Neurotrophin-3 reduces apoptosis induced by 6-OHDA in PC12 cells through Akt signaling pathway

Previous work has demonstrated that 6-hydroxydopamine (6-OHDA) induces apoptosis in PC12 cells. The goal of the present study was to investigate the mechanisms underlying the protection by neurotrophin-3 (NT-3) against 6-OHDA-induced apoptosis in PC12 cells. Treatment of PC12 cells with 6-OHDA resulted in activation of caspase-3 and subsequent apoptosis, as detected by TUNEL staining. In addition, Akt phosphorylation was decreased following 6-OHDA treatment. Pretreatment with NT-3 reduced the percentage of apoptotic cells and caspase-3 activity induced by 6-OHDA and suppressed the cleavage of caspase-3 and Poly(ADP-ribose) polymerase (PARP) with a significant decrease in cell viability. Moreover, Akt phosphorylation was enhanced and 6-OHDA-induced chromatin condensation was suppressed by NT-3. Such NT-3-evoked suppression in chromatin condensation was reversed by anti-TrkA antibody receptor blockade. Further study revealed that LY294002, an inhibitor of PI3-kinase (a molecule upstream of Akt), enhanced 6-OHDA-induced apoptosis. These data indicate that NT-3 prevents 6-OHDA-induced apoptosis in PC12 cells via activation of PI3-kinase/Akt pathway.     

Involvement of caspase-3 and p38 mitogen-activated protein kinase in cobalt chloride-induced apoptosis in PC12 cells

Our previous study showed that cobalt chloride (CoCl2) could induce PC12 cell apoptosis and that the CoCl2-treated PC12 cells may serve as a simple in vitro model for the study of the mechanism of hypoxia-linked neuronal disorders. The aim of this study is to elucidate the mechanism of CoCl2-induced apoptosis in PC12 cells. Caspases are known to be involved in the apoptosis induced by various stimuli in many cell types. To investigate the involvement of caspases in CoCl2-induced apoptosis in PC12 cells, we generated PC12 cells that stably express the viral caspases inhibitor gene p35 and analyzed the effect of p35 on the process of apoptosis induced by CoCl2. We also examined the effect of cell-permeable peptide inhibitors of caspases. The results showed that the baculovirus p35 gene and the general caspases inhibitor Z-VAD-FMK significantly block apoptosis induced by CoCl2, confirming that caspase is involved in CoCl2-induced apoptosis. Further investigation showed that in this process the caspase-3-like activity is increased, as indicated by the cells' ability to cleave the fluorogenic peptide substrate Ac-Asp-Glu-Val-Asp-7-AMC and to degrade the DNA-repairing enzyme poly-(ADP-ribose) polymerase (PARP), an endogenous caspase-3 substrate. At the same time, caspase-3-specific inhibitors, namely, the peptide Ac-DEVD-CHO, Ac-DEVD-FMK, partially inhibit CoCl2-induced apoptosis. These findings suggested that caspase-3 or caspase-3-like proteases are involved in the apoptosis induced by CoCl2 in PC12 cells. Additionally, we have observed that another apoptotic marker, p38 mitogen-activated protein kinase (MAPK), is significantly activated in this process in a time-dependent manner and that a selective p38 MAPK inhibitor, SB203580, partially inhibits this cell death. The addition of SB203580 also partially suppresses caspase-3-like activity. All these results confirm that the CoCl2-treated PC12 cell is a useful in vitro model with which to study hypoxia-linked neuronal disorders. Furthermore, the results showing that the baculovirus p35 gene and caspase inhibitors possess a remarkable ability to rescue PC12 cells from CoCl2-induced cell death may have implications for future neuroprotective therapeutic approaches for the hypoxia-associated disorders.     

