Prostaglandin A1 inhibits rotenone-induced apoptosis in SH-SY5Y cells

The degeneration of nigral dopamine neurons in Parkinson's disease (PD) reportedly involves a defect in brain mitochondrial complex I in association with the activation of nuclear factor-kappaB (NF-kappaB) and caspase-3. To elucidate molecular mechanisms possibly linking these events, as well as to evaluate the neuroprotective potential of the cyclopentenone prostaglandin A1 (PGA1), an inducer of heat shock proteins (HSPs), we exposed human dopaminergic SH-SY5Y cells to the complex I inhibitor rotenone. Dose-dependent apoptosis was preceded by the nuclear translocation of NF-kappaB and then the activation of caspase-3 over the ensuing 24 h. PGA1 increased the expression of HSP70 and HSP27 and protected against rotenone-induced apoptosis, without increasing necrotic death. PGA1 blocked the rotenone-induced nuclear translocation of NF-kappaB and attenuated, but did not abolish, the caspase-3 elevation. Unexpectedly, the caspase-3 inhibitor, Ac-DEVD.CHO (DEVD), at a concentration that completely prevented the caspase-3 elevation produced by rotenone, failed to protect against apoptosis. These results suggest that complex I deficiency in dopamine cells can induce apoptosis by a process involving early NF-kappaB nuclear translocation and caspase-3 activation. PGA1 appears to protect against rotenone-induced cell death by inducing HSPs and blocking nuclear translocation of NF-kappaB in a process that attenuates caspase-3 activation, but is not mediated by its inhibition.     

Loss of caspase-2-dependent apoptosis induces autophagy after mitochondrial oxidative stress in primary cultures of young adult cortical neurons

Mitochondrial dysfunctions have been associated with neuronal apoptosis and are characteristic of neurodegenerative conditions. Caspases play a central role in apoptosis; however, their involvement in mitochondrial dysfunction-induced neuronal apoptosis remains elusive. In the present report using rotenone, a complex I inhibitor that causes mitochondrial dysfunction, we determined the initiator caspase and its role in cell death in primary cultures of cortical neurons from young adult mice (1-2 months old). By pretreating the cells with a cell-permeable, biotinylated pan-caspase inhibitor that irreversibly binds to and traps the active caspase, we identified caspase-2 as an initiator caspase activated in rotenone-treated primary neurons. Loss of caspase-2 inhibited rotenone-induced apoptosis; however, these neurons underwent a delayed cell death by necrosis. We further found that caspase-2 acts upstream of mitochondria to mediate rotenone-induced apoptosis in neurons. The loss of caspase-2 significantly inhibited rotenone-induced activation of Bid and Bax and the release of cytochrome c and apoptosis inducing factor from mitochondria. Rotenone-induced downstream activation of caspase-3 and caspase-9 were also inhibited in the neurons lacking caspase-2. Autophagy was enhanced in caspase-2 knock-out neurons after rotenone treatment, and this response was important in prolonging neuronal survival. In summary, the present study identifies a novel function of caspase-2 in mitochondrial oxidative stress-induced apoptosis in neurons cultured from young adult mice.     

Mitochondrial membrane depolarization and the selective death of dopaminergic neurons by rotenone: protective effect of coenzyme Q10

Chronic exposure to the pesticide rotenone induces a selective degeneration of nigrostriatal dopaminergic neurons and reproduces the features of Parkinson's disease in experimental animals. This action is thought to be relevant to its inhibition of the mitochondrial complex I, but the precise mechanism of this suppression in selective neuronal death is still elusive. Here we investigate the mechanism of dopaminergic neuronal death mediated by rotenone in primary rat mesencephalic neurons. Low concentrations of rotenone (5-10 nM) induce the selective death of dopaminergic neurons without significant toxic effects on other mesencephalic cells. This cell death was coincident with apoptotic events including capsase-3 activation, DNA fragmentation, and mitochondrial membrane depolarization. Pretreatment with coenzyme Q10, the electron transporter in the mitochondrial respiratory chain, remarkably reduced apoptosis as well as the mitochondrial depolarization induced by rotenone, but other free radical scavengers such as N-acetylcysteine, glutathione, and vitamin C did not. Furthermore, the selective neurotoxicity of rotenone was mimicked by the mitochondrial protonophore carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), a cyanide analog that effectively collapses a mitochondrial membrane potential. These data suggest that mitochondrial depolarization may play a crucial role in rotenone-induced selective apoptosis in rat primary dopaminergic neurons.     

