cytochrome-c-t and Nerve-Degeneration

Intermediates play a relevant role in the protein-folding process, because the onset of diseases of genetic nature is usually coupled with protein misfolding and the formation of stable intermediate species. This article describes and briefly discusses the mechanisms considered responsible, at molecular level, for a number of neurodegenerative diseases. In particular, interest is focused on the newly discovered role of cytochrome c in programmed cell death (apoptosis), consisting of acquisition of powerful cardiolipin-specific peroxidase action. Cardiolipin oxidation induces cytochrome c detachment from the mitochondrial membrane and favors the accumulation of products releasing proapoptotic factors. Cytochrome c showing peroxidase activity has non-native structure, and shows enhanced access of the heme catalytic site to small molecules, such as H(2)O(2). The strict correlation linking cytochrome c with the onset of neurodegenerative disorders is described and the therapeutic approach discussed.

Topics: Apoptosis; Cardiolipins; Cytochromes c; Humans; Nerve Degeneration; Peroxidase; Proteostasis Deficiencies

Mitochondria play a pivotal role in the cascade of events associated with cell death pathways that are involved with several forms of neurodegeneration. Recent findings show that in the Bax/Bak-dependent pathway of apoptosis, the release of cytochrome c from mitochondria is a consequence of two carefully coordinated events: opening of crista junctions triggered by OPA1 oligomer disassembly and formation of outer membrane pores. Both steps are necessary for the complete release of pro-apoptotic proteins. The remodeling of mitochondrial structure accompanies this pathway, including mitochondrial fission, and cristae and crista junction alterations. Yet, there is controversy surrounding the timing of certain remodeling events and whether they are necessary early events required for the release of pro-apoptotic factors or are simply a downstream after-effect. Here, we analyze the current knowledge of mitochondrial remodeling during cell death and discuss what structural alterations occur to this organelle during neurodegeneration, focusing on the higher resolution structural correlates obtained by electron microscopy and electron tomography.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Cytochromes c; Electron Microscope Tomography; GTP Phosphohydrolases; Humans; Mitochondria; Nerve Degeneration

The prefrontal cortex is the largest lobe of the brain and is consequently involved in stroke. There is no comprehensive practical pharmacological strategy for ameliorating prefrontal cortex injury induced by cerebral ischemia. Therefore, we studied the neuroprotective properties of verapamil (Ver) on mitochondrial dysfunction and morphological features of apoptosis in transient global ischemia/reperfusion (I/R). Ninety-six Wistar rats were allocated into four groups: control, I/R, I/R+Ver (10 mg/kg twice 1 hour prior to ischemia and 1 hour after reperfusion phase), and I/R+NaCl (vehicle). Animals were sacrificed, and mitochondrial dysfunction parameters (i.e., mitochondrial swelling, mitochondrial membrane potential, ATP concentration, ROS production, and cytochrome c release), antioxidant defense (i.e., superoxide dismutase, malondialdehyde, glutathione peroxidase, catalase, and caspase-3 activation), and morphological features of apoptosis were determined. The results showed that mitochondrial damage, impairment of antioxidant defense system, and apoptosis were significantly more prevalent in the I/R group in comparison with the other groups. Ver decreased mitochondrial damage by reducing oxidative stress, augmented the activity of antioxidant enzymes in the brain, and decreased apoptosis in the I/R neurons. The current study confirmed the role of oxidative stress and mitochondrial dysfunction in I/R progression and indicated the possible antioxidative mechanism of the neuroprotective activities of Ver.

Topics: Adenosine Triphosphate; Animals; Antioxidants; Apoptosis; Brain; Caspase 3; Cell Survival; Cytochromes c; Ischemic Attack, Transient; Male; Malondialdehyde; Mitochondria; Mitochondrial Swelling; Nerve Degeneration; Neurons; Prefrontal Cortex; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; RNA, Messenger; Verapamil

Cytochrome c (Cyt c), a heme-containing mitochondrial protein, has a critical function in both respiration and apoptosis. Consistent with these vital functions, somatic Cyt c mouse knockout is embryonic lethal. In order to investigate the sensitivity of postnatal neurons to Cyt c depletion, we developed a neuron-specific conditional knockout model. Neuron-specific Cyt c KO mouse (nCytc

Topics: Animals; Apoptosis; Cytochromes c; Electron Transport Complex IV; Gene Deletion; Inflammation; Locomotion; Mice, Knockout; Mitochondria; Nerve Degeneration; Neurons; Oxidative Phosphorylation; Oxidative Stress; Phenotype; Prosencephalon

Sinomenine (SIN) has been shown to have protective effects against brain damage following traumatic brain injury (TBI). However, the mechanisms and its role in these effects remain unclear. This study was conducted to investigate the potential mechanisms of the protective effects of SIN.. The weight-drop model of TBI in Institute of Cancer Research (ICR) mice were treated with SIN or a vehicle via intraperitoneal administration 30 min after TBI. All mice were euthanized 24 h after TBI and after neurological scoring, a series of tests were performed, including brain water content and neuronal cell death in the cerebral cortex.. The level of cytochrome. SIN protected neuronal cells by protecting them against apoptosis via mechanisms that involve the mitochondria following TBI.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Brain Edema; Brain Injuries, Traumatic; Cerebral Cortex; Cytochromes c; Cytoprotection; Disease Models, Animal; Dose-Response Relationship, Drug; Glutathione Peroxidase; Male; Malondialdehyde; Mice, Inbred ICR; Mitochondria; Morphinans; Nerve Degeneration; Neurons; Neuroprotective Agents; Oxidative Stress; Signal Transduction; Superoxide Dismutase-1

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

The study aimed to investigate the role of NO and neuronal NO synthase (nNOS) in Zn-induced neurodegeneration. Animals were treated with zinc sulfate (20 mg/kg), twice a week, for 2-12 weeks along with control. In a few sets, animals were also treated with/without a NO donor, sodium nitroprusside (SNP), or S-nitroso-N-acetyl penicillamine (SNAP) for 12 weeks. Moreover, human neuroblastoma (SH-SY-5Y) cells were also employed to investigate the role of nNOS in Zn-induced toxicity in in vitro in the presence/absence of nNOS inhibitor, 7-nitroindazole (7-NI). Zn caused time-dependent reduction in nitrite content and total/nNOS activity/expression. SNP/SNAP discernibly alleviated Zn-induced neurobehavioral impairments, dopaminergic neurodegeneration, tyrosine hydroxylase (TH) expression, and striatal dopamine depletion. NO donors also salvage from Zn-induced increase in lipid peroxidation (LPO), mitochondrial cytochrome c release, and caspase-3 activation. While Zn elevated LPO content, it attenuated nitrite content, nNOS activity, and glutathione level along with the expression of TH and nNOS in SH-SY-5Y cells. 7-NI further augmented Zn-induced changes in the cell viability, oxidative stress, and expression of TH and nNOS. The results obtained thus demonstrate that Zn inhibits nNOS that partially contributes to an increase in oxidative stress, which subsequently leads to the nigrostriatal dopaminergic neurodegeneration.

Topics: Animals; Behavior, Animal; Blotting, Western; Caspase 3; Cell Survival; Cytochromes c; Dopaminergic Neurons; Enzyme Activation; Glutathione; Humans; Indazoles; Lipid Peroxidation; Male; Metabolome; Mitochondria; Neostriatum; Nerve Degeneration; Nitric Oxide Donors; Nitric Oxide Synthase Type I; Nitrites; Nitroprusside; Oxidative Stress; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine; Tyrosine 3-Monooxygenase; Zinc

Accumulation of zinc (Zn) in dopaminergic neurons is implicated in Parkinson's disease (PD), and microglial activation plays a critical role in toxin-induced Parkinsonism. Oxidative stress is accused in Zn-induced dopaminergic neurodegeneration; however, its connection with microglial activation is still not known. This study was undertaken to elucidate the role and underlying mechanism of microglial activation in Zn-induced nigrostriatal dopaminergic neurodegeneration. Male Wistar rats were treated intraperitoneally with/without zinc sulphate (20 mg/kg) in the presence/absence of minocycline (30 mg/kg), a microglial activation inhibitor, for 2-12 weeks. While neurobehavioral and biochemical indexes of PD and number of dopaminergic neurons were reduced, the number of microglial cells was increased in the substantia nigra of the Zn-exposed animals. Similarly, Zn elevated lipid peroxidation (LPO) and activities of superoxide dismutase (SOD) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase; however, catalase activity was reduced. Besides, Zn increased an association of NADPH oxidase subunit p67(phox) with membrane, cytochrome c release from the mitochondria and cleavage of pro-caspase 3. Zn attenuated the expression of tyrosine hydroxylase (TH) and vesicular monoamine transporter-2 (VMAT-2) while augmented the expression of dopamine transporter (DAT) and heme oxygenase-1 (HO-1). Minocycline alleviated Zn-induced behavioural impairments, loss of TH-positive neurons, activated microglial cells and biochemical indexes and modulated the expression of studied genes/proteins towards normalcy. The results demonstrate that minocycline reduces the number of activated microglial cells and oxidative stress, which rescue from Zn-induced changes in the expression of monoamine transporter and nigrostriatal dopaminergic neurodegeneration.

Topics: Animals; Antigens, Nuclear; Behavior, Animal; Caspase 3; CD11b Antigen; Cytochromes c; Dopamine; Dopaminergic Neurons; Gene Expression Regulation; Heme Oxygenase-1; Lipid Peroxidation; Male; Membrane Transport Proteins; Metabolome; Microglia; Minocycline; Mitochondria; Motor Activity; NADPH Oxidases; Nerve Degeneration; Nerve Tissue Proteins; Oxidative Stress; Rats, Wistar; Rotarod Performance Test; Substantia Nigra; Tyrosine 3-Monooxygenase; Zinc

Clinical evidences showing zinc (Zn) accumulation in the post-mortem brain of Parkinson's disease (PD) patients and experimental studies on rodents chronically exposed to Zn suggested its role in PD. While oxidative stress is implicated in Zn-induced neurodegeneration, roles of inflammation and apoptosis in degeneration of the nigrostriatal dopaminergic neurons have yet been elusive. The present study investigated the contribution of the nuclear factor kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and B-cell lymphoma 2 (Bcl-2) family proteins in Zn-induced Parkinsonism. Male Wistar rats were treated with/without zinc sulfate (Zn; 20 mg/kg, intraperitoneally), twice a week, for 2-12 weeks. In a few sets, animals were treated intraperitoneally with a NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC; 100 mg/kg), a TNF-α inhibitor, pentoxyfylline (PTX; 50 mg/kg), and an anti-inflammatory agent, dexamethasone (DEX; 5 mg/kg), prior to Zn exposure along with respective controls. Zn caused neurobehavioral impairments and reduction in dopamine and its metabolites, tyrosine hydroxylase (TH)-positive neurons, catalase activity, and expression of TH, Bcl-2, and NOXA. On the contrary, Zn augmented lipid peroxidation, activity of superoxide dismutase, expression of TNF-α, IL-1β, Bcl-xl, and p53-upregulated modulator of apoptosis (PUMA), and translocation of NF-κB and Bax from the cytosol to the nucleus and mitochondria, respectively, with concomitant increase in the mitochondrial cytochrome c release and activation of procaspase-3 and -9. Pre-treatment with PTX, DEX, or PDTC invariably ameliorated Zn-induced changes in behavioral and neurodegenerative indexes, inflammatory mediators, and apoptosis. Results demonstrate that inflammation regulates Bax expression that subsequently contributes to the nigrostriatal dopaminergic neurodegeneration.

Topics: Animals; Antigens, Nuclear; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; Behavior, Animal; Cytochromes c; Dopamine; Dopaminergic Neurons; Gene Expression Regulation; Inflammation; Interleukin-1beta; Lipid Peroxidation; Male; Metabolome; Mitochondria; Neostriatum; Nerve Degeneration; Nerve Tissue Proteins; NF-kappa B; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Rats, Wistar; RNA, Messenger; Serotonin; Substantia Nigra; Tumor Necrosis Factor-alpha; Tyrosine 3-Monooxygenase; Zinc

Recent evidence suggests that nerve growth factor IB (Nur77) and nuclear receptor related1 (Nurr1) are differentially involved in dopaminergic neurodegeneration. Since memantine has shown clinically relevant efficacy in Parkinson's disease (PD) and displayed a potent protective effect on dopaminergic neurons in experimental PD models, we asked if it exerts its neuroprotection by regulating Nur77 and Nurr1 signaling. We adopted a well-established in vitro PD model, 6-hydroxydopamine (OHDA)-lesioned PC12 cells, to test our hypothesis. Different concentrations of memantine were incubated with 6-OHDA-lesioned PC12 cells, and Nur77/Nurr1 and their related signaling molecules were examined by Western blot and immunocytochemistry. Nur77-deficient PC12 cells were used to verify the influences of Nur77 on neurodegeneration and memantine-mediated neuroprotection. We found that memantine reversed Nur77 upregulation and restored Nurr1 downregulation in 6-OHDA-lesioned PC12 cells. 6-OHDA incubation caused Nur77 translocation from the nucleus to cytosol and induced co-localization of Cyt c/HSP60/Nur77 in the cytosol. Memantine strongly reduced the sub-cellular translocations of Nur77/Cyt c/HSP60 under 6-OHDA-induced oxidative condition. Knockdown of Nur77 enhanced the viability of PC12 cells exposed to 6-OHDA, while memantine-induced neuroprotection was much less in the cells with Nur77 knockdown than in those without it. We conclude that Nur77 plays a crucial role in modulating mitochondrial impairment and contributes to neurodegeneration under the experimental PD condition. Memantine effectively suppresses such Nur77-mediated neurodegeneration and promotes survival signaling through post-translational modification of Nurr1. Nur77 and Nurr1 present a contra-directionally coupling interaction in memantine-mediated neuroprotection.

