson Disease; Ubiquitin-Protein Ligases

In previous work, we investigated dieldrin cytotoxicity and signaling cell death mechanisms in dopaminergic PC12 cells. Dieldrin has been reported to be one of the environmental factors correlated with Parkinson's disease and may selectively destroy dopaminergic neurons.. Here we further investigated dieldrin toxicity in a dopaminergic neuronal cell model of Parkinson's disease, namely N27 cells, using biochemical, immunochemical, and flow cytometric analyses.. In this study, dieldrin-treated N27 cells underwent a rapid and significant increase in reactive oxygen species followed by cytochrome c release into cytosol. The cytosolic cytochrome c activated caspase-dependent apoptotic pathway and the increased caspase-3 activity was observed following a 3 hr dieldrin exposure in a dose-dependent manner. Furthermore, dieldrin caused the caspase-dependent proteolytic cleavage of protein kinase C delta (PKCδ) into 41 kDa catalytic and 38 kDa regulatory subunits in N27 cells as well as in brain slices. PKCδ plays a critical role in executing the apoptotic process in dieldrin-treated dopaminergic neuronal cells because pretreatment with the PKCδ inhibitor rottlerin, or transfection and over-expression of catalytically inactive PKCδ(K)³⁷⁶(R), significantly attenuates dieldrin-induced DNA fragmentation and chromatin condensation.. Together, we conclude that caspase-3-dependent proteolytic activation of PKCδ is a critical event in dieldrin-induced apoptotic cell death in dopaminergic neuronal cells.

Topics: Animals; Apoptosis; Biocatalysis; Brain; Caspase 3; Caspase Inhibitors; Cell Nucleus; Cytochromes c; Dieldrin; DNA Fragmentation; Environmental Pollutants; Enzyme Activation; Male; Mitochondria; Mutant Proteins; Nerve Degeneration; Neurotoxins; Parkinson Disease; Protein Kinase C-delta; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

Oxidative stress and aggregation of the protein alpha-synuclein are thought to be key factors in Parkinson's disease. Previous work shows that cytochrome c with H(2)O(2) causes tyrosine-dependent in vitro peroxidative aggregation of proteins, including alpha-synuclein. Here, we examine the role of each of alpha-synuclein's four tyrosine residues and how the protein's conformation affects covalent oxidative aggregation. When alpha-synuclein adopts a collapsed conformation, tyrosine 39 is essential for wild-type-like covalent aggregation. This lone N-terminal tyrosine, however, is not required for wild-type-like covalent aggregation in the presence of a denaturant or when alpha-synuclein is present in noncovalent fibrils. We also show that preformed oxidative aggregates are not incorporated into noncovalent fibrils. These data provide insight into how dityrosine may be formed in Lewy bodies seen in Parkinson's disease.

Topics: alpha-Synuclein; Cytochromes c; Humans; Hydrogen Peroxide; Molecular Conformation; Mutation; Oxidative Stress; Oxygen; Parkinson Disease; Protein Binding; Protein Denaturation; Protein Structure, Tertiary; Recombinant Proteins; Tyrosine

In the present study mitochondrial respiratory function of fibroblasts from a patient affected by early-onset parkinsonism carrying the homozygous W437X nonsense mutation in the PINK1 gene has been thoroughly characterized. When compared with normal fibroblasts, the patient's fibroblast mitochondria exhibited a lower respiratory activity and a decreased respiratory control ratio with cellular ATP supply relying mainly on enhanced glycolytic production. The quantity, specific activity and subunit pattern of the oxidative phosphorylation complexes were normal. However, a significant decrease of the cellular cytochrome c content was observed and this correlated with a reduced cytochrome c oxidase in situ-activity. Measurement of ROS revealed in mitochondria of the patient's fibroblasts enhanced O(2)(*-) and H(2)O(2) production abrogated by inhibition of complex I. No change in the glutathione-based redox buffering was, however, observed.