Rotenone-induced oxidative stress and apoptosis in human liver HepG2 cells

Rotenone, a commonly used pesticide, is well documented to induce selective degeneration in dopaminergic neurons and motor dysfunction. Such rotenone-induced neurodegenration has been primarily suggested through mitochondria-mediated apoptosis and reactive oxygen species (ROS) generation. But the status of rotenone induced changes in liver, the major metabolic site is poorly investigated. Thus, the present investigation was aimed to study the oxidative stress-induced cytotoxicity and apoptotic cell death in human liver cells-HepG2 receiving experimental exposure of rotenone (12.5–250 μM) for 24 h. Rotenone depicted a dose-dependent cytotoxic response in HepG2 cells. These cytotoxic responses were in concurrence with the markers associated with oxidative stress such as an increase in ROS generation and lipid peroxidation as well as a decrease in the glutathione, catalase, and superoxide dismutase levels. The decrease in mitochondrial membrane potential also confirms the impaired mitochondrial activity. The events of cytotoxicity and oxidative stress were found to be associated with up-regulation in the expressions (mRNA and protein) of pro-apoptotic markers viz., p53, Bax, and caspase-3, and down-regulation of anti-apoptotic marker Bcl-2. The data obtain in this study indicate that rotenone-induced cytotoxicity in HepG2 cells via ROS-induced oxidative stress and mitochondria-mediated apoptosis involving p53, Bax/Bcl-2, and caspase-3.     

Involvement of activated caspase-3-like proteases in N-methyl-D-aspartate-induced apoptosis in cerebrocortical neurons

Excessive activation of glutamate receptors mediates neuronal death in a number of neurodegenerative diseases. The intracellular signaling pathways that mediate this type of neuronal death are only partly understood. Following mild insults via NMDA receptor activation, central neurons undergo apoptosis, but with more fulminant insults, necrosis intervenes. Caspases are important in several forms of apoptosis in vivo and in vitro. Previously, we have demonstrated that caspases are important in excitotoxicity-mediated apoptosis of cerebrocortical neurons. To determine the possible activation of caspase-3 in NMDA-induced neuronal apoptosis, we used an affinity-labeling technique: Biotinylated N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD.CHO) preferentially labels conformationally active caspase-3-like proteases, allowing us to visualize affinity-labeled caspases with streptavidin-fluorescein isothiocyanate under confocal microscopy. NMDA-induced apoptosis of cerebrocortical neurons was associated with a time-dependent increase in conformationally active caspase-3-like proteases. The activation of caspases was apparent within 20 min of NMDA stimulation and was localized primarily in the cytosol. However, following incubation of neurons for 18-24 h, conformationally active caspase-3-like proteases were also detectable in nuclei. Double labeling with propidium iodide to detect chromatin condensation indicated that affinity-labeled caspase-3-like proteases were specifically expressed in apoptotic cells. To further confirm this, we used an antibody specific for the conformationally active fragment of caspase-3 and found largely concordant results. Moreover, preincubation with DEVD.CHO prevented NMDA-induced apoptosis. Our results suggest that caspase-3-like proteases play a major role in excitotoxin-induced neuronal apoptosis.     

Betaine Protects Against Rotenone-Induced Neurotoxicity in PC12 Cells

Rotenone is an inhibitor of mitochondrial complex I-induced neurotoxicity in PC12 cells and has been widely studied to elucidate the pathogenesis of Parkinson’s disease. We investigated the neuroprotective effects of betaine on rotenone-induced neurotoxicity in PC12 cells. Betaine inhibited rotenone-induced apoptosis in a dose-dependent manner, with cell viability increasing from 50 % with rotenone treatment alone to 71 % with rotenone plus 100-μM betaine treatment. Flow cytometric analysis demonstrated cell death in the rotenone-treated cells to be over 50 %; the number of live cells increased with betaine pretreatment. Betaine pretreatment of PC12 cells attenuated rotenone-mediated mitochondrial dysfunction, including nuclear fragmentation, ATP depletion, mitochondrial membrane depolarization, caspase-3/7 activation, and reactive oxygen species production. Western blots demonstrated activation of caspase-3 and caspase-9, and their increased expression levels in rotenone-treated cells; betaine decreased caspase-3 and caspase-9 expression levels and suppressed their activation. Together, these results suggest that betaine may serve as a neuroprotective agent in the treatment of neurodegenerative diseases.     