Topics: Animals; Cell Survival; Cytochromes c; Dopamine Plasma Membrane Transport Proteins; Gene Knockdown Techniques; Glutamic Acid; Inflammation; Interleukin-6; L-Lactate Dehydrogenase; MAP Kinase Signaling System; Memantine; Mitochondria; Nerve Degeneration; Neuroprotection; Nuclear Receptor Subfamily 4, Group A, Member 1; Nuclear Receptor Subfamily 4, Group A, Member 2; Oxidopamine; PC12 Cells; Rats; Subcellular Fractions; Tumor Necrosis Factor-alpha; Tyrosine 3-Monooxygenase

It has been reported in the literature that cholinesterase inhibitors provide protection in Alzheimer's disease (AD). Recent reports have implicated triazine derivatives as cholinesterase inhibitors. These findings led us to investigate anti-cholinestrase property of some novel triazine derivatives synthesized in this laboratory. In vitro cholinesterase inhibition assay was performed using Ellman method. The potent compounds screened out from in vitro assay were further evaluated using scopolamine-induced amnesic mice model. Further, in vitro reactive oxygen species (ROS) scavenging and anti-apoptotic property of the potent compounds were demonstrated against Aβ1-42-induced neurotoxicity in rat hippocampal cells. Their neuroprotective role was assessed using Aβ1-42-induced Alzheimer's-like phenotype in rats. Further, the role of compounds on the activation of the Wnt/β-catenin pathway was studied. The results showed that the chosen compounds are having protective effect in Alzheimer's-like condition; the ex vivo results advocated their anti-cholinestrase and anti-oxidant activities. Treatment with TRZ-15 and TRZ-20 showed neuroprotective ability of the compounds as evidenced from the improved cognitive ability in the animals, and decrease in Aβ1-42 burden and cytochrome c and cleaved caspase-3 levels in the brain. This study also demonstrates positive involvement of the novel triazine derivatives in the Wnt/β-catenin pathway. Immunoblot and immunofluorescence data suggested that ratio of pGSK3/GSK3 and β-catenin got dramatically improved after treatment with TRZ-15 and TRZ-20. TRZ-15 and TRZ-20 showed neuroprotection in scopolamine-induced amnesic mice and Aβ1-42-induced Alzheimer's rat model and also activate the Wnt/β-catenin signaling pathway. These findings conclude that TRZ-15 and TRZ-20 could be a therapeutic approach to treat AD.

Topics: Acetylcholinesterase; Alzheimer Disease; Amyloid beta-Peptides; Animals; Antioxidants; Apoptosis; beta Catenin; Butyrylcholinesterase; Caspase 3; Cholinesterase Inhibitors; Cytochromes c; Disease Models, Animal; Glycogen Synthase Kinase 3; Hippocampus; Male; Maze Learning; Memory; Mice; Molecular Docking Simulation; Nerve Degeneration; Neurons; Neuroprotective Agents; Oxidative Stress; Rats, Wistar; Scopolamine; Triazines; Wnt Signaling Pathway

We have documented that tissue kallikrein (TK) prevents neurons from hypoxia/reoxygenation injury through the B2R-ERK1/2 pathway and the antihypoxic function of TK through Homer1b/c-ERK1/2 signaling pathways. The present study investigates the molecular mechanisms of exogenous TK activation of the B2R-ERK1/2 pathway through the β-arrestin-2 assembled B2R-Raf-MEK1/2 signaling module in vivo. The cresyl violet staining results indicated that exogenous TK protected the rat hippocampal CA1 neurons against cerebral ischemia/reperfusion (I/R) injury. The immunoprecipitation (IP) and immunoblotting (IB) results revealed that exogenous TK upregulated the β-arrestin-2 assembled B2R-Raf-MEK1/2 signaling module and upregulated the phosphorylation of Raf (p-Raf), MEK1/2 (p-MEK1/2), and ERK1/2 (p-ERK1/2). Meanwhile, exogenous TK upregulated the expression of nuclear factor-κB (NF-κB), depressed the release of cytochrome c (Cyt c) and bax from mitochondria to the cytosol, and depressed the activation of caspase-3. Take together, our results suggest that exogenous TK attenuated the cerebral I/R induced rat hippocampal CA1 neurons injury through activating the β-arrestin-2 assembled B2R-Raf-MEK1/2 signaling module and that the activated B2R-Raf-MEK1/2 signaling module could upregulate the expression of NF-κB, decrease the release of cytochrome c and bax from mitochondria to the cytosol, and depress the activation of caspase-3.

Topics: Animals; Arrestins; bcl-2-Associated X Protein; beta-Arrestin 2; beta-Arrestins; Brain Ischemia; CA1 Region, Hippocampal; Caspase 3; Cytochromes c; Disease Models, Animal; Dose-Response Relationship, Drug; Male; MAP Kinase Signaling System; Nerve Degeneration; Neurons; Oligodeoxyribonucleotides, Antisense; raf Kinases; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tissue Kallikreins; Up-Regulation

Neurons can activate pathways that destroy the whole cell via apoptosis or selectively degenerate only the axon (pruning). Both apoptosis and axon degeneration require Bax and caspases. Here we demonstrate that despite this overlap, the pathways mediating axon degeneration during apoptosis versus axon pruning are distinct. While Caspase-6 is activated in axons following nerve growth factor deprivation, microfluidic chamber experiments reveal that Caspase-6 deficiency only protects axons during axon-specific but not whole-cell (apoptotic) nerve growth factor deprivation. Strikingly, axon-selective degeneration requires the apoptotic proteins Caspase-9 and Caspase-3 but, in contrast to apoptosis, not apoptotic protease activating factor-1. Additionally, cell bodies of degenerating axons are protected from caspase activation by proteasome activity and X-linked inhibitor of apoptosis protein. Also, mature neurons restrict apoptosis but remain permissive for axon degeneration, further demonstrating the independent regulation of these two pathways. These results reveal insight into how neurons allow for precise control over apoptosis and axon-selective degeneration pathways, thereby permitting long-term plasticity without risking neurodegeneration.

Topics: Aging; Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Axons; Caspases; Cytochromes c; Enzyme Activation; Mice; Mice, Inbred C57BL; Models, Biological; Nerve Degeneration; Nerve Growth Factors; Proteasome Endopeptidase Complex; Signal Transduction; X-Linked Inhibitor of Apoptosis Protein

Autophagy is the evolutionarily conserved degradation and recycling of cellular constituents. In mammals, autophagy is implicated in the pathogenesis of many neurodegenerative diseases. However, its involvement in acute brain damage is unknown. This study addresses the function of autophagy in neurodegeneration that has been induced by acute focal cerebellar lesions. We provide morphological, ultrastructural, and biochemical evidence that lesions in a cerebellar hemisphere activate autophagy in axotomized precerebellar neurons. Through time course analyses of the apoptotic cascade, we determined mitochondrial dysfunction to be the early trigger of degeneration. Further, the stimulation of autophagy by rapamycin and the employment of mice with impaired autophagic responses allowed us to demonstrate that autophagy protects from damage promoting functional recovery. These findings have therapeutic significance, demonstrating the potential of pro-autophagy treatments for acute brain pathologies, such as stroke and brain trauma.

Topics: Animals; Apoptosis Regulatory Proteins; Autophagy; Axotomy; Beclin-1; Brain Injuries; Cerebellum; Chloroquine; Cytochromes c; Cytoprotection; Mice; Mice, Inbred C57BL; Mitochondria; Nerve Degeneration; Neurons; Neuroprotective Agents; Phagosomes; Sirolimus

GM2 ganglioside in the brain increased during ethanol-induced acute apoptotic neurodegeneration in 7-day-old mice. A small but a significant increase observed 2 h after ethanol exposure was followed by a marked increase around 24 h. Subcellular fractionation of the brain 24 h after ethanol treatment indicated that GM2 increased in synaptic and non-synaptic mitochondrial fractions as well as in a lysosome-enriched fraction characteristic to the ethanol-exposed brain. Immunohistochemical staining of GM2 in the ethanol-treated brain showed strong punctate staining mainly in activated microglia, in which it partially overlapped with staining for LAMP1, a late endosomal/lysosomal marker. Also, there was weaker neuronal staining, which partially co-localized with complex IV, a mitochondrial marker, and was augmented in cleaved caspase 3-positive neurons. In contrast, the control brain showed only faint and diffuse GM2 staining in neurons. Incubation of isolated brain mitochondria with GM2 in vitro induced cytochrome c release in a manner similar to that of GD3 ganglioside. Because ethanol is known to trigger mitochondria-mediated apoptosis with cytochrome c release and caspase 3 activation in the 7-day-old mouse brain, the GM2 elevation in mitochondria may be relevant to neuroapoptosis. Subsequently, activated microglia accumulated GM2, indicating a close relationship between GM2 and ethanol-induced neurodegeneration.

Topics: Animals; Apoptosis; Blotting, Western; Brain; Brain Chemistry; Caspase 3; Central Nervous System Depressants; Cytochromes c; Endosomes; Enzyme Activation; Ethanol; G(M2) Ganglioside; Immunohistochemistry; Lysosomes; Mice; Mice, Inbred C57BL; Microscopy, Electron; Mitochondria; Nerve Degeneration; Subcellular Fractions

Aminochrome, the precursor of neuromelanin, has been proposed to be involved in the neurodegeneration neuromelanin-containing dopaminergic neurons in Parkinson's disease. We aimed to study the mechanism of aminochrome-dependent cell death in a cell line derived from rat substantia nigra. We found that aminochrome (50μM), in the presence of NAD(P)H-quinone oxidoreductase, EC 1.6.99.2 (DT)-diaphorase inhibitor dicoumarol (DIC) (100μM), induces significant cell death (62 ± 3%; p < 0.01), increase in caspase-3 activation (p < 0.001), release of cytochrome C, disruption of mitochondrial membrane potential (p < 0.01), damage of mitochondrial DNA, damage of mitochondria determined with transmission electron microscopy, a dramatic morphological change characterized as cell shrinkage, and significant increase in number of autophagic vacuoles. To determine the role of autophagy on aminochrome-induced cell death, we incubated the cells in the presence of vinblastine and rapamycin. Interestingly, 10μM vinblastine induces a 5.9-fold (p < 0.001) and twofold (p < 0.01) significant increase in cell death when the cells were incubated with 30μM aminochrome in the absence and presence of DIC, respectively, whereas 10μM rapamycin preincubated 24 h before addition of 50μM aminochrome in the absence and the presence of 100μM DIC induces a significant decrease (p < 0.001) in cell death. In conclusion, autophagy seems to be an important protective mechanism against two different aminochrome-induced cell deaths that initially showed apoptotic features. The cell death induced by aminochrome when DT-diaphorase is inhibited requires activation of mitochondrial pathway, whereas the cell death induced by aminochrome alone requires inhibition of autophagy-dependent degrading of damaged organelles and recycling through lysosomes.

Topics: Animals; Autophagy; Caspase 3; Cell Death; Cell Line; Cytochromes c; DNA, Mitochondrial; Indolequinones; Melanins; Membrane Potential, Mitochondrial; Microscopy, Electron, Transmission; Mitochondria; NAD(P)H Dehydrogenase (Quinone); Nerve Degeneration; Rats; Sirolimus; Substantia Nigra; Vinblastine

Breathing-disordered states, such as in obstructive sleep apnea, which are cyclical in nature, have been postulated to induce neurocognitive morbidity in both pediatric and adult populations. The oscillatory nature of intermittent hypoxia, especially when chronic, may mimic the paradigm of ischemia-reperfusion in that tissues and cells are exposed to episodes of low and high O(2) and this may lead to oxidant stress. Therefore, we decided to explore the potential contribution of oxidant stress in our intermittent hypoxia/hypercapnia animal model and the role that mitochondria might play in this stress. Neonatal mice were exposed to intermittent hypoxia/hypercapnia for 10 days and 2 wk. Combined intermittent hypoxia/hypercapnia led to a marked increase in apoptotic cell death in the cerebral cortex. Oxygen consumption studies in isolated mitochondria from intermittent hypoxia/hypercapnia-exposed brains demonstrated significant reductions in both state 4 and state 3 respiratory activities by approximately 60% and 75%, respectively. Electron paramagnetic resonance spectroscopy registered a significant increase in superoxide production during nonphosphorylating state 4 by 37%, although superoxide leakage during state 3 did not increase upon treatment. Neuronal superoxide-specific dihydroethidium oxidation was also greater in exposed animals. These studies indicate that intermittent hypoxia/hypercapnia leads to oxidative stress due to mitochondrial response within the mouse central nervous system.

Topics: Animals; Animals, Newborn; Apoptosis; Body Weight; Cell Death; Cerebral Cortex; Cytochromes c; Disease Models, Animal; Electron Spin Resonance Spectroscopy; Hematocrit; Hypercapnia; Hypoxia; Mice; Mitochondria; Nerve Degeneration; Neurons; Oxidation-Reduction; Oxidative Phosphorylation; Oxidative Stress; Oxygen Consumption; Superoxides; Time Factors

Proteasomal stress and the accumulation of polyubiquitinated proteins are key features of numerous neurodegenerative disorders. Previously we demonstrated that stabilization of p53 and activation of its target gene, puma (p53-upregulated mediator of apoptosis), mediated proteasome inhibitor-induced apoptosis in cancer cells. Here we demonstrated that Puma also contributed to proteasome inhibitor-induced apoptosis in mouse neocortical neurons. Although protection afforded by puma gene deletion was incomplete, we found little evidence indicating contributions from other proapoptotic BH3-only proteins. Attenuation of bax expression did not further reduce Puma-independent apoptosis, suggesting that pathways other than the mitochondrial apoptosis pathway were activated. Real-time imaging experiments in wild-type and puma-deficient neurons using a fluorescence resonance energy transfer (FRET)-based caspase sensor confirmed the involvement of a second cell death pathway characterized by caspase activation prior to mitochondrial permeabilization and, more prominently, a third, caspase-independent and Puma-independent pathway characterized by rapid cell shrinkage and nuclear condensation. This pathway involved lysosomal permeabilization in the absence of autophagy activation and was sensitive to cathepsin but not autophagy inhibition. Our data demonstrate that proteasomal stress activates distinct cell death pathways in neurons, leading to both caspase-dependent and caspase-independent apoptosis, and demonstrate independent roles for Puma and lysosomal permeabilization in this model.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Base Sequence; Bcl-2-Like Protein 11; BH3 Interacting Domain Death Agonist Protein; Caspase 3; Cathepsins; Cytochromes c; DNA Primers; Fluorescence Resonance Energy Transfer; Gene Expression; Lysosomes; Membrane Potential, Mitochondrial; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Neurological; Nerve Degeneration; Neurons; Protease Inhibitors; Proteasome Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Stress, Physiological; Tumor Suppressor Proteins

EUK-207 is a synthetic superoxide dismutase/catalase mimetic that has been shown to reverse age-related learning deficits and brain oxidative stress in mice. In the present experiments, we tested the effects of EUK-207 on oxygen/glucose deprivation (OGD)-induced cell death in cultured hippocampal slices and on several mechanisms that have been postulated to participate in this process. Cultured hippocampal slices were subjected to 1 h OGD followed by 3 or 24 h recovery in regular medium with glucose and oxygen. Lactate dehydrogenase (LDH) release in culture medium and propidium iodide (PI) uptake in slices were used to evaluate cell viability. When EUK-207 was applied either 1 or 2 h before OGD, OGD-induced LDH release was significantly reduced. When EUK-207 was applied 1 h before OGD and during 24 h recovery, PI uptake was also reduced. OGD-induced accumulation of reactive oxygen species (ROS) was evaluated with the fluorescent probe DCF. DCF fluorescence in slices increased steadily during OGD treatment, rapidly disappeared following return to regular medium before slowly increasing again during the 24 h recovery period. When measured 3 h after OGD, increased ROS levels were significantly reduced by EUK-207. OGD also increased lipid peroxidation levels and this effect was also reduced by EUK-207 6 h following OGD. Cytosolic cytochrome c and nuclear apoptosis-inducing factor (AIF) were increased 3 h after OGD, and the translocation of AIF from mitochondria to nucleus was partly blocked by treatment with EUK-207. In conclusion, EUK-207 provides neuroprotection against OGD-induced cell death in cultured hippocampal slices. As EUK-207 prevents free radical formation and lipid peroxidation, the neuroprotection is related to elimination of free radical generation and lipid peroxidation, as well as to decreased activation of pro-apoptotic factors. Our data support the further clinical evaluation of this class of molecules for the prevention of ischemic cell damage.