The intracellular domain of p55 tumor necrosis factor receptor induces apoptosis which requires different caspases in naive and neuronal PC12 cells

Apoptosis is induced in cells via distinct pathways, which may differ according to various stimuli and different cell types. One apoptotic stimulus is the activation of receptors such as the p55 tumor necrosis factor (TNF) receptor. These receptors transduce their apoptotic signals via a cytoplasmic region termed the death domain. Here we investigated the apoptotic pathway induced by overexpression of the intracellular domain of p55 TNF receptor (p55-IC) in a neuronal model system consisting of PC12 cells. Using the tetracycline-regulated transactivator system, which allows controlled gene expression, we show that overexpression of p55-IC induces apoptosis in both naive and neuronal PC12 cells. The apoptosis-inducing effect of p55-IC is blocked by the expression of bcl-2, suggesting that p55-IC induces apoptosis in PC12 cells via a pathway controlled by bcl-2. The need for caspases in the p55-IC-induced cell death effect in naive and neuronal PC12 cells was studied by examining the effects of broad-spectrum and specific inhibitors of caspases as well as expression of antisense caspase-2 RNA. The broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone blocked p55-IC-induced cell death in both naive and neuronal cells, suggesting that caspases are needed for this process in both cell types. Caspase-1-like proteases are most probably not involved in the process since neither expression of crmA nor treatment with the caspase-1-specific peptide inhibitor Ac-Try-Val-Ala-Asp-aldehyde had any protective effect. Interestingly, expression of antisense caspase-2 RNA blocked the p55-IC-induced cell death in naive but not in neuronal PC12 cells, whereas the caspase-3-like specific inhibitor Ac-Asp-Glu-Val-Asp-aldehyde partially inhibited this death in neuronal but not in naive cells. These results suggest that the apoptosis-inducing effect of p55-IC requires different caspases in naive and neuronal PC12 cells. J. Neurosci. Res. 52:380–389, 1998. © 1998 Wiley-Liss, Inc.     

Ultrastructural confirmation of neuronal protection by melatonin against the neurotoxin 6-hydroxydopamine cell damage

6-Hydroxydopamine (6-OHDA) is a neurotoxin used in the induction of experimental Parkinson's disease in both animals and cultured neuronal cells. Biochemical and molecular approaches showed previously that low doses of 6-OHDA induced apoptosis in PC12 cells, while high doses of this neurotoxin induced necrosis. Melatonin has been shown to protect against the neuronal programmed cell death induced by 6-OHDA, although it was not able to prevent the massive necrotic cellular death occurring after the addition of high doses of the neurotoxin. In the present work, we demonstrate by ultrastructural analysis that although low doses of 6-OHDA induced apoptosis in PC12 cells, it also damaged the non-apoptotic cells, morphologically corresponding this damage to incipient and reversible necrotic lesions. When the doses of the neurotoxin increase, there are still apoptotic cells, although most of the cells show necrotic irreversible lesions. We also found that melatonin partially prevents the incipient necrotic lesions caused by low doses of 6-OHDA. The fact that melatonin was shown in previous work to prevent apoptosis caused by low doses of 6-OHDA, but not necrosis induced by high doses of the neurotoxin, seemed to indicate that this agent is only able to protect against apoptosis. However, our present results, melatonin preventing also the incipient necrotic neuronal lesions, suggest that this hormone may provide a general protection against cell death, suggesting that higher doses should be tried in order to prevent the necrotic cell death induced by high doses of the neurotoxin.     

Down-regulation of Bcl-2, activation of caspases,and involvement of reactive oxygen species in 6-hydroxydopamine-induced thymocyte apoptosis

OBJECTIVE: Our previous work showed that 6-hydroxydopamine (6-OHDA) induced mouse thymocytes to undergo apoptosis both in vivo and in vitro. In the present study, we further investigated the mechanisms of 6-OHDA-induced apoptosis in vitro. METHODS: Naive mouse thymocytes were cultured with 6-OHDA. The percentages of apoptotic cells were quantified by propidium iodide staining, and DNA fragmentation was detected by agarose gel electrophoresis. Intracellular Bcl-2 was analyzed by immunofluorescence staining. Cu/Zn superoxide dismutase (Cu/Zn-SOD) activities were measured by the SOD-525 method. RESULTS: The apoptotic effect of 6-OHDA was blocked by desipramine, a catecholamine uptake blocker. Treatment with 6-OHDA caused a reduction in Bcl-2 expression. VAD-FMK, a broad-spectrum caspase inhibitor, and DEVD-CHO, a potent inhibitor of caspase-3, could block 6-OHDA-induced thymocyte apoptosis. However, the specific caspase-1 (ICE) inhibitor YVAD-CMK had no effect. This cell death process was prevented by the protein synthesis inhibitor cycloheximide and by antioxidants. The level of Cu/Zn-SOD activities also decreased after cells were exposed to 6-OHDA. CONCLUSION: These results suggest an apoptotic effect of 6-OHDA via the uptake of this neurotoxin by thymocytes, and that down-regulation of Bcl-2, activation of caspases, such as caspase-3 but not caspase-1, generation of reactive oxygen species, and new synthesis of proteins are involved in this process.     