p38/p53-Mediated Bax induction contributes to neurons degeneration in rotenone-induced cellular and rat models of Parkinson’s disease

Rotenone is an environmental neurotoxin that induces degeneration of dopaminergic (DA) neurons in substantia nigra pars compacta (SNpc), which ultimately results in parkinsonism, but the molecular mechanisms of selective degeneration of nigral DA neurons are not fully understood. In the present study, we investigated the induction of p38^MAPK/p53 and Bax in SNpc of Lewis rats after chronic treatment with rotenone and the contribution of Bax to rotenone-induced apoptotic commitment of differentiated PC12 cells. Lewis rats were subcutaneously treated with rotenone (1.5 mg/kg) twice a day for 50 days and the loss of tyrosine hydroxylase (THase), motor function impairment, and expression of p38^MAPK, P-p38^MAPK, p53, and Bax were assessed. After differentiated PC cells were treated with rotenone (500 nM) for 6–36 h, protein levels of p38^MAPK and P-p38^MAPK, p53 nuclear translocation, Bax induction and cell death were measured. The results showed that rotenone administration significantly reduced motor activity and caused a loss of THase immunoreactivity in SNpc of Lewis rats. The degeneration of nigral DA neurons was accompanied by the increases in p38^MAPK, P-p38^MAPK, p53, and Bax protein levels. In cultured PC12 cells, rotenone also induced an upregulation of p38^MAPK, P-p38^MAPK, p53 and Bax. Pharmacological inhibition of p38^MAPK with SB203580 (25 μM) blunted rotenone-induced cell apoptosis. Treatment with SB203580 prevented the p53 nuclear translocation and upregulation of Bax. Inhibition of p53 with pifthrin-alpha or Bax with siRNAs significantly reduced rotenone-induced Bax induction and apoptotic cell death. These results suggest that the p38^MAPK/p53-dependent induction of Bax contributes to rotenone’s neurotoxicity in PD models.     

Detoxified Extract of <Emphasis Type="Italic">Rhus verniciflua</Emphasis> Stokes Inhibits Rotenone-Induced Apoptosis in Human Dopaminergic Cells,SH-SY5Y

Rhus verniciflua Stokes (RVS), traditionally used as a food supplement and in traditional herbal medicine for centuries in Korea, is known to possess various pharmacological properties. Environmental neurotoxins such as rotenone, a specific inhibitor of complex I provide models of Parkinson’s disease (PD) both in vivo and in vitro. In this study, we investigated the neuroprotective effect of RVS against rotenone-induced toxicity in human dopaminergic cells, SH-SY5Y. Cells exposed to rotenone for 24 h-induced cellular injury and apoptotic cell death. Pretreatment of cells with RVS provided significant protection to SH-SY5Y cells. Further, RVS offered remarkable protection against rotenone-induced oxidative stress and markedly inhibited mitochondrial membrane potential (MMP) disruption. RVS also attenuated the up-regulation of Bax, Caspase-9 and Caspase-3 and down-regulation of Bcl-2. Moreover, pretreatment with RVS prevented the decrease in tyrosine hydroxylase (TH) levels in SH-SY5Y cells. Interestingly, RVS conferred profound protection to human dopaminergic cells by preventing the downregulation of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). These results suggest that RVS may protect dopaminergic neurons against rotenone-induced apoptosis by multiple functions and contribute to neuroprotection in neurodegenerative diseases, such as PD.     