Topics: Animals; Apoptosis Inducing Factor; Catalase; Cell Death; Cell Survival; Cytochromes c; Fluorescent Dyes; Free Radical Scavengers; Hippocampus; Hypoxia-Ischemia, Brain; L-Lactate Dehydrogenase; Lipid Peroxidation; Nerve Degeneration; Neurons; Neuroprotective Agents; Organ Culture Techniques; Organometallic Compounds; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Superoxide Dismutase

Secondary brain damage plays a critical role in the outcome of patients with traumatic brain injury (TBI). The multiple mechanisms underlying secondary brain damage, including posttraumatic cerebral ischemia, glutamate excitotoxicity, oxidative stress, calcium overload and inflammation, are associated with increased mortality and morbidity after head injury. TBI is documented to have detrimental effects on mitochondria, such as alterations in glucose utilization and the depression of mitochondrial oxidative phosphorylation. Studies on mitochondrial metabolism have provided evidence for dysfunction of the cytochrome oxidase complex of the electron transport chain (complex IV) after TBI. A growing body of evidence indicates that cytochrome c oxidase is vital for mitochondrial oxidative phosphorylation. Therefore, this study aimed to detect the expression of cytochrome c oxidase (CO) mRNA in a rat weight-dropping trauma model and to clarify the differences between injured cortex (IC) and contralateral cortex (CC) after TBI. A total of forty-four rats were randomly assigned to 7 groups: control groups (n=4), sham-operated group (n=20), 6 h, 1 d, 3 d, 5 d and 7 d postinjury groups (n=4 for each group). The group consisted of sham-operated animals underwent parietal craniotomy without TBI. The rats in postinjury groups were subjected to TBI. The rats of control group were executed immediately without TBI or craniotomy after anesthesia. The brain-injured and sham-operated animals were killed on 6 h, 1 d, 3 d, 5 d and 7 d, respectively. Tissue sections from IC and CC were obtained and the expression of cytochrome c oxidase I, II, and III (CO I, II, III) mRNA, three mitochondrial encoded subunits of complex IV, were assessed by Real-time quantitative PCR. A reduction of CO I, II, and III mRNA expression was detected from IC and reduced to the lowest on 3 d. By contrast, the mRNA expression from CC suggested a slight elevation. The differences may indicate the degree of metabolic and physiologic dysfunction. Our results will better define the roles of gene expression and metabolic function in long-term prognosis and outcome after TBI. With a considerable understanding of post-injury mitochondrial dysfunction, therapeutic interventions targeted to the mitochondria may prevent secondary brain damage that leads to long-term cell death and neurobehavioral disability.

Topics: Animals; Brain Injuries; Cell Death; Cell Respiration; Cerebral Cortex; Cytochromes c; Disease Models, Animal; Down-Regulation; Electron Transport Chain Complex Proteins; Energy Metabolism; Functional Laterality; Gene Expression Regulation, Enzymologic; Male; Mitochondria; Nerve Degeneration; Neurons; Oxidative Phosphorylation; Protein Subunits; Rats; Rats, Sprague-Dawley; RNA, Messenger; Up-Regulation

Traumatic, infectious, metabolic, and chemical noxa to the nervous system are the etiology of a crippling disease generally termed neuropathy. Motor disorders, altered sensibility, and pain are the pathognomonic traits. Cellular alterations induced by this chronic pathology include mitochondrial dysfunctions that lead to the activation of the apoptotic cascade. Energy imbalance can compromise the maintenance of mitochondrial membrane potential, furthering the release of cytochrome C and the subsequent cleavage and activation of caspases. Chronic constriction injury (CCI) of the rat sciatic nerve is a neuropathy model able to induce a strong mitochondrial impairment with a consequent apoptotic induction. In this model, the acetylcholinesterase inhibitor physostigmine is administered at 0.125 mg/kg i.p. (twice per day) starting from the operation and for 15 days after. The cholinergic activation reduces cytosolic levels of cytochrome C, suggesting an improved stability of the mitochondrial membrane, and the expression level of the active caspase 3 fragments (19, 16 kDa) is reduced significantly with respect to saline treatment. Accordingly, physostigmine impairs caspase 3 protease activity. In fact, the target of the activated caspase 3, the 89-kDa PARP fragment, is significantly less expressed in the ligated nerve of physostigmine-treated rats, reaching levels that are comparable to those in the contralateral unligated nerve. Finally, this natural acetylcholinesterase inhibitor reduces DNA fragmentation both in the proximal and in the distal parts of the nerve. This protection correlates with the induction of XIAP. Therefore, apoptosis, central to tissue degeneration, is prevented by repeated physostigmine treatment of CCI animals.

Topics: Acetylcholine; Animals; Apoptosis; Axons; Caspase 3; Cholinesterase Inhibitors; Cytochromes c; Cytoprotection; Disease Models, Animal; DNA Fragmentation; Energy Metabolism; Male; Mitochondria; Nerve Degeneration; Neuroprotective Agents; Peripheral Nervous System Diseases; Physostigmine; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Rats; Rats, Sprague-Dawley; Sciatic Neuropathy; Sensory Receptor Cells

The present study was undertaken to evaluate possible neuroprotective effect of bradykinin against delayed neuronal death in hippocampal CA1 neurons if applied two days after transient forebrain ischemia in the rat.. Transient forebrain ischemia was induced in male Wistar rats by four-vessel occlusion for 8 min. To assess efficacy of bradykinin as a new stressor for delayed postconditioning we used two experimental groups of animals: ischemia 8 min and 3 days of survival, and ischemia 8 min and 3 days of survival with i.p. injection of bradykinin (150 microg/kg) applied 48 h after ischemia.. We found extensive neuronal degeneration in the CA1 region at day 3 after ischemia/reperfusion. The postischemic neurodegeneration was preceded by increased activity of mitochondrial enzyme MnSOD in cytoplasm, indicating release of MnSOD from mitochondria in the process of delayed neuronal death. Increased cytosolic cytochrome c and subsequently caspase-3 activation are additional signs of neuronal death via the mitochondrial pathway. Bradykinin administration significantly attenuated ischemia-induced neuronal death, and also suppressed the release of MnSOD, and cytochrome c, and prevented caspase-3 activation.. Bradykinin can be used as an effective stressor able to prevent mitochondrial failure leading to apoptosis-like delayed neuronal death in postischemic rat hippocampus.

Topics: Animals; Apoptosis; Bradykinin; Brain; Caspase 3; Cell Count; Cell Death; Cytochromes c; Hippocampus; Immunohistochemistry; Ischemic Attack, Transient; Male; Nerve Degeneration; Neurons; Rats; Rats, Wistar; Superoxide Dismutase

Amyloid beta-peptide (Abeta) has been implicated in the pathogenesis of AD. It can cause cell death in AD by evoking a cascade of oxidative damage to neurons. So antioxidant compounds may throw a light on the treatment of AD. In the present study, we investigated the protective effect of acteoside (AS), an antioxidative phenylethanoid glycoside, on Abeta(25-35)-induced SH-SY5Y cell injury. Exposure of cells to 25 muM Abeta(25-35) for 24 h caused viability loss, apoptotic increase and reactive oxygen species (ROS) increase, pre-treatment with acteoside for 1.5 h significantly reduced the viability loss, apoptotic rate and attenuated Abeta-mediated ROS production. In addition, AS strikingly inhibited Abeta(25-35)-induced mitochondrial dysfunctions, including lowered membrane potential, increased Bax/Bcl-2 ratio, cytochrome c release and the cleavage of caspase-3. Taken together, these results indicated that acteoside could protect SH-SY5Y cells against beta-amyloid-induced cell injury by the attenuating ROS production and the modulating apoptotic signal pathway through Bcl-2 family, cytochrome c, and caspase-3.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Cell Line, Tumor; Cell Survival; Cytochromes c; Glucosides; Humans; Membrane Potential, Mitochondrial; Nerve Degeneration; Neurons; Neuroprotective Agents; Oxidative Stress; Peptide Fragments; Phenols; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

Epilepsy is a prominent sign of neurological dysfunction in children with various fetal and maternal deficiencies. However, the detailed mechanism and influences underlying epileptic disorders are still unrevealed. The hippocampal neurons are vulnerable to epilepsy-induced pathologic changes and often manifests as neuronal death. The present study was designed to investigate the effect of maternal epileptic seizure on apoptotic neuronal death, modulation of GABAB1 receptor (R), and protein kinase A-alpha (PKA) in prenatal rat hippocampal neurons at gestational days (GD) 17.5. Seizure was induced in pregnant rat using intraperitoneal injection of pentylenetetrazol (PTZ) (40 mg/kg for 15 days). To confirm the seizure electroencephalography (EEG) data was obtained by the Laxtha EEG-monitoring device in the EEG recording room and EEG were monitored 5 min and 15 min after PTZ injection. The RT-PCR and Western blot results showed significant increased expression of cytochrome-c and caspases-3, while decreased levels of GABAB1R, and PKA protein expression upon ethanol, PTZ and ethanol plus PTZ exposure in primary neuronal cells cultured from PTZ-induced seizure model as compare to non-PTZ treated maternal group. Apoptotic neurodegeneration was further confirmed with Fluoro-Jade B and propidium iodide staining, where neurons were scattered and shrunken, with markedly condensed nuclei in PTZ treated group compared with control. This study for the first time indicate that PTZ-induced seizures triggered activation of caspases-3 to induce widespread apoptotic neuronal death and decreased GABAB1R expression in hippocampal neurons, providing a possible mechanistic link between maternal epilepsy induced neurodegeneration alteration of GABAB1R and PKA expression level during prenatal brain development. This revealed new aspects of PTZ and ethanol's modulation on GABAB1R, learning and memory. Further, explain the possibility that children delivered by epileptic mothers may have higher risk of developmental disturbances and malformations.

Topics: Animals; Apoptosis; Brain; Caspase 3; Cyclic AMP-Dependent Protein Kinases; Cytochromes c; Electroencephalography; Epilepsy; Female; Fetus; Gene Expression Regulation, Developmental; Hippocampus; Maternal Exposure; Mitochondria; Nerve Degeneration; Neurons; Pentylenetetrazole; Pregnancy; Rats; Receptors, GABA-B; RNA, Messenger

Lead, a ubiquitous and potent neurotoxicant causes oxidative stress which leads to numerous neurobehavioral and physiological alterations. The ability of lead to bind sulfhydryl groups or compete with calcium could be one of the reasons for its debilitating effects. In the present study, we addressed: i) if chelation therapy could circumvent the altered oxidative stress and prevent neuronal apoptosis in chronic lead-intoxicated rats, ii) whether chelation therapy could reverse biochemical and behavioral changes, and iii) if mono or combinational therapy with captopril (an antioxidant) and thiol chelating agents (DMSA/MiADMSA) is more effective than individual thiol chelator in lead-exposed rats. Results indicated that lead caused a significant increase in reactive oxygen species, nitric oxide, and intracellular free calcium levels along with altered behavioral abnormalities in locomotor activity, exploratory behavior, learning, and memory that were supported by changes in neurotransmitter levels. A fall in membrane potential, release of cytochrome c, and DNA damage indicated mitochondrial-dependent apoptosis. Most of these alterations showed significant recovery following combined therapy with captopril with MiADMSA and to a smaller extend with captopril+DMSA over monotherapy with these chelators. It could be concluded from our present results that co-administration of a potent antioxidant (like captopril) might be a better treatment protocol than monotherapy to counter lead-induced oxidative stress. The major highlight of the work is an interesting experimental evidence of the efficacy of combinational therapy using an antioxidant with a thiol chelator in reversing neurological dystrophy caused due to chronic lead exposure in rats.

Topics: Animals; Antioxidants; Apoptosis; Behavior, Animal; Biogenic Amines; Calcium; Captopril; Chelating Agents; Cytochromes c; Disease Models, Animal; DNA Damage; Drug Therapy, Combination; Exploratory Behavior; Lead Poisoning, Nervous System; Learning; Male; Membrane Potential, Mitochondrial; Memory; Mitochondria; Motor Activity; Nerve Degeneration; Neurons; Nitric Oxide; Organometallic Compounds; Oxidative Stress; Rats; Rats, Wistar; Reactive Oxygen Species; Succimer

Pramipexole, an agonist for dopamine (DA) D2/D3-receptors, has been used to treat both early and advanced Parkinson's disease (PD). In this study, we examined the effect of pramipexole on DA neurons in a PD model of C57BL/6 mice, which were treated with rotenone (30 mg/kg, p.o.) daily for 28 days. Pramipexole (1 mg/kg, i.p.) was injected daily 30 min before each oral administration of rotenone. Chronic oral administration of rotenone caused a loss of DA neurons in the substantia nigra pars compacta (SNpc), motor deficits and the up-regulation of alpha-synuclein immunoreactivity in some surviving DA neurons. Pramipexole inhibited rotenone-induced DA neuronal death and motor deficits, and reduced immunoreactivity for alpha-synuclein. In addition, pramipexole inhibited the in vitro oligomerization of human wild-type alpha-synuclein by H(2)O(2)plus cytochrome c. To examine the neuroprotective effect of pramipexole against oxidative stress, we used a DJ-1-knockdown SH-SY5Y cell line and electron spin resonance (ESR) spectrometry. Simultaneous treatment with H(2)O(2) and pramipexole resulted in the significant protection of DJ-1-knockdown cells against cell death in a concentration-dependent manner. A high concentration of pramipexole directly scavenged hydroxyl radical (*OH) generated from H(2)O(2) and Fe(2+). Furthermore, pramipexole increased Bcl-2 immunoreactivity in DA neurons in the SNpc. These results suggest that pramipexole may protect DA neurons against exposure to rotenone by chronic oral administration, and this effect is mediated by multiple functions including scavenging of *OH and induction of Bcl-2 protein.