Topics: Adenosine Triphosphate; Adult; Cells, Cultured; Cytochromes c; Glutathione; Humans; Immunohistochemistry; Microscopy, Confocal; Mitochondria; Mutation; Oxidative Phosphorylation; Parkinson Disease; Protein Kinases; Reactive Oxygen Species; Subcellular Fractions

Mitochondria likely play a role in Parkinson's disease (PD) neurodegeneration. We modelled PD by creating cytoplasmic hybrid (cybrid) cell lines in which endogenous mitochondrial DNA (mtDNA) from PD or control subject platelets was expressed within human teratocarcinoma (NT2) cells previously depleted of endogenous mtDNA. Complex I activity was reduced in both PD cybrid lines and in the platelet mitochondria used to generate them. Under basal conditions PD cybrids had less ATP, more LDH release, depolarized mitochondria, less mitochondrial cytochrome c, and higher caspase 3 activity. Equivalent MPP+ exposures are more likely to trigger programmed cell death in PD cybrid cells than in control cybrid cells. Our data support a relatively upstream role for mitochondrial dysfunction in idiopathic PD.

Topics: 1-Methyl-4-phenylpyridinium; Adenosine Triphosphate; Aged; Blood Platelets; Case-Control Studies; Caspase 3; Cell Line, Tumor; Cell Nucleus; Cell Survival; Citrate (si)-Synthase; Cytochromes c; DNA, Mitochondrial; Dose-Response Relationship, Drug; Electron Transport Complex IV; Enzyme Activation; Humans; Hybrid Cells; L-Lactate Dehydrogenase; Membrane Potential, Mitochondrial; Middle Aged; Mitochondria; Neurons; Parkinson Disease; Teratocarcinoma

We determined whether tetrahydrobiopterin(BH4), an endogenous cofactor for dopamine(DA) synthesis, causes preferential damage to DArgic neurons among primary cultured rat mesencephalic neurons and whether the death mechanism has relevance to Parkinson's disease (PD). DArgic neurons were more vulnerable to BH4 than non-DArgic neurons, exhibiting sensitivity at lower concentrations, evident by morphological and neurotransmitter uptake studies. BH4-exposed DArgic neurons showed (1) increased TUNEL staining and activated caspase-3 immunoreactivity, indicative of apoptotic death; (2) mitochondrial membrane potential loss and increased cytosolic cytochrome c, suggesting mitochondrial dysfunction; (3) increased level of oxidized proteins and protection by antioxidants, indicative of oxidative stress; and (4) increased ubiquitin immunoreactivity, suggesting alteration of protein degradation pattern. Percent of cells positive for these parameters were much higher for DArgic neurons, demonstrating preferential vulnerability. Therefore, the DArgic neuronal damage induced by BH4, the molecule synthesized and readily upregulated in DArgic neurons and activated microglia, suggests physiological relevance to the pathogenesis of PD.

Topics: Animals; Apoptosis; Biopterins; Caspase 3; Cell Count; Cells, Cultured; Coloring Agents; Cytochromes c; Dopamine; Dose-Response Relationship, Drug; Electrophysiology; gamma-Aminobutyric Acid; Immunohistochemistry; In Situ Nick-End Labeling; Membrane Potentials; Mesencephalon; Neurons; Oxidative Stress; Parkinson Disease; Rats

Compound FLZ is a synthetic novel derivative of squamosamide. Previous pharmacological study found that FLZ improved the abnormal behavior and the decrease of dopamine content in striatum in 6-hydroxydopamine (6-OHDA) model mice. 6-OHDA can cause Parkinsonism in experimental animals. The purpose of this paper was to study the protective action of FLZ against 6-OHDA-induced apoptosis and alternations of related signal transduction. The results indicated that FLZ at concentrations of 0.1 microM and 1 microM prevented 6-OHDA-induced apoptosis of SH-SY5Y cells, and inhibited the increase of cytochrome c and Apoptosis-inducing factor (AIF) release and activation of Caspase 3 and NF-kappaB. The results suggested that FLZ has potential neuroprotective effect on 6-OHDA-induced apoptosis of SH-SY5Y cells through regulating related signal-transduction.