Rutin from Dendropanax morbifera Leveille Protects Human Dopaminergic Cells Against Rotenone Induced Cell Injury Through Inhibiting JNK and p38 MAPK Signaling

Dendropanax morbifera Leveille (Araliaceae) is well known in Korean traditional medicine for a variety of diseases. Rotenone is a commonly used neurotoxin to produce in vivo and in vitro Parkinson’s disease models. This study was designed to elucidate the processes underlying neuroprotection of rutin, a bioflavonoid isolated from D. morbifera Leveille in cellular models of rotenone-induced toxicity. We found that rutin significantly decreased rotenone-induced generation of reactive oxygen species levels in SH-SY5Y cells. Rutin protected the increased level of intracellular Ca²⁺ and depleted level of mitochondrial membrane potential (ΔΨm) induced by rotenone. Furthermore, it prevented the decreased ratio of Bax/Bcl-2 caused by rotenone treatment. Additionally, rutin protected SH-SY5Y cells from rotenone-induced caspase-9 and caspase-3 activation and apoptotic cell death. We also observed that rutin repressed rotenone-induced c-Jun N-terminal kinase and p38 mitogen-activated protein kinase phosphorylation. These results suggest that rutin may have therapeutic potential for the treatment of neurodegenerative diseases associated with oxidative stress.     

Loss of mitochondrial complex I activity potentiates dopamine neuron death induced by microtubule dysfunction in a Parkinson's disease model

Mitochondrial complex I dysfunction is regarded as underlying dopamine neuron death in Parkinson's disease models. However, inactivation of the Ndufs4 gene, which compromises complex I activity, does not affect the survival of dopamine neurons in culture or in the substantia nigra pars compacta of 5-wk-old mice. Treatment with piericidin A, a complex I inhibitor, does not induce selective dopamine neuron death in either Ndufs4(+/+) or Ndufs4(-/-) mesencephalic cultures. In contrast, rotenone, another complex I inhibitor, causes selective toxicity to dopamine neurons, and Ndufs4 inactivation potentiates this toxicity. We identify microtubule depolymerization and the accumulation of cytosolic dopamine and reactive oxygen species as alternative mechanisms underlying rotenone-induced dopamine neuron death. Enhanced rotenone toxicity to dopamine neurons from Ndufs4 knockout mice may involve enhanced dopamine synthesis caused by the accumulation of nicotinamide adenine dinucleotide reduced. Our results suggest that the combination of disrupting microtubule dynamics and inhibiting complex I, either by mutations or exposure to toxicants, may be a risk factor for Parkinson's disease.     

Dopamine Selectively Sensitizes Dopaminergic Neurons to Rotenone-Induced Apoptosis

Among various types of neurons affected in Parkinson’s disease, dopamine (DA) neurons of the substantia nigra undergo the most pronounced degeneration. Products of DA oxidation and consequent cellular damage have been hypothesized to contribute to neuronal death. To examine whether elevated intracellular DA will selectively predispose the dopaminergic subpopulation of nigral neurons to damage by an oxidative insult, we first cultured rat primary mesencephalic cells in the presence of rotenone to elevate reactive oxygen species. Although MAP2⁺ neurons were more sensitive to rotenone-induced toxicity than type 1 astrocytes, rotenone affected equally both DA (TH⁺) neurons and MAP2⁺ neurons. In contrast, when intracellular DA concentration was elevated, DA neurons became selectively sensitized to rotenone. Raising intracellular DA levels in primary DA neurons resulted in dopaminergic neuron death in the presence of subtoxic concentrations of rotenone. Furthermore, mitochondrial superoxide dismutase mimetic, manganese (III) meso-tetrakis (4-benzoic acid) porphyrin, blocked activation of caspase-3, and consequent cell death. Our results demonstrate that an inhibitor of mitochondrial complex I and increased cytosolic DA may cooperatively lead to conditions of elevated oxidative stress and thereby promote selective demise of dopaminergic neurons.     