Topics: alpha-Synuclein; Animals; Antiparkinson Agents; Apoptosis; Benzothiazoles; Cell Line, Tumor; Corpus Striatum; Cytochromes c; Dopamine; Dose-Response Relationship, Drug; Humans; Hydrogen Peroxide; Hydroxyl Radical; Male; Mice; Mice, Inbred C57BL; Nerve Degeneration; Neural Pathways; Neurotoxins; Oxidative Stress; Parkinsonian Disorders; Pramipexole; Proto-Oncogene Proteins c-bcl-2; Rotenone; Substantia Nigra; Uncoupling Agents

Intracerebral hemorrhage (ICH) remains a major medical problem and currently has no effective treatment. Hemorrhaged blood is highly toxic to the brain, and catabolism of the pro-oxidant heme, mainly released from hemoglobin, is critical for the resolution of hematoma after ICH. The degradation of the pro-oxidant heme is controlled by heme oxygenase (HO). We have previously reported a neuroprotective role for HO2 in early brain injury after ICH; however, in vivo data that specifically address the role of HO2 in brain edema and neuroinflammation after ICH are absent. Here, we tested the hypothesis that HO2 deletion would exacerbate ICH-induced brain edema, neuroinflammation, and oxidative damage. We subjected wild-type (WT) and HO2 knockout ((-/-)) mice to the collagenase-induced ICH model. Interestingly, HO2(-/-) mice had enhanced brain swelling and neuronal death, although HO2 deletion did not increase collagenase-induced bleeding; the exacerbation of brain injury in HO2(-/-) mice was also associated with increases in neutrophil infiltration, microglial/macrophage and astrocyte activation, DNA damage, peroxynitrite production, and cytochrome c immunoreactivity. In addition, we found that hemispheric enlargement was more sensitive than brain water content in the detection of subtle changes in brain edema formation in this model. Combined, these novel findings extend our previous observations and demonstrate that HO2 deficiency increases brain swelling, neuroinflammation, and oxidative damage. The results provide additional evidence that HO2 plays a critical protective role against ICH-induced early brain injury.

Topics: Analysis of Variance; Animals; Brain Edema; Calcium-Binding Proteins; Cerebral Hemorrhage; Cytochromes c; Disease Models, Animal; Encephalitis; Fluoresceins; Functional Laterality; Glial Fibrillary Acidic Protein; Granulocyte Colony-Stimulating Factor; Heme Oxygenase (Decyclizing); Interleukin-3; Mice; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Nerve Degeneration; Organic Chemicals; Recombinant Fusion Proteins; Recombinant Proteins; Spectrophotometry; Time Factors; Tyrosine

Parkinson disease is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. It has been proposed that dysfunction of the ubiquitin proteasome system plays an important role in the pathogenesis of Parkinson disease, but the mechanisms underlying ubiquitin proteasome system-related neuron degeneration are unknown. Here, we demonstrate that the proteasome inhibitor lactacystin induces phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun, the release of cytochrome c, activation of both caspase-9 and caspase-3, and sequential apoptosis of dopaminergic neurons in vitro. Most of these effects can be attenuated by the JNK inhibitor SP600125. Furthermore, infusion of lactacystin in rats in vivo also leads to phosphorylation of JNK before nigral neuron loss; chronic administration of SP600125 also blocks this loss. These results indicate that JNK is involved in proteasome inhibition-induced dopaminergic neuron degeneration through caspase-3-mediated apoptotic pathways, suggesting that this kinase may be a therapeutic target for the prevention of substantia nigra pars compacta degeneration in Parkinson disease patients.

Topics: Acetylcysteine; Animals; Apoptosis; Blotting, Western; Caspase 3; Cell Line; Chromatography, High Pressure Liquid; Cysteine Proteinase Inhibitors; Cytochromes c; Dopamine; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; Male; Nerve Degeneration; Neurons; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Substantia Nigra; Tetrazolium Salts; Thiazoles

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

Platelet-derived growth factor (PDGF) has been implicated in promoting survival and proliferation of immature neurons, and even protecting neurons from gp120-induced cytotoxicity. However, the mechanisms involved in neuroprotection are not well understood. In the present study we demonstrate the role of phosphatidylinositol 3-kinase (PI3K)/Akt signaling in PDGF-mediated neuroprotection. Pharmacological inhibition of PI3K greatly reduced the ability of PDGF-BB to block gp120 IIIB-mediated apoptosis and cell death in human neuroblastoma cells. The role of Akt in PDGF-mediated protection was further corroborated using a dominant-negative mutant of Akt, which was able to block the protective effect of PDGF. We next sequentially examined the signals downstream of Akt in PDGF-mediated protection in human neuroblastoma cells. In cells pretreated with PDGF prior to gp120 there was increased phosphorylation of both GSK-3beta and Bad, an effect that was inhibited by PI3-kinase inhibitor. Nuclear translocation of NF-kappaB, which lies downstream of GSK-3beta, however, remained unaffected in cells treated with PDGF. In addition to inducing phosphorylation of Bad, PDGF-mediated protection also involved down-regulation of the proapoptotic protein Bax. Furthermore, PDGF-mediated protection also involved the inhibition of gp120-induced release of mitochondrial cytochrome C. Our findings thus underscore the roles of both PI3K/Akt and Bcl family pathways in PDGF-mediated neuroprotection.

Topics: Active Transport, Cell Nucleus; AIDS Dementia Complex; Animals; Apoptosis Regulatory Proteins; bcl-Associated Death Protein; Cell Line, Tumor; Cells, Cultured; Cytochromes c; Cytoprotection; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HIV Envelope Protein gp120; Humans; Nerve Degeneration; Neuroprotective Agents; NF-kappa B; Phosphatidylinositol 3-Kinases; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction

Deposition of amyloid-beta protein (Abeta) is one of the most important pathologic features in Alzheimer's disease. It is well known that Abeta induces neuronal cell death through several pathogenic mechanisms. Although the role of glycogen synthase kinase (GSK)-3beta in the neurotoxicity of Abeta has been highlighted, there has been no report evaluating the effect of direct GSK-3beta inhibition on Abeta-induced neurotoxicity. Thus, in this study, the relationship between GSK-3beta activity and Abeta-induced neurotoxicity was explored. To investigate the role of GSK-3beta in Abeta-induced neurotoxicity, neurons were treated with amyloid beta-protein (1-42) (Abeta42) oligomers with or without the addition of a GSK-3beta inhibitor for 72 h. An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, trypan blue staining, and DAPI staining all showed that Abeta42 treatment alone resulted in decreased neuronal cell viability in a concentration-dependent manner. Abeta42 treatment significantly increased the activity of GSK-3beta and cell death signals such as phosphorylated Tau (pThr231), cytosolic cytochrome c, and activated caspase-3. Abeta42 treatment also resulted in decreased survival signals, including that of heat shock transcription factor-1. Treatment with a GSK-3beta inhibitor prevented Abeta-induced cell death. These results suggest that the neurotoxic effect of Abeta42 is mediated by GSK-3beta activation and that inhibition of GSK-3beta can reduce Abeta42-induced neurotoxicity.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Caspase 3; Cell Survival; Cells, Cultured; Coloring Agents; Cytochromes c; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Heat Shock Transcription Factors; Indicators and Reagents; Nerve Degeneration; Neurons; Neurotoxins; Peptide Fragments; Rats; Rats, Sprague-Dawley; Signal Transduction; tau Proteins; Tetrazolium Salts; Transcription Factors

The evolving role of mitochondria as a target for different death-inducing noxae prompted us to investigate trimethyltin (TMT)-dependent effects on mitochondrial functionality. For this purpose, we used a homogeneous cell culture model represented by undifferentiated PC12 cells. Mitochondria isolated from PC12 cells treated with TMT for 6, 12 and 24h, showed a time-dependent inhibition of ADP-stimulated oxygen consumption using succinate or glutamate/malate as substrate. Using a fluorescent assay, the effect of TMT on mitochondrial membrane potential (delta Psi) in PC12 cells was also determined. After 24h in culture, a strong loss of mitochondrial membrane potential (delta Psi) was observed in TMT-treated cells. Collapse of mitochondrial membrane potential correlated with an increased expression of bax/bcl-2 ratio, as evaluated by polymerase chain reaction. Western blotting and spectrophotometric analysis showed that cytochrome c release and activation of caspase 3 were concurrently induced. Our findings suggest that inhibition of mitochondrial respiration represents the early toxic event for cell death in PC12 due to trimethyltin.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Respiration; Cytochromes c; Gene Expression Regulation; Membrane Potential, Mitochondrial; Mitochondria; Nerve Degeneration; Neurotoxins; Oxygen Consumption; PC12 Cells; Proto-Oncogene Proteins c-bcl-2; Rats; Trimethyltin Compounds

We have demonstrated that kainate (KA) induces a reduction in mitochondrial Mn-superoxide dismutase (Mn-SOD) expression in the rat hippocampus and that KA-induced oxidative damage is more prominent in senile-prone (SAM-P8) than senile-resistant (SAM-R1) mice. To extend this, we examined whether KA seizure sensitivity contributed to mitochondrial degeneration in these mouse strains. KA-induced seizure susceptibility in SAM-P8 mice paralleled prominent increases in lipid peroxidation and protein oxidation and was accompanied by significant impairment in glutathione homeostasis in the hippocampus. These findings were more pronounced in the mitochondrial fraction than in the hippocampal homogenate. Consistently, KA-induced decreases in Mn-SOD protein expression, mitochondrial transmembrane potential, and uncoupling protein (UCP)-2 expression were more prominent in SAM-P8 than SAM-R1 mice. Marked release of cytochrome c from mitochondria into the cytosol and a higher level of caspase-3 cleavage were observed in KA-treated SAM-P8 mice. Additionally, electron microscopic evaluation indicated that KA-induced increases in mitochondrial damage and lipofuscin-like substances were more pronounced in SAM-P8 than SAM-R1 animals. These results suggest that KA-mediated mitochondrial oxidative stress contributed to hippocampal degeneration in the senile-prone mouse.

Topics: Aging, Premature; Animals; Caspase 3; Cytochromes c; Disease Models, Animal; Enzyme Activation; Glutathione; Glutathione Disulfide; Hippocampus; Ion Channels; Kainic Acid; Lipid Peroxidation; Lipofuscin; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred Strains; Mitochondria; Mitochondrial Proteins; Nerve Degeneration; Neurons; Oxidation-Reduction; Oxidative Stress; Proteins; Proto-Oncogene Proteins c-fos; Seizures; Superoxide Dismutase; Time Factors; Uncoupling Protein 2

Amyloid-beta protein (A beta) and the scrapie isoform of prion protein (PrPSs) have a central role in the pathogenesis of Alzheimer's disease (AD) and prion-related encephalopathies (PRE), respectively. In both disorders, the deposition of these misfolded proteins is accompanied by apoptotic neuronal loss. However, the pathogenesis and molecular basis of A beta- and PrPSc-neurotoxic effects are not completely understood. The Ca2+/calmodulin-dependent phosphatase calcineurin (CaN), through the dephosphorylation of the proapoptotic protein BAD, may be the link between Ca2+homeostasis deregulation and apoptotic neuronal death. In this study we used primary cultures of rat brain cortical neurons in order to investigate whether A beta and PrP affect CaN activity. We observed that synthetic peptides of A beta (A beta 25-35 and A beta 1-40) and PrP (PrP106-126) increased CaN activity, but did not affect the levels of this protein phosphatase. Moreover, we found that these peptides reduced the levels of BAD phosphorylated at serine residue 112, and this effect was prevented by the CaN inhibitor FK506. Since dephosphorylated BAD translocates to mitochondria, where it triggers cytochrome c release, we determined the levels of BAD in mitochondrial and cytosolic fractions. The data obtained showed that A beta- and PrP-treated neurons had higher levels of BAD in mitochondria than control neurons. This increase in mitochondrial BAD levels was matched by a decrease in cytochrome c. FK506 prevented the alterations of mitochondrial BAD and cytochrome c levels induced by A beta and PrP peptides. Taken together the data suggest that A beta and PrP increased CaN activity, inducing BAD dephosphorylation and translocation to mitochondria and, subsequently, cytochrome c release that may trigger an apoptotic cascade. Therefore, therapeutic strategies targeting CaN might be valuable for these neurodegenerative disorders.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Apoptosis; bcl-Associated Death Protein; Brain; Calcineurin; Cells, Cultured; Cytochromes c; Enzyme Activation; Mitochondria; Nerve Degeneration; Neurons; Peptides; Prion Diseases; Protein Transport; PrPSc Proteins; Rats; Rats, Wistar

Kynurenic acid (KYNA), a tryptophan metabolite in the kynurenine pathway, is protective against various insults. However, the molecular mechanism of this protective effect has not been identified. In this study, we examined the protective effects of KYNA against 1-methyl-4-phenylpyridinium (MPP(+)), the best-characterized toxin inducing pathological changes resembling Parkinson's disease (PD), using SH-SY5Y and SK-N-SH human neuroblastoma cells. Pre-treatment of KYNA attenuated MPP(+)-induced neuronal cell death in SH-SY5Y and SK-N-SH cells. MPP(+)-induced cell death was preceded by increases in Bax expression and mitochondrial dysfunction, such as collapse of mitochondrial membrane potential (DeltaPsi(m)), release of cytochrome c from mitochondria into the cytoplasm, and increases in caspase-9/-3 activities. KYNA effectively inhibited all of these mitochondrial apoptotic processes. Our results indicate that KYNA plays a protective role by down-regulating Bax expression and maintaining mitochondrial function in MPP(+)-induced neuronal cell death, and suggest that KYNA may have therapeutic potential in PD.

Topics: 1-Methyl-4-phenylpyridinium; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Dopamine; Enzyme Activation; Humans; Kynurenic Acid; Membrane Potential, Mitochondrial; Mitochondria; Nerve Degeneration; Neurons; Time Factors

Oxidative stress is believed to contribute to neuronal damage induced by cerebral ischemia/reperfusion (I/R) injury. The present study was undertaken to evaluate the possible antioxidant neuroprotective effect of genistein against neuronal death in hippocampal CA1 neurons following transient global cerebral ischemia in the rat. Transient global cerebral ischemia was induced in male Sprague-Dawley rats by four-vessel-occlusion for 10min. At various times of reperfusion, the histopathological changes and the levels of mitochondria-generated reactive oxygen species (ROS), malondialdehyde (MDA), cytosolic cytochrome c and caspase-3 activity in hippocampus were measured. We found extensive neuronal death in the CA1 region at day 5 after I/R. The ischemic changes were preceded by increases in ROS generation and MDA concentration and followed by increased cytosolic cytochrome c, and subsequently caspase-3 activation and apoptosis. Treatment with genistein (15mg/kg, i.p.) significantly attenuated ischemia-induced neuronal death. Genistein administration also decreased ROS generation, MDA concentration and the apoptotic indices. These results suggest that genistein protects neurons from transient global cerebral I/R injury in rat hippocampus by attenuating oxidative stress, lipid peroxidation and the signaling cascade leading to apoptotic cell death.