D-beta-hydroxybutyrate inhibits the apoptosis of PC12 cells induced by 6-OHDA in relation to up-regulating the ratio of Bcl-2/Bax mRNA

D-beta-hydroxybutyrate (DbetaHB) is a predominant member of ketone bodies produced by hepatocytes and, to a lesser extent, by astrocytes. It is an alternative source of energy in the brain when glucose supply is depleted such as during starvation. It has been reported that ketone bodies could protect dopaminergic culture. However, the biological function of DbetaHB in Parkinson disease (PD) is still unclear. In the present work, we investigated the role of DbetaHB in protecting rat pheochromocytoma (PC12) cells from apoptosis induced by 6-Hydroxydopamine (6-OHDA). DbetaHB rescued PC12 cells from apoptotic death induced by 6-OHDA by MTT assay, acridine orange (AO) staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining and the activity of caspase-3. DbetaHB prevented the decrease of cell viability and the increase of caspase-3 activity induced by 6-OHDA in a dose-dependent manner in PC12 cells. AO and TUNEL staining showed that DbetaHB prevented the apoptosis of PC12 cells induced by 6-OHDA. The ratio of Bcl-2/Bax at mRNA levels, which regulates the apoptosis of PC12 cells when exposed to 6-OHDA, increased when DbetaHB was preincubated. The data showed that DbetaHB inhibited the apoptosis of PC12 cells induced by 6-OHDA in relation to up-regulating the ratio of Bcl-2/Bax mRNA.     

尿酸减轻6-羟基多巴胺对PC12细胞的毒性作用

目的：评价尿酸减轻6．羟基多巴胺（6-OHDA）对PC12细胞的毒性作用。方法：应用PCI2细胞制作帕金森细胞模型,分为对照组、尿酸组、6-OHDA组、尿酸＋6-OHDA组。采用MTT测定各组PC12细胞活性,免疫荧光法观察各组PCI2细胞caspase-3激活情况,流式细胞术检测各组PC12细胞凋亡率。尿酸100～400μmol·L^-1不影响PCI2细胞生存率,尿酸100～400μmol·L-1可显著提高6-OHDA50gmol-L。作用6、12和24h造成的PCI2细胞生存率的下降（P〈0．01）;尿酸能减少6-OHDA导致的PCI2细胞caspase-3激活,降低6-OHDA导致的凋亡率（P〈0．05）。结论：尿酸具有减轻6-OHDA对PC12细胞的毒性作用。     

6-Hydroxydopamine induces dopaminergic cell degeneration via a caspase-9-mediated apoptotic pathway that is attenuated by caspase-9dn expression

This study showed that primary dopaminergic neurons or the dopaminergic cell line MN9D, when exposed to 15 min of the parkinsonian toxin 6-hydroxydopamine (6-OHDA) in the range of 30-100 microM, underwent delayed degeneration and exhibited hallmarks of apoptosis. These results, along with the absence of any increase in lactate dehydrogenase (LDH) release from the degenerated cells, imply that apoptosis was the dominant mode of cell death. Moreover, a distinct elevation in the measured cellular activities of caspase-9 and -3 but not of caspase-8 points to the caspase-9/caspase-3 cascade as the predominant apoptotic pathway in the degeneration of dopaminergic neurons and MN9D cells. In addition, the presence of caspase-9 or -3 peptide inhibitors but not of caspase-8 inhibitor attenuated cell death significantly, supporting the notion that only the intrinsic apoptotic pathway is utilized to achieve cell death. Finally, overexpression of a mutant caspase-9 with dominant negative phenotype (caspase-9dn) in MN9D cells and primary dopaminergic neurons via the adenovirus and adenoassociated virus gene delivery system, respectively, conferred marked increases in tolerance to the toxicity of 6-OHDA. These results point to the intrinsic caspase-9/caspase-3 cascade as the predominant signaling pathway underlying dopaminergic cell death induced by 6-OHDA and suggest that gene delivery of caspase-9dn can attenuate this pathway and its degenerative consequences.     