1-Methyl-4-phenylpyridinium accumulates in cerebellar granule neurons via organic cation transporter 3

1-Methyl-4-phenylpyridinium (MPP+), the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, induces apoptosis in cerebellar granule neurons (CGNs). We have tested the hypothesis that organic cation transporter (OCT) 3 mediates the accumulation and, hence, the toxicity of MPP+ in CGNs. CGNs in primary culture express OCT3 but do not express mRNA for OCT1, OCT2 or the dopamine transporter. Cerebellar astrocytes are negative for OCT3 protein by immunocytochemistry. [3H]MPP+ accumulation by CGNs exhibits first-order kinetics, and a Kt value of 5.3 +/- 1.2 micro m and a Tmax of 0.32 +/- 0.02 pmol per min per 106 cells. [3H]MPP+ accumulation is inhibited by corticosterone, beta-estradiol and decynium 22 with Ki values of 0.25 micro m, 0.17 micro m and 4.0 nm respectively. [3H]MPP+ accumulation is also inhibited by desipramine, dopamine, serotonin and norepinephrine, but is not affected by carnitine (10 mm), mazindol (9 micro m) or GBR 12909 (1 micro m). MPP+-induced caspase-3-like activation and cell death are prevented by pretreatment with 5 micro mbeta-estradiol. In contrast, the neurotoxic effects of rotenone are unaffected by beta-estradiol. Interestingly, GBR 12909 protects CGNs from both MPP+ and rotenone toxicity. In summary, CGNs accumulate MPP+ in manner that is consistent with uptake via OCT3 and the presence of this protein in CGNs explains their sensitivity to MPP+ toxicity.     

Neurodegeneration of mouse nigrostriatal dopaminergic system induced by repeated oral administration of rotenone is prevented by 4-phenylbutyrate, a chemical chaperone

Parkinson's disease (PD) is a progressive neurodegenerative disorder that is primarily characterized by the degeneration of dopaminergic neurons in the nigrostriatal pathway. Previous studies have demonstrated that chronic systemic exposure of Lewis rats to rotenone produced many features of PD, and cerebral tauopathy was also detected in the case of severe weight loss. The present study was designed to assess the neurotoxicity of rotenone after daily oral administration for 28 days at several doses in C57BL/6 mice. In addition, we examined the protective effects of 4-phenylbutyrate (4-PBA) on nigral dopamine (DA) neurons in rotenone-treated mice. 4-PBA was injected intraperitoneally daily 30 min before each oral administration of rotenone. Chronic oral administration of rotenone at high doses induced specific nigrostriatal DA neurodegeneration, motor deficits and the up-regulation of alpha-synuclein in the surviving DA neurons. In contrast to the Lewis rat model, cerebral tauopathy was not detected in this mouse model. 4-PBA inhibited rotenone-induced neuronal death and decreased the protein level of alpha-synuclein. These results suggest that this rotenone mouse model may be useful for understanding the mechanism of DA neurodegeneration in PD, and that 4-PBA has a neuroprotective effect in the treatment of PD.     

Rotenone induces cell death in primary dopaminergic culture by increasing ROS production and inhibiting mitochondrial respiration

Although the definite etiology of Parkinson's disease is still unclear, increasing evidence has suggested an important role for environmental factors such as exposure to pesticides in increasing the risk of developing Parkinson's disease. In the present study, primary cultures prepared from embryonic mouse mesencephala were applied to investigate the toxic effects and underlying mechanisms of rotenone-induced neuronal cell death relevant to Parkinson's disease. Results revealed that rotenone destroyed dopaminergic neurons in a dose- and time-dependent manner. Consistent with the cytotoxic effect of rotenone as evidenced by dopaminergic cell loss, it significantly increased the release of lactate dehydrogenase into the culture medium, the number of necrotic cells in the culture and the number of nuclei showing apoptotic features. Rotenone exerted toxicity by decreasing the mitochondrial membrane potential, increasing reactive oxygen species production and shifting respiration to a more anaerobic state.     