Topics: Animals; Antioxidants; Apoptosis; Brain Ischemia; Caspase 3; Cerebral Infarction; Cytochromes c; Disease Models, Animal; Down-Regulation; Genistein; Lipid Peroxidation; Male; Malondialdehyde; Nerve Degeneration; Neuroprotective Agents; Oxidative Stress; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Treatment Outcome

Loss-of-function mutations of attractin (Atrn) in animals result in age-dependent progressive neurodegeneration including neuronal cell death, hypomyelination and vacuolation. The mechanisms of how age-dependent neurodegeneration occurs in these animals are not clear. In this study, we found that reducing the endogenous expression level of Atrn exacerbated, whereas overexpressing Atrn protected against, the neuronal cell death caused by the neurotoxins, 1-methyl-4-phenylpyridinium (MPP+) and lactacystin. In addition, both MPP+ and lactacystin-induced cytochrome c and apoptosis inducing factor (AIF) release, which was inhibited by overexpressing Atrn and enhanced by knocking down Atrn, indicating that Atrn may be involved in regulating the mitochondrial function. Furthermore, we found that vast majority of the dopaminergic neurons in mice express Atrn and its expression decreases with age. Our findings demonstrated that Atrn may play a protective role against environmental toxins, and implied a potential therapeutic effect of Atrn for neurodegenerative diseases.

Topics: 1-Methyl-4-phenylpyridinium; Acetylcysteine; Analysis of Variance; Apoptosis Inducing Factor; Cell Line, Tumor; Cell Survival; Cytochromes c; Gene Expression Regulation; Humans; In Situ Hybridization; Membrane Proteins; Nerve Degeneration; Neurotoxins; RNA, Small Interfering; Transfection; Tyrosine 3-Monooxygenase

Both the neurotransmitter dopamine (DA) and a neurotoxic metabolite, 6-hydroxy DA, can be oxidized to generate hydrogen peroxide and other reactive species (ROS). ROS promote oxidative stress and have been implicated in dopaminergic neurodegeneration, e.g., Parkinson's disease (PD). There is also evidence for a relation between catecholamine-mediated oxidative damage in dopaminergic neurons and the effects of these neurotransmitters on the redox state of cytochrome c (Cytc). In neurons and other cells, oxidative stress may be enhanced by abnormal release of Cytc and other mitochondrial proteins into the cytoplasm. Cytc release can result in apoptosis; but sub-apoptogenic-threshold release can also occur, and may be highly damaging in the presence of DA metabolites. Loss of mitochondrial membrane integrity, a pathological situation of relevance to several aging-related neurodegenerative disorders including PD, contributes to release of Cytc; and the level of such release is known to be indicative of the extent of mitochondrial dysfunction. In this context, we have used a Cytc-enhanced 6-hydroxy DA oxidation reaction to gauge dietary antioxidant activities. Anthocyanin-rich preparations of Vaccinium species (Vaccinium myrtillus, Vaccinium corymbosum, and Vaccinium oxycoccus) as well as a purified glycosylated anthocyanidin were compared. The most potent inhibition of oxidation was observed with V. myrtillus preparation: 50% inhibition with 7 microM of total anthocyanins. This activity was 1.5-4 times higher than that for the other preparations or for the purified anthocyanin. Ascorbate (Vitamin C), at up to 4-fold higher concentrations, did not result in significant inhibition in this assay. Antioxidant activity in the assay correlated strongly (r2>0.91, P<0.01) with reported Vaccinium content of anthocyanins and total cyanidins, but not quercetin or myricetin. The results provide evidence for the high potency of anthocyanins towards a potentially neurotoxic reaction, and provide a basis for in vivo testing of these flavonoids and their physiological metabolites in the context of neuro- and mitochondrio-protective effects.

Topics: Animals; Anthocyanins; Antioxidants; Ascorbic Acid; Cytochromes c; Dopamine; Flavonoids; Glucosides; Kinetics; Mitochondria; Nerve Degeneration; Neurotoxicity Syndromes; Oxidation-Reduction; Oxidopamine; Sympatholytics; Vaccinium

The present study investigated the effect of 5-hydroxydecanoate, a selective mitochondrial K(ATP) channel blocker, on the cytotoxicity of neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) in differentiated PC12 cells. 5-Hydroxydecanoate and glibenclamide (a cell surface and mitochondrial K(ATP) channel inhibitor) reduced the MPP(+)-induced cell death and GSH depletion and showed a maximal inhibitory effect at 5 and 10 microM, respectively. Addition of 5-hydroxydecanoate attenuated the MPP(+)-induced nuclear damage, changes in the mitochondrial membrane permeability and increase in the reactive oxygen species formation in PC12 cells. The results show that 5-hydroxydecanote may prevent the MPP(+)-induced viability loss in PC12 cells by suppressing formation of the mitochondrial permeability transition, leading to the cytochrome c release and caspase-3 activation. This effect appears to be accomplished by the inhibitory action on the formation of reactive oxygen species and the depletion of GSH. The blockade of mitochondrial K(ATP) channels seems to prevent the MPP(+)-induced neuronal cell damage.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Caspase 3; Cell Death; Cell Membrane Permeability; Cell Nucleus; Cell Survival; Cytochromes c; Decanoic Acids; Glutathione; Glyburide; Herbicides; Hydroxy Acids; Mitochondria; Mitochondrial Membranes; Nerve Degeneration; Neurons; Neuroprotective Agents; Oxidative Stress; PC12 Cells; Potassium Channel Blockers; Potassium Channels; Rats; Reactive Oxygen Species

Glycogen synthase kinase-3, especially the beta form (GSK-3beta), plays key roles in oxidative stress-induced neuronal cell death, an important pathogenic mechanism of various neurodegenerative diseases. Although the neuroprotective effects of GSK-3beta inhibitors have been described, the optimal level of GSK-3beta inhibition for neuronal cell survival has not yet been determined. We investigated the effect of varying GSK-3beta activity on the viability of oxidative stress-injured neuronally differentiated PC12 (nPC12) cells and intracellular signals related with the GSK-3beta and caspase-3 pathways. Compared to the nPC12 control cells treated with only 100 microM H(2)O(2), treatment of 50-200 nM GSK-3beta inhibitor II or 25-500 nM GSK-3beta inhibitor VIII reduced the increased enzyme activity by about 50% and protected the cells against H(2)O(2)-induced oxidative stress. The optimal concentration of GSK-3beta inhibitor II enhanced heat shock transcription factor-1 levels, decreased levels of phosphorylated tau (Ser202) and cytosolic cytochrome c, activated caspase-3, and cleaved poly (ADP-ribose) polymerase. In contrast, higher concentrations of GSK-3beta inhibitor II (more than 500 nM) induced neuronal cell death and showed opposite effects relative to the above described intracellular signals. These results suggest that optimized inhibitor levels for modulating GSK-3beta activity may prevent apoptosis induced by oxidative stress associated with neurodegenerative diseases.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Cytochromes c; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Heat Shock Transcription Factors; Nerve Degeneration; Neurons; Neuroprotective Agents; Oxidative Stress; PC12 Cells; Poly(ADP-ribose) Polymerases; Rats; Signal Transduction; tau Proteins; Transcription Factors

Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The aim of the present study was to assess the effect of antiepileptic lamotrigine against the cytotoxicity of mitochondrial respiratory complex I inhibitors rotenone and 1-methyl-4-phenylpyridinium (MPP+) in relation to the mitochondria-mediated cell death process and oxidative stress. Both rotenone and MPP+ induced the nuclear damage, the changes in the mitochondrial membrane permeability, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in differentiated PC12 cells. Lamotrigine significantly attenuated the rotenone- or MPP+-induced mitochondrial damage leading to caspase-3 activation, increased oxidative stress and cell death. The preventive effect of lamotrigine against the toxicity of rotenone was greater than its effect on that of MPP+. The results show that lamotrigine seems to reduce the cytotoxicity of rotenone and MPP+ by suppressing the mitochondrial permeability transition formation, leading to cytochrome c release and subsequent activation of caspase-3. The preventive effect may be ascribed to its inhibitory action on the formation of reactive oxygen species and depletion of GSH. Lamotrigine seems to exert a protective effect against the neuronal cell injury due to the mitochondrial respiratory complex I inhibition.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Apoptosis; Caspase 3; Cell Death; Cell Membrane Permeability; Cytochromes c; Electron Transport Complex I; Enzyme Activation; Excitatory Amino Acid Antagonists; Glutathione; Lamotrigine; Membrane Potential, Mitochondrial; Mitochondria; Nerve Degeneration; Neurons; Neurotoxins; Oxidative Stress; PC12 Cells; Rats; Reactive Oxygen Species; Rotenone; Triazines

There is evidence showing impaired mitochondrial energy production and increased oxidative damage to mitochondria in amyotrophic lateral sclerosis (ALS). It is known that a lack of cytochrome c (CyC) in the mitochondrial intermembrane space can increase free radical release from mitochondria through interruption of the electron transport. CyC also plays a role in the apoptotic cell death which is suspected in ALS. The aim of the study was to measure cerebrospinal fluid (CSF) and serum CyC levels in patients with ALS.. Forty ALS patients were diagnosed according to the El Escorial criteria of ALS. The clinical state of the patients was measured using the Amyotrophic Lateral Sclerosis Functional Rating Scale [ALSFRS].. It was shown that overall CyC levels were significantly decreased by 46 % in the CSF of patients with ALS compared with controls (p<0.05), and not affected in serum of patients with ALS (p>0.05). There was no significant difference in CyC levels in relation to the clinical parameters of the disease (p>0.05).. The study indicates that CyC may play a role in the pathogenesis of ALS. A possible mechanism is that increased neurodegeneration in ALS caused by free radical production decreases the concentrations of CyC.

Topics: Adult; Age Factors; Aged; Amyotrophic Lateral Sclerosis; Apoptosis; Case-Control Studies; Cerebrospinal Fluid Proteins; Cytochromes c; Down-Regulation; Electron Transport; Female; Humans; Male; Middle Aged; Mitochondria; Nerve Degeneration; Reference Values

Cerebral ischemia followed by reperfusion activates numerous pathways that lead to cell death. One such pathway involves the release of large quantities of the excitatory amino acid glutamate into the synapse and activation of N-methyl-D-aspartate receptors. This causes an increase in mitochondrial calcium levels ([Ca(2+)](m)) and a production of reactive oxygen species (ROS), both of which may induce the mitochondrial permeability transition (MPT). As a consequence, there is eventual mitochondrial failure culminating in either apoptotic or necrotic cell death. Thus, agents that inhibit MPT might prove useful as therapeutic interventions in cerebral ischemia. In this study, we have investigated the neuroprotective efficacy of the novel compound NIM811. Similar in structure of its parent compound cyclosporin A, NIM811 is a potent inhibitor of the MPT. Unlike cyclosporin A, however, it is essentially void of immunosuppressive actions, allowing the role of MPT to be clarified in ischemia/reperfusion injury. The results of these studies demonstrate that NIM811 provides almost 40% protection in a model of transient focal cerebral ischemia. This was associated with a nearly 10% reduction in mitochondrial reactive species formation and 34% and 38% reduction of cytochrome c release in core and penumbra, respectively. Treatment with NIM811 also increased calcium retention capacity by approximately 20%. Interestingly, NIM811 failed to improve ischemia-induced impairment of bioenergetics. The neuroprotective effects of NIM811 were not due to drug-induced alterations in cerebral perfusion after ischemia. Activation of MPT appears to be an important process in ischemia/reperfusion injury and may be a therapeutic target.

Topics: Animals; Brain Infarction; Brain Ischemia; Calcium Signaling; Cell Death; Cyclosporine; Cytochromes c; Disease Models, Animal; Energy Metabolism; Male; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Nerve Degeneration; Neurons; Neuroprotective Agents; Oxidative Stress; Rats; Rats, Inbred SHR; Reactive Oxygen Species; Treatment Outcome

The mechanisms underlying neurologic impairment in celiac disease remain unknown. We tested whether antineuronal antibody-positive sera of patients with celiac disease evoke neurodegeneration via apoptosis in vitro.. SH-Sy5Y cells were exposed to crude sera, isolated immunoglobulin (Ig) G and IgG-depleted sera of patients with and without celiac disease with and without neurologic disorders, and antineuronal antibodies. Adsorption studies with gliadin and tissue transglutaminase (tTG) were performed in celiac disease sera. Apoptosis activated caspase-3, apaf-1, Bax, cytochrome c, cleaved caspase-8 and caspase-9 and mitochondrial respiratory chain complexes were evaluated with different methods.. SH-Sy5Y cells exposed to antineuronal antibody-positive sera and isolated IgG from the same sera exhibited a greater percentage of TUNEL-positive nuclei than that of antineuronal antibody-negative sera. Neuroblasts exposed to antineuronal antibody-negative celiac disease sera also showed greater TUNEL positivity and apaf-1 immunolabeled cells than controls. Antigliadin- and anti-tTG-depleted celiac disease sera had an apoptotic effect similar to controls. Anti-caspase-3 immunostained cells were greater than controls when exposed to positive sera. The mitochondrial respiratory chain complex was reduced by positive sera. Western blot demonstrated only caspase-9 cleavage in positive sera. Cytochrome c and Bax showed reciprocal translocation (from mitochondria to cytoplasm and vice versa) after treatment with positive sera.. Antineuronal antibodies and, to a lower extent, combined antigliadin and anti-tTG antibodies in celiac disease sera contribute to neurologic impairment via apoptosis. Apaf-1 activation with Bax and cytochrome c translocation suggest a mitochondrial-dependent apoptosis.

Topics: Adult; Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; Bisbenzimidazole; Caspase 3; Caspase 9; Celiac Disease; Cell Line, Tumor; Citrate (si)-Synthase; Cytochromes c; Electron Transport Complex I; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Dyes; Humans; Immunoglobulin G; In Situ Nick-End Labeling; In Vitro Techniques; Male; Middle Aged; Mitochondria; Nerve Degeneration; Neuroblastoma

Peripheral neuropathies are widespread disorders induced by autoimmune diseases, drug or toxin exposure, infections, metabolic insults or trauma. Nerve damage may cause muscle weakness, altered functionalities and sensitivity, and a chronic pain syndrome characterized by allodynia and hyperalgesia. Pathophysiological mechanisms related to neuropathic disease are associated with mitochondrial dysfunctions that lead to the activation of the apoptotic cascade. In a model of peripheral neuropathy, obtained by the loose ligation of the rat sciatic nerve (CCI), we describe a nerve apoptotic state that encompasses the release of cytochrome C in the cytosol, the activation of caspase 3, and the fragmentation of the genome. Animal treatment with acetyl-L-carnitine (ALCAR), but not with L-carnitine (L-Carn) or Gabapentin, prevents apoptosis induction. ALCAR reduces cytosolic cytochrome C and caspase 3 active fragments expression in a significant manner with respect to saline treatment. Accordingly, ALCAR treatment impairs caspase 3 protease activity, as demonstrated by reduced levels of cleaved PARP. Finally, ALCAR decreases the number of piknotic nuclei. This protection correlates with the induction of X-linked inhibitor apoptosis protein (XIAP). Taken together these results show that CCI is a valuable model to investigate neuropathies-related apoptosis phenomena and that ALCAR is able to prevent regulated cell death in the damaged sciatic nerve.