Progressive degeneration of dopamine neurons in 6-hydroxydopamine rat model of Parkinson's disease does not involve activation of caspase-9 and caspase-3

6-Hydroxydopamine (6-OHDA), a neurotoxin that causes the death of dopamine (DA) neurons, is commonly used to produce experimental models of Parkinson's disease (PD) in rodents. In the rat model of PD first described by Sauer and Oertel, DA neurons progressively die over several weeks following a striatal injection of 6-OHDA. It is generally assumed that DA neurons die through apoptosis after exposure to 6-OHDA, but data supporting activation of a caspase enzymatic cascade are lacking. In this study, we sought to determine if caspases involved in the intrinsic apoptotic cascade play a role in the initial stages of 6-OHDA-induced death of DA neurons in the progressively lesioned rat model of PD. We found that injection of 6-OHDA into adult rat striatum did not activate caspase-9 or caspase-3 or increase levels of caspase-dependent cleavage products in the substantia nigra at various survival times up to 7 days after the lesion, even though this paradigm produced DA neuronal loss. These data suggest that in the adult rat brain DA neurons whose terminals are challenged with 6-OHDA do not die through a classical caspase-dependent apoptotic mechanism.     

Caspase-3 is required for apoptosis-associated DNA fragmentation but not for cell death in neurons deprived of potassium

Caspases are crucial effectors of the cell death pathway activated by virtually all apoptosis-inducing stimuli within neurons and nonneuronal cells. Among the caspases, caspase-3 (CPP32) appears to play a pivotal role and has been found to be necessary for developmentally regulated cell death in the brain. We have used mice lacking caspase-3 (-/-CPP32) to examine its involvement in cultured cerebellar granule neurons induced to undergo apoptosis by potassium deprivation (K+). We find that, following K+ deprivation, neurons from -/-CPP32 mice die to the same extent as those from normal (+/+) mice. Although a small delay in the induction of cell death is observed in -/-CPP32 neurons, the rate of cell death is generally comparable to that of +/+ cultures. Though not critical for neuronal death, caspase-3 is required for DNA fragmentation and chromatin condensation as judged by the absence of these apoptotic features in -/-CPP32 neurons. Boc.Asp.fmk, a pan caspase inhibitor, partially protects +/+ neurons from low-K+-mediated cell death and does so to the same extent in -/-CPP32 cultures, suggesting the involvement of a caspase other than caspase-3 in cell death. However, the protective effect of boc.Asp.fmk is not seen beyond 24 hr, suggesting that the effect of caspase inhibition is one of delaying rather than preventing apoptosis. The more selective caspase inhibitors DEVD.fmk, IETD.fmk, and VEID.fmk fail to affect cell death, indicating that members inhibited by these agents (such as caspases - 6 ,7, 8, 9 and 10) are also not involved in low-K+-mediated apoptosis.     

Nerve growth factor inhibits apoptosis induced by tumor necrosis factor in PC12 cells.

Tumor necrosis factor-alpha (TNFalpha) may play a role in at least some of the neuronal death that occurs following brain insults or in neurodegenerative diseases. It is therefore important to characterize the mechanism underlying apoptosis induced by TNFalpha in neuronal cells and to identify factors capable of protecting neurons from this death. In the present study, we characterized the apoptotic effect of TNFalpha in PC12 cells, a model system commonly used for studying neuronal apoptosis, and examined the role of Bcl-2 and caspases in this process. We show that TNFalpha induces apoptosis in both naive and primed PC12 cells. The TNFalpha-induced apoptosis was inhibited by nerve growth factor (NGF) but not by insulin. These findings suggest that the apoptotic effect of TNFalpha can be inhibited by trophic factors and that the survival-promoting effect of NGF is mediated by a specific pathway not shared by all tyrosine kinase receptors. The effect of Bcl-2 on TNFalpha-induced apoptosis was examined in PC12 cells overexpressing Bcl-2. These cells were resistant to TNFalpha-induced apoptosis, suggesting that the apoptotic effect of TNFalpha in PC12 cells is mediated via a pathway controlled by Bcl-2. Examination of the role of caspase-3 like activity in TNFalpha-induced apoptosis showed that caspase-3-like proteases are activated, and their substrate, poly (ADP-ribose) polymerase, is cleaved following TNFalpha treatment. In addition, the broad-spectrum inhibitor of caspases, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), was found to inhibit the TNFalpha-induced apoptosis of PC12 cells. These results suggest that caspases are activated following TNFalpha treatment and are needed for TNFalpha-induced apoptosis in PC12 cells.     