L-deprenyl protects against rotenone-induced, oxidative stress-mediated dopaminergic neurodegeneration in rats

The present study investigated oxidative damage and neuroprotective effect of the antiparkinsonian drug, L-deprenyl in neuronal death produced by intranigral infusion of a potent mitochondrial complex-I inhibitor, rotenone in rats. Unilateral stereotaxic intranigral infusion of rotenone caused significant decrease of striatal dopamine levels as measured employing HPLC-electrochemistry, and loss of tyrosine hydroxylase immunoreactivity in the perikarya of ipsilateral substantia nigra (SN) neurons and their terminals in the striatum. Rotenone-induced increases in the salicylate hydroxylation products, 2,3- and 2,5-dihydroxybenzoic acid indicators of hydroxyl radials in mitochondrial P2 fraction were dose-dependently attenuated by L-deprenyl. L-deprenyl (0.1-10mg/kg; i.p.) treatment dose-dependently attenuated rotenone-induced reductions in complex-I activity and glutathione (GSH) levels in the SN, tyrosine hydroxylase immunoreactivity in the striatum or SN as well as striatal dopamine. Amphetamine-induced stereotypic rotations in these rats were also significantly inhibited by deprenyl administration. The rotenone-induced elevated activities of cytosolic antioxidant enzymes superoxide dismutase and catalase showed further significant increase following L-deprenyl. Our findings suggest that unilateral intranigral infusion of rotenone reproduces neurochemical, neuropathological and behavioral features of PD in rats and L-deprenyl can rescue the dopaminergic neurons from rotenone-mediated neurodegeneration in them. These results not only establish oxidative stress as one of the major causative factors underlying dopaminergic neurodegeneration as observed in Parkinson's disease, but also support the view that deprenyl is a potent free radical scavenger and an antioxidant.     

Role of nitric oxide in rotenone-induced nigro-striatal injury

Rotenone, a widely used pesticide, causes a syndrome in rats that mimics, both behaviorally and pathologically, the symptoms of Parkinson's disease. The present study evaluated the role of nitric oxide in rotenone-induced nigro-striatal injury. After administration of rotenone in rats for 40 days, there was a moderate but significant injury of the nigro-striatal pathway indicated by a 47% decrease in striatal dopamine levels and a 28% loss of substantia nigra tyrosine hydroxylase-immunopositive neurons. Furthermore, a significant (37%) increase in the number of cells positive for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in the striatum was observed, accompanied by a 83% increase in nitric oxide synthase (NOS) activity and a significant increase in the production of 3-nitrotyrosine (3-NT). There was a significant increase (45%) in the optical density of NADPH-d staining and an increase (72%) in NOS activity in the substantia nigra. Moreover, administration of the neuronal NOS inhibitor 7-nitroindazole significantly attenuated the increased NOS activity and 3-NT production, and provided significant protection against rotenone-induced nigro-striatal injury. Our data suggest that chronic rotenone administration can lead to significant injury to the nigro-striatal system, mediated by increased generation of nitric oxide.     

Edaravone guards dopamine neurons in a rotenone model for Parkinson's disease

3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone), an effective free radical scavenger, provides neuroprotection in stroke models and patients. In this study, we investigated its neuroprotective effects in a chronic rotenone rat model for Parkinson's disease. Here we showed that a five-week treatment with edaravone abolished rotenone's activity to induce catalepsy, damage mitochondria and degenerate dopamine neurons in the midbrain of rotenone-treated rats. This abolishment was attributable at least partly to edaravone's inhibition of rotenone-induced reactive oxygen species production or apoptotic promoter Bax expression and its up-regulation of the vesicular monoamine transporter 2 (VMAT2) expression. Collectively, edaravone may provide novel clinical therapeutics for PD.     

Apoptotic signaling in dopamine-induced cell death: the role of oxidative stress, p38 mitogen-activated protein kinase, cytochrome c and caspases

Oxidative stress generated by dopamine (DA) oxidation could be one of the factors underlying the selective vulnerability of nigral dopaminergic neurons in Parkinson's diseases. Here we show that DA induces apoptosis in SH-SY5Y neuroblastoma cells demonstrated by activation of caspase-9 and caspase-3, cleavage of poly(ADP-ribose) polymerase as well as nuclear condensation. We also show that p38 mitogen-activated protein kinase is activated within 10 min of DA treatment, which precedes the onset of apoptosis because the potent p38 kinase inhibitor SB203580 protects against DA-induced cell death as well as against caspase-9 and caspase-3 activation. In addition, the antioxidant N-acetyl-L-cysteine (NAC) effectively blocks DA-induced p38 kinase activation, caspase-9 and caspase-3 cleavage and subsequent apoptosis, indicating that DA triggers apoptosis via a signaling pathway that is initiated by the generation of reactive oxygen species (ROS). Dopamine exerts its toxicity principally intracellularly as the DA uptake inhibitor, nomifensine significantly reduces DA-induced cell death as well as activation of p38 kinase and caspase-3. Furthermore, DA induces mitochondrial cytochrome c release, which is dependent on p38 kinase activation and precedes the cleavage of caspases. These observations indicate that DA induces apoptosis primarily by generating ROS, p38 kinase activation, cytochrome c release followed by caspase-9 and caspase-3 activation.     