Topics: Acetylcarnitine; Animals; Apoptosis; Blotting, Western; Caspase 3; Cytochromes c; Cytosol; DNA Fragmentation; Hyperalgesia; In Situ Nick-End Labeling; Male; Nerve Degeneration; Nootropic Agents; Peripheral Nervous System Diseases; Poly(ADP-ribose) Polymerases; Rats; Rats, Sprague-Dawley; Sciatic Neuropathy

Previously, we demonstrated neuroprotection with 2-iminobiotin (2-IB) after cerebral hypoxia-ischemia (HI) in female, but not in male P7 rats. Given the different patterns of brain injury in more immature rats, we examined whether these gender differences could also be observed in P3 rats. HI was induced by unilateral carotid ligation and FiO2 reduction, followed by 2-IB administration. HSP70 protein expression and cytochrome c release from the mitochondria, markers of short-term outcome, were induced by HI to the same extent in male and female animals. However, reduction in HSP70 production and cytochrome c release by 2-IB was seen in female rats only. Long-term cerebral injury after HI, assessed with histology, was similar in male and female P3 rats, but long-term neuroprotection by 2-IB was observed in female rats only. In conclusion, 2-IB provides neuroprotection after cerebral HI in female, but not in male immature P3 rats.

Topics: Aging; Animals; Animals, Newborn; Biotin; Birth Injuries; Brain; Cell Death; Cytochromes c; Cytoprotection; Disease Models, Animal; Female; HSP70 Heat-Shock Proteins; Hypoxia-Ischemia, Brain; Male; Mitochondria; Nerve Degeneration; Neuroprotective Agents; Oxidative Stress; Rats; Rats, Wistar; Sex Characteristics; Time; Treatment Outcome

The present study elucidates a possible mechanism by which chronic organophosphate exposure (dichlorvos 6 mg/kg bw, s.c. for 12 weeks) causes neuronal degeneration. Mitochondria, as a primary site of cellular energy generation and oxygen consumption represent itself a likely target for organophosphate poisoning. Therefore, the objective of the current study was planned with an aim to investigate the effect of chronic dichlorvos exposure on mitochondrial calcium uptake, oxidative stress generation and its implication in the induction of neuronal apoptosis in rodent model. Mitochondrial preparation from dichlorvos (DDVP) treated rat brain demonstrated significant increase in mitochondrial Ca(2+) uptake (644.2 nmol/mg protein). Our results indicated decreased mitochondrial electron transfer activities of cytochrome oxidase (complex IV) along with altered mitochondrial complex I, and complex II activity, which might have resulted from elevated mitochondrial calcium uptake. The alterations in the mitochondrial calcium uptake and mitochondrial electron transfer enzyme activities in turn might have caused an increase in malondialdehyde, protein carbonyl and 8-hydroxydeoxyguanosine formation as a result of enhanced lipid peroxidation, and as well as protein and mtDNA oxidation. All this could have been because of enhanced oxidative stress, decreased GSH levels and also decreased Mn-SOD activity in the mitochondria isolated from dichlorvos treated rat brain. Thus, chronic organophosphate exposure has the potential to disrupt cellular antioxidant defense system which in turn triggers the release of cytochrome c from mitochondria to cytosol as well as caspase-3 activation in dichlorvos treated rat brain as revealed by immunoblotting experiments. Low-level long-term organophosphate exposure finally resulted in oligonucleosomal DNA fragmentation, a hallmark of apoptosis. These studies provide an evidence of impaired mitochondrial bioenergetics and apoptotic neuronal degeneration after chronic low-level exposure to dichlorvos.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Apoptosis; Brain; Calcium; Caspase 3; Cytochromes c; Deoxyguanosine; Dichlorvos; DNA, Mitochondrial; Dose-Response Relationship, Drug; Electron Transport Complex I; Electron Transport Complex II; Electron Transport Complex IV; Energy Metabolism; Enzyme Activation; Glutathione; Insecticides; Lipid Peroxidation; Male; Mitochondria; Nerve Degeneration; Neurons; Oxidative Stress; Protein Carbonylation; Rats; Rats, Wistar; Reactive Oxygen Species; Superoxide Dismutase; Time Factors

In previous studies, we have shown that cerebral hypoxia results in increased activity of caspase-9, the initiator caspase, and caspase-3, in the cytosolic fraction of the cerebral cortex of newborn piglets. The present study examines the mechanism of caspase-9 activation during hypoxia and tests the hypothesis that the ATP and cytochrome c-dependent activation of caspase-9 increases in the cytosol of the cerebral cortex of newborn piglets. Newborn piglets were divided into normoxic (Nx, n=4), and hypoxic (Hx, n=4) groups. Anesthetized, ventilated animals were exposed to an FiO(2) of 0.21 (Nx) or 0.07 (Hx) for 60 min. Cerebral tissue hypoxia was documented biochemically by determining levels of ATP and phosphocreatine (PCr). Cytosolic fraction was isolated and passed through a G25-Sephadex column to remove endogenous ATP and cytochrome c. Fractions were collected and protein determined by UV spectrophotometry at 280 nm. Eluted high-molecular weight samples from normoxic and hypoxic animals were divided into four subgroups: subgroup 1 (control), incubated without added ATP and cytochrome c; subgroup 2, incubated with added ATP; subgroup 3, incubated with added cytochrome c; and subgroup 4, incubated with added ATP and cytochrome c. The incubation was carried out at 37 degrees C for 30 min. Following incubation, the protein was separated by 12% SDS-PAGE and active caspase-9 was detected using specific active caspase-9 antibody. Protein bands were detected by enhanced chemiluminescence. Protein density was determined by imaging densitometry and expressed as absorbance (OD x mm(2)). ATP (mumol/g brain) level was 4.7 +/- 0.18 in normoxic, as compared to 1.53 +/- 0.16 in hypoxic (p < 0.05 vs. Nx). PCr (mumol/g brain) level was 4.03 +/- 0.11 in the normoxic and 1.1 +/- 0.3 in the hypoxic brain (p < 0.05 vs. Nx). In the normoxic preparations, active caspase-9 density increased by 9, 4 and 20% in the presence of ATP, cytochrome c and ATP+cytochrome c, respectively. In the hypoxic preparations, active caspase-9 density increased by 30, 45 and 60% in the presence of ATP, cytochrome c and ATP+cytochrome c, respectively. These results show that incubation with ATP, cytochrome c and ATP+cytochrome c result in a significantly increased activation of caspase-9 in the hypoxic group (p < 0.05). We conclude that the ATP and cytochrome c dependent activation of caspase-9 is increased during hypoxia. We propose that the ATP and cytochrome c sites of apoptotic protease activat

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Apoptosis; Apoptotic Protease-Activating Factor 1; Brain Chemistry; Brain Infarction; Caspase 9; Cell Death; Cerebral Cortex; Cytochromes c; Enzyme Activation; Hypoxia, Brain; Nerve Degeneration; Sus scrofa; Up-Regulation

Several studies have indicated that apoptotic pathways are responsible for the loss of motor neurons that constitute the hallmark of amyotrophic lateral sclerosis (ALS). In this study, we demonstrate that apoptosis induced by the expression of several mutant Cu,Zn superoxide dismutases (SOD1) typical of familial ALS is mediated by Apaf1, a scaffold protein involved in neural development. Using different cell lines of neuronal origin and modulating the expression of both mutant SOD1s and Apaf1, we show that the removal of Apaf1 prevents cells death. Interestingly, intercepting activation of the caspases cascade is also effective in preventing both the mitochondrial damage and the increase in the production of reactive oxygen species induced by fALS-SOD1, even in the presence of cytochrome c release. This death pathway may be crucial also for the pathogenesis of the sporadic form of the disease, where markers of increased oxidative stress and mitochondria damage have been found.

Topics: Amyotrophic Lateral Sclerosis; Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Cell Line, Tumor; Cytochromes c; Gene Expression; Humans; Intracellular Signaling Peptides and Proteins; Mice; Mice, Knockout; Mitochondria; Nerve Degeneration; Neuroblastoma; Oxidative Stress; Proteins; Superoxide Dismutase; Superoxide Dismutase-1

The present study was undertaken to evaluate whether in a neonatal model of stroke a prophylactic neuroprotective treatment with simvastatin modulates hypoxia-ischemia-induced inflammatory and apoptotic signaling. Procaspase-3 and cleaved caspase-3 expression showed a peak at 24 h and returned to control values after 5 days. Caspase-3 activity followed the same pattern of caspase-3 proteolytic cleavage. In simvastatin-treated ischemic animals, the expression of these proteins and caspase-3 activity were significantly lower when compared to that of ischemic animals. alpha-Spectrin and protein kinase C-alpha (PKCalpha) cleavages were not affected by the treatment. Poly (ADP-ribose) polymerase fragmentation, caspase-1 activation, and IL-1beta and ICAM-1 mRNA expression were increased by hypoxia-ischemia and significantly reduced in simvastatin-treated animals. The results indicate that simvastatin-induced attenuation of hypoxia-ischemia brain injury in the newborn rat occurs through reduction of the inflammatory response, caspase-3 activation, and apoptotic cell death.

Topics: Animals; Animals, Newborn; Apoptosis; Biomarkers; Calpain; Caspase 1; Caspase 3; Caspases; Cytochromes c; Enzyme Activation; Gene Expression; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypoxia-Ischemia, Brain; Intercellular Adhesion Molecule-1; Interleukin-1; Nerve Degeneration; Poly(ADP-ribose) Polymerases; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Simvastatin

General anesthetics cause widespread apoptotic neurodegeneration in many regions of the developing rat brain. The activation of mitochondria-dependent apoptotic pathway is important in the early stages of anesthesia-induced developmental neuroapoptosis. To investigate potential means of protecting against this type of damage, we studied melatonin, a sleep-promoting agent and antioxidant known to inhibit apoptotic-type neuronal damage by improving mitochondrial homeostasis and stabilizing the inner mitochondrial membrane. When 7-day-old rats (the peak of synaptogenesis) were exposed to a commonly used and highly pro-apoptotic anesthesia cocktail (midazolam, isoflurane, nitrous oxide) in combination with the escalating doses of melatonin (from 1 to 20 mg/kg, s.c.), the severity of anesthesia-induced damage was reduced in a dose-dependent manner in two most vulnerable brain regions--the cerebral cortex and anterior thalamus. Melatonin-induced neuroprotection was mediated, at least in part, via the inhibition of mitochondria-dependent apoptotic pathway since melatonin caused an up-regulation of the anti-apoptotic protein, bcl-X(L), reduction in anesthesia-induced cytochrome c release into the cytoplasm and a decrease in anesthesia-induced activation of caspase-3, an important step in the activation of DNAses and the formation of the apoptotic bodies.

Topics: Anesthetics, General; Animals; Apoptosis; bcl-X Protein; Blotting, Western; Brain; Caspase 3; Caspases; Cytochromes c; Dose-Response Relationship, Drug; Melatonin; Mitochondria; Nerve Degeneration; Neuroprotective Agents; Rats; Rats, Sprague-Dawley

To decipher the pathway of apoptosis induction downstream to caspase-8 activation by exogenous expression of Hippi, an interactor of huntingtin-interacting protein Hip1, we studied apoptosis in HeLa and Neuro2A cells expressing GFP-tagged Hippi. Nuclear fragmentation, caspase-1, caspase-8, caspase-9/caspase-6 and caspase-3 activation were increased significantly in Hippi expressing cells. Cleavage of Bid, release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria were also increased in GFP-Hippi expressing cells. It was observed that caspase-1 and caspase-8 activation was earlier than caspase-3 activation and nuclear fragmentation. Expression of caspase-1, caspase-3 and caspase-7 was increased while anti-apoptotic gene Bcl-2 and mitochondrial genes ND1 and ND4 were reduced in Hippi expressing cells. Besides, the expression SDHA and SDHB, nuclear genes, subunits of mitochondrial complex II were decreased in GFP-Hippi expressing cells. Taken together, we concluded that Hippi expression induced apoptosis by releasing AIF and cytochrome c from mitochondria, activation of caspase-1 and caspase-3, and altering the expression of apoptotic genes and genes involved in mitochondrial complex I and II.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Apoptosis Inducing Factor; Brain; Caspases; Cytochromes c; DNA-Binding Proteins; Electron Transport Complex I; Electron Transport Complex II; HeLa Cells; Humans; Huntington Disease; Intracellular Signaling Peptides and Proteins; Iron-Sulfur Proteins; Mice; Mitochondria; Nerve Degeneration; Neurons; Protein Subunits; Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Succinate Dehydrogenase

An elevated level of cholesterol in mitochondrial membranes of Niemann-Pick disease type C1 (NPC1) mouse brains and neural cells has been found to cause mitochondrial dysfunction. In this study, we demonstrate that inhibition of intracellular cholesterol trafficking in primary neurons by class 2 amphiphiles, which mimics the major biochemical and cellular feature of NPC1, led to not only impaired mitochondrial function but also activation of the mitochondrial apoptosis pathway. In activation of this pathway both cytochrome c and Smac/Diablo were released but apoptosis-inducing factor (AIF) was not involved. Treatment of the neurons with taurine, a caspase 9-specific inhibitor, could prevent the amphiphile-induced apoptotic cell death, suggesting that formation of apoptosome, followed by caspase 9 and caspase 3 activation, might play a critical role in the neuronal death pathway. Taken together, the mitochondria-dependent death cascade induced by blocking intracellular cholesterol trafficking was caspase dependent. The findings provide clues for both understanding the molecular basis of neurodegeneration in NPC1 disease and developing therapeutic strategies for treatment of this disorder.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Carrier Proteins; Caspase 3; Caspase 9; Caspase Inhibitors; Caspases; Cells, Cultured; Cerebral Cortex; Cholesterol; Cytochromes c; Enzyme Activation; Mice; Mitochondria; Mitochondrial Proteins; Nerve Degeneration; Neurons; Niemann-Pick Diseases; Protein Transport; Signal Transduction; Surface-Active Agents

Drug abuse is associated with brain dysfunction and neurodegeneration, and various recreational drugs induce apoptotic cell death. This study examined the role of the mitochondrial apoptotic pathway in psychostimulant-induced neuronal dysfunction. Using primary neuronal cultures, we observed that amphetamine (IC50=1.40 mM) was more potent than cocaine (IC50=4.30 mM) in inducing cell toxicity. Apoptotic cell death was further evaluated using cocaine and amphetamine concentrations that moderately decreased cell reduction capacity but did not affect plasma membrane integrity. Compared to cocaine, amphetamine highly decreased the mitochondrial membrane potential, as determined using the fluorescent probe rhodamine-123, whereas both drugs decreased mitochondrial cytochrome c. In contrast to amphetamine, cocaine cytotoxicity was partly mediated through effects on the electron transport chain, since cocaine toxicity was ameliorated in mitochondrial DNA-depleted cells lacking mitochondrially encoded electron transport chain subunits. Cocaine and amphetamine induced activation of caspases-2, -3 and -9 but did not affect activity of caspases-6 or -8. In addition, amphetamine, but not cocaine, was associated with the appearance of evident nuclear apoptotic morphology. These events were not accompanied by differences in the release of the apoptosis-inducing factor (AIF) from mitochondria. Our results demonstrate that although both amphetamine and cocaine activate the mitochondrial apoptotic pathway in cortical neurons, amphetamine is more likely to promote apoptosis.