半夏总生物碱对6-OHDA诱导PC12细胞损伤的保护作用及机制

目的探讨半夏总生物碱(TAPT)对6羟基多巴胺(6-OHDA)诱导的大鼠肾上腺嗜铬细胞瘤细胞PC12细胞株损伤的保护作用及其机制。方法通过不同剂量TAPT预处理PC12细胞后,加入6-OHDA诱导氧化应激损伤。采用甲基噻唑基四唑比色法(MTT法)检测PC12细胞活力、天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)活性检测试剂盒测定caspase-3活性、黄嘌呤氧化酶法检测细胞总超氧化物歧化酶(T-SOD)活力,硫代巴比妥法检测丙二醛(MDA)的含量、按Fenton反应原理检测抑制羟自由基(·OH)的能力。结果浓度为100μmol/L的6-OHDA作用PC12细胞24h后,与正常对照组相比,细胞活力明显降低(P<0.05),T-SOD活性和抑制·OH的能力明显下降(P<0.05),而MDA含量和caspase-3活性明显升高(P<0.05);与6-OHDA组相比,TAPT处理组的PC12细胞活力逐渐升高(P<0.05),T-SOD活性和抑制·OH的能力明显上升(P<0.05),而MDA含量和caspase-3活性明显降低(P<0.05)。结论 TAPT对6-OHDA诱导的PC12细胞损伤具有一定的保护作用;提高T-SOD活性和抑制·OH的能力及降低MDA含量和caspase-3活性可能是其作用机制之一。     

Need for caspases in apoptosis of trophic factor-deprived PC12 cells

PC12 cells are a useful model system for studying neuronal apoptosis. Like neurons, they undergo apoptosis when deprived of trophic support. Involvement of caspases [interleukin 1β-converting enzyme (ICE)-related proteases] has been implicated in apoptosis induced by various stimuli in many cell types, including neurons. In the present study we investigated the need for caspases participation in apoptosis induced by growth factor deprivation in naive and neuronal PC12 cells. For this purpose we generated PC12 cell lines that consistently express the viral caspases inhibitor genes p35 or crmA, and analyzed their susceptibility to trophic factor deprivation. We also examined the effects of cell-permeable peptide inhibitors of caspases. Our results showed that broad-spectrum inhibitors of the caspases, namely the baculovirus p35 gene and the peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, effectively inhibit the death of both naive and neuronal PC12 cells. However, caspase-1 (ICE)-specific inhibitors, namely the peptides Ac-Try-Val-Ala-Asp-chloromethylketone and Ac-Try-Val-Ala-Asp-aldehyde, as well as crmA, were much less effective. These findings demonstrate that caspases, but not caspase-1, are needed for apoptosis induced by trophic factor deprivation in both naive and neuronal PC12 cells. Northern and Western blot analyses showed that PC12 cells express caspase-3. We therefore examined the involvement of caspase-3 in the death process of trophic factor-deprived PC12 cells. Our results showed that the pro-caspase-3 and its substrate poly(ADP-ribose)polymerase are cleaved at similar rates in serum-deprived PC12 cells. Moreover, cell lysates prepared from these cells possess caspase-3-like activity, as determined by their ability to cleave the fluorogenic peptide substrate Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin. These findings strongly suggest that caspase-3 or caspase-3-like proteases are activated in trophic factor-deprived PC12 cells. J. Neurosci. Res. 50:69–80, 1997. © 1997 Wiley-Liss, Inc.     

Activation of caspase-9 and -3 during H2O2-induced apoptosis of PC12 cells independent of ceramide formation

The treatment of PC12 cells with H2O2 (100-500 microM) resulted in typical apoptotic changes including fragmentation and condensation of nuclei, and DNA fragmentation observed as DNA ladder. H2O2-induced apoptosis was associated with activation of caspase-3 as assessed by cleavage of specific fluorogenic substrate peptide and processing of procaspase-3 and poly(ADP-ribose) polymerase. However, formation of ceramide, which often locates upstream of caspase-3, was not observed. The inhibitory peptide relatively specific for caspase-3, z-DEVD-FMK and non-selective caspase inhibitor z-VAD-FMK inhibited activation of caspase-3 and apoptotic cell death. However, the relatively specific inhibitors, Ac-YVKD for caspase-1 and Ac-IETD for caspase-8/6, did not affect the occurrence of apoptotic cell death. As an upstream activation of caspase-3, induction of cytochrome c release followed by processing of procaspase-9 was observed by Western blotting, although the formation of intracellular ceramide was not observed. On the other hand, in PC12 cells overexpressing Bcl-2, the number of apoptotic cells was markedly decreased and activation of both caspases-9 and -3 was prevented. These results suggest that cytochrome c and caspase-9 initiate the activation of executor caspase-3 in H2O2-treated PC12 cells, and that Bcl-2 inhibits H2O2-induced release of cytochrome c from mitochondria and then proteolytic processing of procaspase-9.     