Topics: Apoptosis; Apoptosis Inducing Factor; Benzeneacetamides; Caspase 3; Cell Line; Cell Survival; Cytochromes c; Drug Delivery Systems; Models, Biological; Neuroprotective Agents; NF-kappa B; Oxidopamine; Parkinson Disease; Phenols; Signal Transduction

Parkinson's disease (PD) is a neurodegenerative disorder associated with a selective loss of dopaminergic neurons in the substantia nigra. While the underlying cause of PD is not clearly understood, oxidative stress and mitochondrial dysfunction are thought to play a role. We have previously suggested tetrahydrobiopterin (BH4), an obligatory cofactor for the dopamine synthesis enzyme tyrosine hydroxylase and present selectively in monoaminergic neurons in the brain, as an endogenous molecule that contributes to the dopaminergic neurodegeneration. In the present study, we show that BH4 leads to inhibition of activities of complexes I and IV of the electron transport chain (ETC) and reduction of mitochondrial membrane potential. BH4 appears to be different from rotenone and MPP(+), the synthetic compounds used to generate Parkinson models, in its effect on complex IV. BH4 also induces the release of mitochondrial cytochrome c. Pretreatment with the sulfhydryl antioxidant N-acetylcysteine or the quinone reductase inducer dimethyl fumarate prevents the ETC inhibition and cytochrome c release following BH4 exposure, suggesting the involvement of quinone products. Together with our previous observation that BH4 leads to generation of oxidative stress and selective dopaminergic neurodegeneration both in vitro and in vivo via inducing apoptosis, the mitochondrial involvement in BH4 toxicity further suggests possible relevance of this endogenous molecule to pathogenesis of PD.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Biopterins; Cell Line; Cytochromes c; Dopamine; Humans; Membrane Potentials; Mitochondria; Oxidative Stress; Parkinson Disease; Rotenone

Cellular stress may stimulate cell survival pathways or cell death depending on its severity. 6-Hydroxydopamine (6-OHDA) is a neurotoxin that targets dopaminergic neurons that is often used to induce neuronal cell death in models of Parkinson's disease. Here we present evidence that 6-OHDA induces apoptosis in rat PC12 cells that involves release of cytochrome c and Smac/Diablo from mitochondria, caspase-3 activation, cleavage of PARP, and nuclear condensation. 6-OHDA also induced the heat shock response, leading to increased levels of Hsp25 and Hsp70. Increased Hsp25 expression was associated with cell survival. Prior heat shock or overexpression of Hsp27 (human homologue of Hsp25) delayed cytochrome c release, caspase activation, and reduced the level of apoptosis caused by 6-OHDA. We conclude that 6-OHDA induces a variety of responses in cultured PC12 cells ranging from cell survival to apoptosis, and that induction of stress proteins such as Hsp25 may protect cells from undergoing 6-OHDA-induced apoptosis.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Carrier Proteins; Caspase 3; Caspase Inhibitors; Caspases; Complement Membrane Attack Complex; Complement System Proteins; Cytochromes c; Cytosol; Glycoproteins; Heat-Shock Proteins; Mitochondria; Mitochondrial Proteins; Neurons; Neurotoxins; Oxidopamine; Parkinson Disease; PC12 Cells; Rats; Time Factors

We show that human wild-type alpha synuclein (WT alpha-syn), and the inherited mutants A53T or A30P, when expressed in the yeast Saccharomyces cerevisiae triggers events that are diagnostic of apoptosis: loss of membrane asymmetry due to the externalization of phosphatidylserine, accumulation of reactive oxygen species (ROS), and the release of cytochrome c from mitochondria. A brief heat shock was strikingly protective in that alpha-syn-expressing cells receiving a heat shock exhibited none of these apoptotic markers. Because the heat shock did not decrease the expression level of alpha-syn, a protective protein or proteins, induced by the heat shock, must be responsible for inhibition of alpha-syn-induced apoptosis. Using ROS accumulation as a marker of apoptosis, the role of various genes and various drugs in controlling alpha-syn-induced apoptosis was investigated. Treatment with geldanamycin or glutathione, overexpression of Ssa3 (Hsp70), or deletion of the yeast metacaspase gene YCA1 abolishes the ability of alpha-syn to induce ROS accumulation. Deletion of YCA1 also promotes vigorous growth of alpha-syn-expressing cells compared to cells that contain a functional copy of YCA1. These findings indicate that alpha-syn-induced ROS generation is mediated by the caspase, according to alpha-syn-->caspase-->ROS-->apoptosis. It is shown by co-immunoprecipitation that Ssa3 binds to alpha-syn in a nucleotide-dependent manner. Thus, we propose that Hsp70 chaperones inhibit this sequence of events by binding and sequestering alpha-syn.