Topics: Amphetamine; Amphetamine-Related Disorders; Animals; Apoptosis; Caspases; Cell Line, Tumor; Central Nervous System Stimulants; Cerebral Cortex; Cocaine; Cocaine-Related Disorders; Cytochromes c; Disease Models, Animal; Electron Transport Chain Complex Proteins; Energy Metabolism; Humans; Membrane Potentials; Mitochondria; Mitochondrial Membranes; Nerve Degeneration; Neurons; Rats

Our previous studies and the others have strongly suggested that JNK signaling pathway plays a critical role in ischemic brain injury. Here, we reported that SP600125, a potent, cell-permeable, selective, and reversible inhibitor of c-Jun N-terminal kinase (JNK), potently decrease neuronal apoptosis induced by global ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. As a result, SP600125 diminished the increased phosphorylation of c-Jun and the increased expression of FasL induced by ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. At the same time, through inhibiting phosphorylation of Bcl-2 and the release of Bax from Bcl-2/Bax dimers, SP600125 attenuated Bax translocation to mitochondria and the release of cytochrome c induced by ischemia/reperfusion (I/R). Furthermore, the activation of caspase-3 induced by ischemia/reperfusion was also significantly suppressed by preinfusion of SP600125. Importantly, the same neuropotective effect was showed by administration of SP600125 both before and after ischemia. Thus, our findings imply that SP600125 can inhibit the activation of JNK signaling pathway and induce neuroprotection against ischemia/reperfusion in rat hippocampal CA1 region via suppressing the extrinsic and intrinsic pathways of apoptosis. Taken together, these results indicate that targeting the JNK pathway provides a promising therapeutic approach for ischemic brain injury.

Topics: Animals; Anthracenes; Apoptosis; bcl-2-Associated X Protein; Brain Ischemia; Caspase 3; Caspases; Cerebral Infarction; Cytochromes c; Enzyme Inhibitors; Fas Ligand Protein; Hippocampus; JNK Mitogen-Activated Protein Kinases; Male; Membrane Glycoproteins; Nerve Degeneration; Neuroprotective Agents; Proto-Oncogene Proteins c-jun; Rats; Rats, Sprague-Dawley; Signal Transduction; Tumor Necrosis Factors

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity is one of the experimental models most commonly used to study the pathogenesis of Parkinson's disease (PD). Although the biochemical mechanisms underlying the cell death induced by MPTP remain to be clarified, it has been found that the mitochondrial apoptotic signaling pathway plays an important role in the neurotoxicity of MPTP. Nucling is a novel type of apoptosis-associated molecule, essential for cytochrome c, apoptosis protease activating factor 1 (Apaf-1), pro-caspase-9 apoptosome induction and caspase-9 activation following pro-apoptotic stress. Here we found that Nucling-deficient mice treated with MPTP did not exhibit locomotor dysfunction in an open-field test. The substantia nigra dopaminergic neurons of Nucling-deficient mice were resistant to the damaging effects of the neurotoxin MPTP. Up-regulated expression of apoptosome was attenuated in Nucling-deficient mice treated with MPTP. These results indicate an important role for Nucling in MPTP-induced neuronal degeneration and suggest that the suppression of Nucling would be of therapeutic benefit for the treatment of neurodegeneration in PD.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Caspase 9; Caspases; Cytochromes c; Disease Models, Animal; Dopamine; Drug Resistance; Genetic Predisposition to Disease; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Motor Activity; MPTP Poisoning; Nerve Degeneration; Neurons; Parkinsonian Disorders; Proteins; Signal Transduction; Substantia Nigra; Up-Regulation

Cytochrome c translocation from the inner mitochondrial membrane into the cytosol is the initial step of the intrinsic apoptotic pathway. As no evidence was ever presented for cytochrome c translocation during cerebellar degeneration in Lurcher (Lc/+) and weaver (wv/wv) mutant mice, we searched for the presence of such a process in cerebellar homogenates of mutant and wild-type mice from postnatal day (P)1 to P56. Here we present the first documented time course of cytochrome c translocation spanning the entire period of neurodegeneration in both mutant types. We identified cytochrome c with Western blotting and monitored cell loss in the cerebellum with Calbindin D-28k immunohistochemistry, Nissl-staining and morphometry. No cytochrome c translocation was ever detected in wild-types at any age investigated. Translocated cytochrome c appeared between P13 and P21 in Lc/+ and between P5 and P6 in wv/wv. These two intervals precisely coincide with the respective periods of maximal neuronal death in the cerebellum. Secondary translocation was also observed at a later stage between P42 and P49 in Lc/+ and from P22 onwards in wv/wv. Since no substantial neuronal loss has ever been observed in Lc/+ and wv/wv mutants at these postnatal ages, the delayed translocation may correspond to cytochrome c of extraneuronal, presumably glial origin. Observations of an increased expression of glial fibrillary acidic protein and sustained remodeling of the astrocytic network in the cerebellum of both mutants, long after the cessation of neuronal death make this assumption rather plausible.

Topics: Animals; Animals, Newborn; Apoptosis; Biological Transport, Active; Calbindins; Cerebellum; Cytochromes c; Disease Models, Animal; Female; Genetic Predisposition to Disease; Glial Fibrillary Acidic Protein; Gliosis; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Mice, Neurologic Mutants; Mitochondria; Mitochondrial Membranes; Nerve Degeneration; Neuroglia; Neurons; S100 Calcium Binding Protein G; Spinocerebellar Degenerations

Protein kinase C (PKC) isozymes have been known to mediate a variety of complex and diverse cellular functions. deltaPKC has been implicated in mediating apoptosis. Using two models of cerebral ischemia, cardiac arrest in rats and oxygen glucose deprivation (OGD) in organotypic hippocampal slices, we tested whether an ischemic insult promoted deltaPKC cleavage during the reperfusion and whether the upstream pathway involved release of cytochrome c and caspase 3 cleavage. We showed that cardiac arrest/OGD significantly enhanced deltaPKC translocation and increased its cleavage at 3 h of reperfusion. Since deltaPKC is one of the substrates for caspase 3, we next determined caspase 3 activation after cardiac arrest and OGD. The maximum decrease in levels of procaspase 3 was observed at 3 h of reperfusion after cardiac arrest and OGD. We also determined cytochrome c release, since it is upstream of caspase 3 activation. Cytochrome c in cytosol increased at 1 h of reperfusion after cardiac arrest/OGD. Inhibition of either deltaPKC/caspase 3 during OGD and early reperfusion resulted in neuroprotection in CA1 region of hippocampus. Our results support the deleterious role of deltaPKC in reperfusion injury. We propose that early cytochrome c release and caspase 3 activation promote deltaPKC translocation/cleavage.

Topics: Animals; Blood Pressure; Brain Ischemia; Caspase 3; Caspases; Cell Death; Cytochromes c; Electrocardiography; Glucose; Heart Arrest; Hippocampus; Nerve Degeneration; Organ Culture Techniques; Oxygen; Protein Kinase C; Protein Kinase C-delta; Rats; Rats, Sprague-Dawley; Signal Transduction

Down's syndrome (DS) is characterized by mental retardation and development of Alzheimer's disease (AD). Oxidative stress and mitochondrial dysfunction are both related to neurodegeneration in DS. Several genes in chromosome 21 have been linked to neuronal death, including the transcription factor ets-2. Cortical cultures derived from normal and DS fetal brains were used to study the role of ets-2 in DS neuronal degeneration. ets-2 was expressed in normal human cortical neurons (HCNs) and was markedly upregulated by oxidative stress. When overexpressed in normal HCNs, ets-2 induced a stereotyped sequence of apoptotic changes leading to neuronal death. DS HCNs exhibit intracellular oxidative stress and increased apoptosis after the first week in culture (Busciglio and Yankner, 1995). ets-2 levels were increased in DS HCNs, and, between 7 and 14 d in vitro, DS HCNs showed increased bax, cytoplasmic translocation of cytochrome c and apoptosis inducing factor, and active caspases 3 and 7, consistent with activation of an apoptotic mitochondrial death pathway. Degeneration of DS neurons was reduced by dominant-negative ets-2, suggesting that increased ets-2 expression promotes DS neuronal apoptosis. In the human brain, ets-2 expression was found in neurons and astrocytes. Strong ets-2 immunoreactivity was observed in DS/AD and sporadic AD brains associated with degenerative markers such as bax, intracellular Abeta, and hyperphosphorylated tau. Thus, in DS/AD and sporadic AD brains, converging pathological mechanisms leading to chronic oxidative stress and ets-2 upregulation in susceptible neurons may result in increased vulnerability by promoting the activation of a mitochondrial-dependent proapoptotic pathway of cell death.

Topics: Alzheimer Disease; Animals; Apoptosis Inducing Factor; Astrocytes; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Caspases; Cell Death; Cell Survival; Cerebral Cortex; Chlorocebus aethiops; COS Cells; Cytochromes c; Diagnostic Imaging; DNA-Binding Proteins; Down Syndrome; Fetus; Fluorescent Antibody Technique; Gene Expression Regulation; Green Fluorescent Proteins; Humans; Hydrogen Peroxide; Mitochondria; Nerve Degeneration; Neurofilament Proteins; Neurons; Polycomb-Group Proteins; Protein Transport; Signal Transduction; tau Proteins; Telomerase; Time Factors; Transcription Factors; Transfection; Tumor Suppressor Protein p53

Ceramide accumulates in neurons during various disorders associated with acute or chronic neurodegeneration. In these studies, we investigated the mechanisms of ceramide-induced apoptosis in primary cortical neurons using exogenous C(2) ceramide as well as inducing endogenous ceramide accumulation using inhibitors of glucosylceramide synthetase. Ceramide induced the translocation of certain, but not all, pro-apoptotic mitochondrial proteins: cytochrome c, Omi, SMAC, and AIF were released from the mitochondria, whereas Endonuclease G was not. Ceramide also selectively altered the phosphorylation state of members of the MAPK superfamily, causing dephosphorylation of ERK1/2 and hyperphosphorylation of p38 MAP kinases, but not affecting the phosphorylation of JNK or ERK5. Inhibitors of the p38 MAP kinase pathway (SB-202190 or SB-203580) and an inhibitor of the ERK1/2 pathway (U0126) reduced ceramide-induced neuronal death. These p38 and ERK1/2 inhibitors appear to block ceramide-activated apoptotic signaling upstream of the mitochondria, as they attenuated mitochondrial release of cytochrome c, Omi, AIF, and SMAC, as well as reducing ceramide-induced caspase-3 activation.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; Carrier Proteins; Caspase 3; Caspase Inhibitors; Caspases; Cells, Cultured; Ceramides; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Flavoproteins; Glucosyltransferases; High-Temperature Requirement A Serine Peptidase 2; MAP Kinase Signaling System; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Mitogen-Activated Protein Kinase 3; Nerve Degeneration; Nerve Tissue Proteins; Neurodegenerative Diseases; Neurons; p38 Mitogen-Activated Protein Kinases; Rats; Serine Endopeptidases

Ischemic stroke is caused by acute neuronal degeneration provoked by interruption of cerebral blood flow. Although the mechanisms contributing to ischemic neuronal degeneration are myriad, mitochondrial dysfunction is now recognized as a pivotal event that can lead to either necrotic or apoptotic neuronal death. Lack of suitable 'upstream' targets to prevent loss of mitochondrial homeostasis has, so far, restricted the development of mechanistically based interventions to promote neuronal survival. Here, we show that the uncoupling agent 2,4 dinitrophenol (DNP) reduces infarct volume approximately 40% in a model of focal ischemia-reperfusion injury in the rat brain. The mechanism of protection involves an early decrease in mitochondrial reactive oxygen species formation and calcium uptake leading to improved mitochondrial function and a reduction in the release of cytochrome c into the cytoplasm. The observed effects of DNP were not associated with enhanced cerebral perfusion. These findings indicate that compounds with uncoupling properties may confer neuroprotection through a mechanism involving stabilization of mitochondrial function.

Topics: 2,4-Dinitrophenol; Animals; Calcium Signaling; Cerebral Infarction; Cytochromes c; Cytoprotection; Disease Models, Animal; Homeostasis; Ischemic Attack, Transient; Male; Mitochondria; Nerve Degeneration; Neurons; Neuroprotective Agents; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Uncoupling Agents

An in vitro ischemia model was used to determine the molecular mechanisms responsible for the ischemia-induced neuronal cell death. Additionally, the neuronal protective mechanisms of anti-apoptotic drugs against ischemia were also evaluated. In this study, the primary neuronal cultures were incubated in an anoxic chamber with 95% of N2 and 5% of CO2 for various times. The death rate, degree of the apoptotic damage, reduction of mitochondrial membrane potential, translocation of Bax, release of cytochrome C and activation of caspase-9 and -3 were determined at each time point. Results showed that a Bax-regulated mitochondria- mediated apoptosis is responsible for the in vitro ischemia-induced neuronal death. Reduction in mitochondrial membrane potential plays no role in triggering this apoptosis. Furthermore, the anti-apoptotic drugs: furosemide (a Bax blocker) and ZVAD-fmk (caspase inhibitor) but not cyclosporine A (a MPT pore blocker), significantly protected the neurons against ischemia-induced damage. This provides an additional consideration in the future selection of new anti-ischemic drugs.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Brain Ischemia; Caspases; Cerebral Infarction; Cytochromes c; Disease Models, Animal; Enzyme Inhibitors; Intracellular Membranes; Membrane Potentials; Mitochondria; Nerve Degeneration; Protein Synthesis Inhibitors; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Signal Transduction

Polyglutamine tract-binding protein-1 (PQBP-1) is a nuclear protein that interacts and colocalizes with mutant polyglutamine proteins. We previously reported that PQBP-1 transgenic mice show a late-onset motor neuron disease-like phenotype and cell death of motor neurons analogous to human neurodegeneration. To investigate the molecular mechanisms underlying the motor neuron death, we performed microarray analyses using the anterior horn tissues of the spinal cord and compared gene expression profiles between pre-symptomatic transgenic and age-matched control mice. Surprisingly, half of the spots changed more than 1.5-fold turned out to be genes transcribed from the mitochondrial genome. Northern and western analyses confirmed up-regulation of representative mitochondrial genes, cytochrome c oxidase (COX) subunit 1 and 2. Immunohistochemistry revealed that COX1 and COX2 proteins are increased in spinal motor neurons. Electron microscopic analyses revealed morphological abnormalities of mitochondria in the motor neurons. PQBP-1 overexpression in primary neurons by adenovirus vector induced abnormalities of mitochondrial membrane potential from day 5, while cytochrome c release and caspase 3 activation were observed on day 9. An increase of cell death by PQBP-1 was also confirmed on day 9. Collectively, these results indicate that dysfunction of PQBP-1 induces mitochondrial stress, a key molecular pathomechanism that is shared among human neurodegenerative disorders.