Mechanisms to prevent caspase activation in rotenone-induced dopaminergic neurodegeneration: role of ATP depletion and procaspase-9 degradation

The evidence implicating a mode of cell death that either favors or argues against caspase-dependent apoptosis is available in studies that used experimental models of Parkinson’s disease. We sought to investigate the mechanisms by which release of cytochrome c is not linked to caspase activation during rotenone-induced dopaminergic (DA) neurodegeneration. Unlike caspase activation in 6-hydroxydopamine-treated cells, both MN9D DA neuronal cells and primary cultures of mesencephalic neurons showed no obvious signs of caspase activation upon exposure to rotenone. We found that intracellular levels of ATP significantly decreased at the early phase of neurodegeneration (<~24 h) and therefore external addition of ATP to the lysates obtained at this stage reconstituted caspase-3 activity. At a later phase of cell death (>~24 h), both decreased levels of ATP and procaspase-9 contributed to the lack of caspase-3 activation. Under this condition, calpain and the proteasome system were responsible for the degradation of procaspase-9. Consequently, external addition of ATP and procaspase-9 to the lysates harvested at the later phase was required for activation of caspase-3. Similarly, caspase-3 activity was also reconstituted in the lysates harvested from cells co-treated with inhibitors of these proteases and incubated in the presence of external ATP. Taken together, our findings provided a sequential mechanism underlying how DA neurons may undergo caspase-independent cell death, even in the presence of cytoplasmic cytochrome c following inhibition of mitochondrial complex I.     

Apoptosis induced by interferon-alpha and antagonized by EGF is regulated by caspase-3-mediated cleavage of gelsolin in human epidermoid cancer cells

We have previously reported that interferon-alpha (IFNalpha) induces apoptosis and EGF can antagonize this effect in human epidermoid cancer KB cells. Since apoptosis occurs together with cytoskeleton reorganization we have evaluated if IFNalpha and EGF could modulate cell remodeling in our experimental conditions. We have found that 48 h 1,000 IU/ml IFNalpha induced structural reorganization of stress fibers and membrane delocalization and partial capping of the actin severing protein gelsolin. The transfection of KB cells with both a wild type (WT) or a C-terminal truncated form of gelsolin caused overexpression of the protein and an increase of both the spontaneous and IFNalpha-induced apoptosis and cell cytoskeletal modifications. In fact, after 48 h of treatment IFNalpha induced 45% of apoptotic cell death in parental cells while an approximately 80% of cell population was apoptotic in transfected cells. These effects occurred together with an increase of the expression and consequent degradation of gelsolin. Again the addition of EGF to IFNalpha-treated transfected cells caused a recovery of the apoptosis. Notably, IFNalpha and EGF did not modify the expression of other molecules associated to cytoskeleton such as focal adhesion kinase and vinculin. In the same experimental conditions IFNalpha induced also gelsolin cleavage that occurred together with caspase-3 activation and release of cytochrome c. All these effects were antagonized by the exposure of IFNalpha-treated KB to 10 nM EGF for the last 12 h. Moreover, the specific inhibition of caspase-3 with 20 microM DEVD completely abrogated apoptosis and gelsolin cleavage induced by IFNalpha. In conclusion, our data are the first demonstration that IFNalpha can induce morphological cell changes that are peculiar of apoptosis onset through the caspase-3-mediated cleavage of gelsolin. Furthermore, we have demonstrated that EGF is able to antagonize these effects through the inhibition of caspase-3 activation.