Topics: alpha-Synuclein; Animals; Annexin A5; Apoptosis; Benzoquinones; Blotting, Western; Caspases; Cell Membrane; Cell Separation; Cysteine Proteinase Inhibitors; Cytochromes c; Flow Cytometry; Gene Deletion; Genotype; Glutathione; Green Fluorescent Proteins; Hot Temperature; HSP70 Heat-Shock Proteins; Humans; Hydrogen Peroxide; Lactams, Macrocyclic; Mitochondria; Mutation; Nerve Tissue Proteins; Parkinson Disease; Plasmids; Quinones; Reactive Oxygen Species; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Synucleins; Time Factors

Mutations in the PTEN-induced kinase 1 (PINK1) gene have recently been implicated in autosomal recessive early onset Parkinson Disease (1, 2). To investigate the role of PINK1 in neurodegeneration, we designed human and murine neuronal cell lines expressing either wild-type PINK1 or PINK1 bearing a mutation associated with Parkinson Disease. We show that under basal and staurosporine-induced conditions, the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive cells was lower in wild-type PINK1 expressing SH-SY5Y cells than in mock-transfected cells. This phenotype was due to a PINK1-mediated reduction in cytochrome c release from mitochondria, which prevents subsequent caspase-3 activation. We show that overexpression of wild-type PINK1 strongly reduced both basal and staurosporine-induced caspase 3 activity. Overexpression of wild-type PINK1 also reduced the levels of cleaved caspase-9, caspase-3, caspase-7, and activated poly(ADP-ribose) polymerase under both basal and staurosporine-induced conditions. In contrast, Parkinson disease-related mutations and a kinase-inactive mutation in PINK1 abrogated the protective effect of PINK1. Together, these results suggest that PINK1 reduces the basal neuronal pro-apoptotic activity and protects neurons from staurosporine-induced apoptosis. Loss of this protective function may therefore underlie the degeneration of nigral dopaminergic neurons in patients with PINK1 mutations.

Topics: Aged; Amino Acid Sequence; Apoptosis; Caspase 3; Caspases; Cell Culture Techniques; Cell Line; Cytochromes c; DNA Mutational Analysis; Fibroblasts; Gene Expression Profiling; Humans; Male; Mitochondria; Molecular Sequence Data; Neurons; Parkinson Disease; Phenotype; Protein Kinases; Receptors, Dopamine; Reverse Transcriptase Polymerase Chain Reaction

Parkinson's disease (PD) is a neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. Alpha-synuclein is a major component of Lewy bodies in sporadic PD, and mutations in alpha-synuclein cause autosomal-dominant hereditary PD. Here, we generated A53T mutant alpha-synuclein-inducible PC12 cell lines using the Tet-off regulatory system. Inducing expression of A53T alpha-synuclein in differentiated PC12 cells decreased proteasome activity, increased the intracellular ROS level and caused up to approximately 40% cell death, which was accompanied by mitochondrial cytochrome C release and elevation of caspase-9 and -3 activities. Cell death was partially blocked by cyclosporine A [an inhibitor of the mitochondrial permeability transition (MPT) process], z-VAD (a pan-caspase inhibitor) and inhibitors of caspase-9 and -3 but not by a caspase-8 inhibitor. Furthermore, induction of A53T alpha-synuclein increased endoplasmic reticulum (ER) stress and elevated caspase-12 activity. RNA interference to knock down caspase-12 levels or salubrinal (an ER stress inhibitor) partially protected against cell death and further reduced A53T toxicity after treatment with z-VAD. Our results indicate that both ER stress and mitochondrial dysfunction contribute to A53T alpha-synuclein-induced cell death. This study sheds light into the pathogenesis of alpha-synuclein cellular toxicity in PD and provides a cell model for screening PD therapeutic agents.

Topics: alpha-Synuclein; Animals; Caspase Inhibitors; Caspases; Cell Death; Cysteine Proteinase Inhibitors; Cytochromes c; Cytosol; Endoplasmic Reticulum; Enzyme Activation; Humans; Mitochondria; Mutation; Parkinson Disease; PC12 Cells; Proteasome Endopeptidase Complex; Rats; Reactive Oxygen Species; Signal Transduction