Topics: Animals; Anterior Horn Cells; Carrier Proteins; Caspases; Cell Death; Cytochromes c; DNA-Binding Proteins; Electron Transport Complex IV; Humans; Membrane Potentials; Mice; Mice, Transgenic; Microscopy, Electron; Mitochondria; Motor Neuron Disease; Motor Neurons; Nerve Degeneration; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Oxidative Stress; Up-Regulation

An important aspect of Huntington's disease (HD) pathogenesis which may have important therapeutic implications is that the cellular events leading to cell death may be different in cortical and striatal neurons. In the present study, we characterized cellular changes in cortical and striatal neurons treated with the mitochondrial toxin 3-nitropropionic acid (3NP) in culture. Degeneration induced by 3NP was similar in both striatal and cortical neurons as observed using markers of cell viability and DNA fragmentation. However, in striatal neurons, 3NP produced a marked delocalization of Bad, Bax, cytochrome c and Smac while this was not observed in cortical neurons. Death of striatal neurons was preceded by activation of calpain and was blocked by calpain inhibitor I. In cortical neurons, calpain was not activated and calpain inhibitor I was without effect. In both cell types, caspase-9 and -3 were not activated by 3NP and the caspase inhibitor zVAD-fmk did not provide neuroprotective effect. Interestingly, treatment with staurosporine (STS) triggered caspase-9 and -3 in cortical and striatal cells, suggesting that the molecular machinery related to caspase-dependent apoptosis was functional in both cell types even though this machinery was not involved in 3NP toxicity. The present results clearly demonstrate that under mitochondrial inhibition, striatal and cortical neurons die through different pathways. This suggests that mitochondrial defects in HD may trigger the death of cortical and striatal neurons through different molecular events.

Topics: Animals; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-Associated Death Protein; Carrier Proteins; Caspase Inhibitors; Caspases; Cell Death; Cell Respiration; Cells, Cultured; Cerebral Cortex; Cytochromes c; Disease Models, Animal; Enzyme Inhibitors; Fetus; Huntington Disease; Mitochondria; Mitochondrial Proteins; Neostriatum; Nerve Degeneration; Neurotoxins; Nitro Compounds; Propionates; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Signal Transduction

Age-related changes and regional differences in caspase expression were determined in the primate brain. Using immunoblot analysis, the levels of endogenous caspase-3, caspase-9 and cytochrome c-triggered activated caspase-3 were examined in brain homogenates from the prefrontal, motor and visual cortices, cerebellum, hippocampus and amygdala of 4-year-old and 20-year-old rhesus macaques. Procaspase-3 was detected in similar quantities in all brain regions of both young and aging macaques. Being found in all brain regions, caspase-9 was significantly elevated in old macaques as compared to young ones. After incubation with cytochrome c, active forms of caspase-3 were detected in all brain regions of young and old macaques. In almost all brain regions of old monkeys, the levels of cytochrome c-dependent caspase-3 activation were higher than those of young macaques. These results suggest that the aging rhesus macaque brain has a lower threshold to apoptotic stimuli.

Topics: Adenosine Triphosphate; Aging; Animals; Apoptosis; Brain; Caspase 3; Caspase 9; Caspases; Cytochromes c; Enzyme Activation; Female; Immunity, Innate; Macaca mulatta; Nerve Degeneration; Neurodegenerative Diseases; Up-Regulation

Lidocaine is a local anesthetic and antiarrhythmic agent. Although clinical and experimental studies have shown that an antiarrhythmic dose of lidocaine can protect the brain from ischemic damage, the underlying mechanisms are unknown. In the present study, we examined whether lidocaine inhibits neuronal apoptosis in the penumbra in a rat model of transient focal cerebral ischemia. Male Wistar rats underwent a 90-min temporary occlusion of middle cerebral artery. Lidocaine was given as an i.v. bolus (1.5 mg/kg) followed by an i.v. infusion (2 mg/kg/h) for 180 min, starting 30 min before ischemia. Rats were killed and brain samples were collected at 4 and 24 h after ischemia. Apoptotic changes were evaluated by immunohistochemistry for cytochrome c release and caspase-3 activation and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) for DNA fragmentation. Cytochrome c release and caspase-3 activation were detected at 4 and 24 h after ischemia and DNA fragmentation was detected at 24 h. Double-labeling with NeuN, a neuronal marker, demonstrated that cytochrome c, caspase-3, and TUNEL were confined to neurons. Lidocaine reduced cytochrome c release and caspase-3 activation in the penumbra at 4 h and diminished DNA fragmentation in the penumbra at 24 h. Lidocaine treatment improved early electrophysiological recovery and reduced the size of the cortical infarct at 24 h, but had no significant effect on cerebral blood flow in either the penumbra or core during ischemia. These findings suggest that lidocaine attenuates apoptosis in the penumbra after transient focal cerebral ischemia. The infarct-reducing effects of lidocaine may be due, in part, to the inhibition of apoptotic cell death in the penumbra.

Topics: Animals; Apoptosis; Caspase 3; Caspases; Cerebral Infarction; Cerebrovascular Circulation; Cytochromes c; Disease Models, Animal; DNA Fragmentation; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Ischemic Attack, Transient; Lidocaine; Male; Nerve Degeneration; Rats; Rats, Wistar; Reaction Time; Recovery of Function; Treatment Outcome

Human immunodeficiency virus-1 (HIV-1) infection affects the striatum, resulting in gliosis and neuronal losses. To determine whether HIV-1 proteins induce striatal neurotoxicity through an apoptotic mechanism, mouse striatal neurons isolated on embryonic day 15 and the effects of HIV-1 Tat(1-72) and gp120 on survival were assessed in vitro. Mitochondrial release of cytochrome c, caspase-3 activation, and neuron survival, as well as an alternative apoptotic pathway involving endonuclease G (endo G), were assessed at 4 h, 24 h, 48 h, and/or 72 h using enzyme assays and immunoblotting. Both HIV-1 Tat and gp120 significantly increased caspase-3 activation in a concentration-dependent manner in striatal neurons at 4 h following continuous exposure in vitro. Tat(1-72) and gp120 caused significant neuronal losses at 48 h and/or 72 h. Tat(1-72) increased cytochrome c release, and caspase-3 and endo G activation at 4 h, 24 h, and/or 72 h. By contrast, gp120 increased caspase-3 activation, but failed to increase cytochrome c or endo G levels in the cytoplasm at 4 h, 24 h, and/or 72 h. The cell permeant caspase inhibitor Z-DEVD-FMK significantly attenuated gp120-induced, but not Tat(1-72)-induced, neuronal death, suggesting that gp120 acts in large part through the activation of caspase(s), whereas Tat(1-72)-induced neurotoxicity was accompanied by activating an alternative pathway involving endo G. Thus, although Tat(1-72) and gp120 induced significant neurotoxicity, the nature of the apoptotic events preceding death differed. Collectively, our findings suggest that HIV-1 proteins are intrinsically toxic to striatal neurons and the pathogenesis is mediated through separate actions involving both caspase-3 and endo G.

Topics: Animals; Apoptosis; Caspase 3; Caspases; Cells, Cultured; Corpus Striatum; Cytochromes c; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Endodeoxyribonucleases; Enzyme Activation; Enzyme Inhibitors; Gene Products, tat; HIV Envelope Protein gp120; HIV-1; Immunoblotting; Mice; Mice, Inbred ICR; Nerve Degeneration; Neurons; tat Gene Products, Human Immunodeficiency Virus

1-Methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), induces apoptosis in dopaminergic neurons; however, the cellular mechanisms underlying the degenerative process are not well understood. In the present study, we demonstrate that caspase-3 mediated proteolytic activation of protein kinase C delta (PKC delta) is critical in MPP+-induced oxidative stress and apoptosis. MPP+ exposure in rat dopaminergic neuronal cells resulted in time-dependent increases in reactive oxygen species generation, cytochrome c release, and caspase-9 and caspase-3 activation. Interestingly, MPP+ induced proteolytic cleavage of PKC delta (72-74 kDa) into a 41-kDa catalytic and a 38-kDa regulatory subunit, resulting in persistently increased kinase activity. The caspase-3 inhibitor Z-DEVD-fmk effectively blocked MPP+-induced PKC delta cleavage and kinase activity, suggesting that the proteolytic activation is caspase-3 mediated. Similar results were seen in MPP+-treated rat midbrain slices. Z-DEVD-fmk and the PKC delta specific inhibitor rottlerin almost completely blocked MPP+-induced DNA fragmentation. The superoxide dismutase mimetic, MnTBAP also effectively attenuated MPP+-induced caspase-3 activation, PKC delta cleavage, and DNA fragmentation. Furthermore, rottlerin attenuated MPP+-induced caspase-3 activity without affecting basal activity, suggesting positive feedback activation of caspase-3 by PKC delta. Intracellular delivery of catalytically active recombinant PKC delta significantly increased caspase-3 activity, further indicating that PKC delta regulates caspase-3 activity. Finally, over-expression of a kinase inactive PKC delta K376R mutant prevented MPP+-induced caspase activation and DNA fragmentation, confirming the pro-apoptotic function of PKC delta in dopaminergic cell death. Together, we demonstrate for the first time that MPP+-induced oxidative stress proteolytically activates PKC delta in a caspase-3-dependent manner to induce apoptosis and up-regulate the caspase cascade in dopaminergic neuronal cells.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Apoptosis; Benzimidazoles; Caspase 3; Caspase 9; Caspase Inhibitors; Caspases; Cell Line; Coumarins; Cytochromes c; DNA Fragmentation; Dopamine; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Flow Cytometry; Free Radical Scavengers; Gene Expression; Herbicides; In Vitro Techniques; Manganese Compounds; Mesencephalon; Metalloporphyrins; Nerve Degeneration; Organometallic Compounds; Oxidative Stress; Precipitin Tests; Protein Kinase C; Protein Kinase C-delta; Rats; Reactive Oxygen Species; Time Factors; Transfection

The length and thinness of neurites render them greatly susceptible to a variety of insults. Accumulating evidence suggests that neurite degeneration is not a passive, but an active and causative, event in some neurodegenerative diseases. Nonetheless, the mechanisms underlying neurite degeneration remain largely unknown. To elucidate the relevant mechanisms, we employed a mutant C57BL/Wld mouse with a unique phenotype of resistance to Wallerian degeneration, and separately analyzed the destruction of cell soma and neurites following treatment with vinblastine, a microtubule-disrupting agent, in superior cervical ganglion neurons. Vinblastine induced macromolecular synthesis-dependent cell death, which was indistinguishable between the wild-type and mutant mice. Evidence for a loss of mitochondrial cytochrome c, caspase activation, and nuclear fragmentation, has indicated that this type of cell death is entirely apoptotic. Consistent with this, the ATP level in the cell soma was well maintained and indistinguishable between wild-type and mutant mice. In neurites of wild-type neurons, vinblastine induced an early loss of mitochondrial membrane potential (MMP) and ATP depletion preceding caspase-independent degeneration, suggesting that this type of neurite degeneration is principally non-apoptotic. In contrast, neurites of mutant neurons were markedly resistant to vinblastine-induced degeneration, and both the MMP and the ATP content in the neurites were well maintained. Exposure of mutant neurons to carbonyl cyanide m-chlorophenyl-hydrazone, an uncoupler, caused extreme neurite degeneration following rapid MMP loss. Collectively, our findings suggest that: 1) neurite degeneration is regulated through a non-apoptotic process achieved by mitochondrial dysfunction in neurites; 2) the mitochondrial functional status is controlled separately in neurites and in the neuronal soma.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Antineoplastic Agents, Phytogenic; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Caspases; Cell Count; Cell Death; Cells, Cultured; Cytochromes c; Fluoresceins; Immunohistochemistry; In Situ Nick-End Labeling; In Vitro Techniques; Ionophores; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Microscopy, Confocal; Mitochondria; Nerve Degeneration; Nerve Growth Factor; Nerve Tissue Proteins; Neurites; Neurons; Propidium; Superior Cervical Ganglion; Time Factors; Trichloroacetic Acid; Tubulin; Vinblastine; Xanthenes

Cerebellar Purkinje neurons (PNs) are selectively vulnerable to AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepriopionic acid)-induced delayed neurotoxicity known as dark cell degeneration (DCD) that is expressed as cytoplasmic and nuclear condensation, neuron shrinkage, and failure of physiology. The present study was initiated to determine whether AMPA-receptor-induced DCD in PNs is associated with Bax translocation to the mitochondria, cytochrome C release from the mitochondria, changes in mitochondrial potential, and activation of representative initiator and executor caspases that include caspase-9, caspase-3, and caspase-7. AMPA consistently and rapidly hyperpolarized mitochondria as reflected by an increase in MitoTracker Red CMS Ros fluorescence. Increases in Bax immunoreactivity were quantitatively and temporally variable and Bax failed to localize to mitochondria. Additionally, we observed a marked increase in immunoreactivity of cytochrome C although its release from mitochondria was not apparent. Mitochondrial membrane hyperpolarization and increases in cytochrome C immunoreactivity preceded caspase activation. Immunohistochemical analyses revealed the active form of caspases-3 and -9 were markedly and significantly increased in PNs following 30 microM AMPA, and caspase-9 activation preceded caspase-3. Increases in active caspase-7 immunoreactivity were less frequently encountered in PNs. Thus DCD shares some characteristics of apoptotic programmed cell death, but lacks typical mitochondrial pathophysiology associated with classic apoptosis. These findings suggest that AMPA-induced DCD is a form of active PCD that lies on a spectrum between classical apoptosis and passive necrosis.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Animals, Newborn; bcl-2-Associated X Protein; Caspases; Cell Count; Cerebellum; Cytochromes c; Electron Transport Complex IV; Enzyme Activation; Fluorescent Dyes; Immunohistochemistry; In Vitro Techniques; Microscopy, Confocal; Mitochondrial Diseases; Nerve Degeneration; Organic Chemicals; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Purkinje Cells; Rats; Rats, Sprague-Dawley; Time Factors