Dysfunction of mitochondrial complex I is a feature of human neurodegenerative diseases such as Leber hereditary optic neuropathy and Parkinson's disease. This mitochondrial defect is associated with a recruitment of the mitochondrial-dependent apoptotic pathway in vivo. However, in isolated brain mitochondria, complex I dysfunction caused by either pharmacological or genetic means fails to directly activate this cell death pathway. Instead, deficits of complex I stimulate intramitochondrial oxidative stress, which, in turn, increase the releasable soluble pool of cytochrome c within the mitochondrial intermembrane space. Upon mitochondrial permeabilization by the cell death agonist Bax, more cytochrome c is released to the cytosol from brain mitochondria with impaired complex I activity. Given these results, we propose a model in which defects of complex I lower the threshold for activation of mitochondrial-dependent apoptosis by Bax, thereby rendering compromised neurons more prone to degenerate. This molecular scenario may have far-reaching implications for the development of effective neuroprotective therapies for these incurable illnesses.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Ascorbic Acid; bcl-2-Associated X Protein; Brain; Cardiolipins; Cell Death; Chromatography, High Pressure Liquid; Cytochromes c; Electron Transport Complex I; Genetic Techniques; Hydrogen Peroxide; Male; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Mitochondria; Models, Biological; Neurodegenerative Diseases; Neurons; Oxidative Stress; Oxygen; Parkinson Disease; Reactive Oxygen Species; Subcellular Fractions; Submitochondrial Particles; Time Factors

Many models of Parkinson's disease (PD) have succeeded in replicating dopaminergic neuron loss or alpha-synuclein aggregation but not the formation of classical Lewy bodies, the pathological hallmark of PD. Our cybrid model of sporadic PD was created by introducing the mitochondrial genes from PD patients into neuroblastoma cells that lack mitochondrial DNA. Previous studies using cybrids have shown that information encoded by mitochondrial DNA in patients contributes to many pathogenic features of sporadic PD. In this paper, we report the generation of fibrillar and vesicular inclusions in a long-term cybrid cell culture model that replicates the essential antigenic and structural features of Lewy bodies in PD brain without the need for exogenous protein expression or inhibition of mitochondrial or proteasomal function. The inclusions generated by PD cybrid cells stained with eosin, thioflavin S, and antibodies to alpha-synuclein, ubiquitin, parkin, synphilin-1, neurofilament, beta-tubulin, the proteasome, nitrotyrosine, and cytochrome c. Future studies of these cybrids will enable us to better understand how Lewy bodies form and what role they play in the pathogenesis of PD.

Topics: Aged; alpha-Synuclein; Blotting, Western; Carrier Proteins; Case-Control Studies; Cell Line; Cysteine Endopeptidases; Cytochromes c; DNA, Mitochondrial; Electron Transport Complex I; Female; Humans; Immunohistochemistry; Lewy Bodies; Male; Microscopy, Confocal; Microscopy, Electron; Middle Aged; Multienzyme Complexes; Nerve Tissue Proteins; Neuroblastoma; Neurofilament Proteins; Neurons; Parkinson Disease; Precipitin Tests; Proteasome Endopeptidase Complex; Staining and Labeling; Synucleins; Transgenes; Tubulin; Tyrosine; Ubiquitin; Ubiquitin-Protein Ligases

Trichloroethylene, a common industrial solvent and a metabolic precursor of chloral hydrate, occurs widely in the environment. Chloral hydrate, which is also used as a hypnotic, has been found to condense spontaneously with tryptamine, in vivo, to give rise to a highly unpolar 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) that has a structural analogy to the dopaminergic neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Earlier studies have revealed the relative permeability of the molecule through the blood-brain barrier and its ability to induce Parkinson-like symptoms in rats. In this study, we report that TaClo induces an apoptotic pathway in the human neuroblastoma cell line, SK-N-SH, involving the translocation of mitochondrial cytochrome c to the cytosol and activation of caspase 3. TaClo-induced apoptosis shows considerable differences from that mediated by other Parkinson-inducing agents such as MPTP, rotenone and manganese. Although it is not clear if the clinically administered dosage of chloral hydrate or the relatively high environmental levels of trichloroethylene could lead to an onset of Parkinson's disease, the spontaneous in vivo formation of TaClo and its pro-apoptotic properties, as shown in this report, should be considered.

Topics: Apoptosis; Carbolines; Caspase 3; Caspases; Cell Line, Tumor; Cell Survival; Cytochromes c; Enzyme Induction; Gene Expression; Humans; Mitochondria; Neuroblastoma; Neurons; Parkinson Disease; Protein Kinase C