Enhanced cystatin C and lysosomal protease expression following 6-hydroxydopamine exposure

6-Hydroxydopamine (6-OHDA) is a selective neurotoxin used to induce apoptosis in catecholamine-containing neurons. Although biochemical products and reactive oxygen species (ROS) of 6-OHDA have been well documented, the activation of cellular pathways following exposure are not well understood. Apoptosis in PC12 (Pheochromocytoma) cells was induced by 6-OHDA in a dose (10-150 microM) and time-dependent (24-72 h) manner compared to experimental controls (no treatment). PC 12 cells exposed to 50 microM 6-OHDA demonstrated the involvement of caspase 3 and lysosomal protease alterations. Following 6-OHDA exposure, the caspase 3-like inhibitor Ac-DEVD-CHO significantly decreased 6-OHDA induced cell death. In addition, alterations in expression of the lysosomal cysteine and aspartic proteases, cathepsin B (CB) and cathepsin D (CD) and the endogenous cysteine protease inhibitor cystatin C were observed utilizing immunocytochemical analysis at 24, 48, and 72 h following 6-OHDA exposure. Furthermore, CB and CD and cystatin C immuno-like reactivity was more pronounced in TUNEL positive cells. Moreover, Western blot analysis confirmed a significant increase in protein expression for CB and CD at 72 h and a temporal and concentration dependent increase in cystatin C in response to 6-OHDA. Cells treated with pepstatin A, an inhibitor for CD, showed a significant decrease in cell death, however, CA-074ME, a specific inhibitor for CB, failed to protect cells from 6-OHDA induced cell death. Thus, these results suggest that apoptosis induced by 6-OHDA exposure is mediated in part through caspase 3 activation and lysosomal protease CD.     

Differential activation of PKC delta in the substantia nigra of rats following striatal or nigral 6-hydroxydopamine lesions

Parkinsonian neurodegeneration is associated with heightened levels of oxidative stress and the activation of apoptotic pathways. In an in vitro cellular model, we reported that 6-hydroxydopamine (6-OHDA) induces apoptotic cell death via the induction of mitochondrial dysfunction, the activation of caspase 3 and the consequent proteolytic activation of the redox-sensitive kinase, protein kinase C (PKC)δ, in PC12 cells. Here we have investigated the involvement of PKCδ in 6-OHDA-induced cell death in vivo . The nigrostriatal pathway of rats was lesioned by unilateral infusion of 6-OHDA into either the striatum or substantia nigra pars compacta (SNpc). Infusion into the SNpc resulted in rapid loss of tyrosine hydroxylase (TH)-positive cells (87% decrease after 4 days), consistent with a necrotic-like mode of cell death. In contrast, striatal infusion initiated a slower, progressive decline in TH immunoreactivity (25% decrease in the SNpc after 4 days); cell appearance was characteristic of apoptosis. This is consistent with a transient increase in active caspase 3 immunoreactivity at 4 days post-infusion, and a concomitant proteolytic activation of PKCδ in the SNpc of striatal-lesioned rats. Cleavage of PKCδ did not occur in the striatum or cerebellum of lesioned animals, or in the SNpc of sham-operated controls. No increase in caspase 3 immunoreactivity or proteolytic activation of PKCδ was detected in nigral-lesioned rats. These results suggest that after 6-OHDA infusion into the striatum, retrograde neurotoxicity induces caspase 3-dependent PKCδ proteolytic activation in the cell bodies of the SNpc, implicating this kinase in the neurodegenerative process.     

Erythropoietin attenuates 6-hydroxydopamine-induced apoptosis via glycogen synthase kinase 3β-mediated mitochondrial translocation of Bax in PC12 cells

The aim of this study was to determine the mechanism by which erythropoietin (EPO) suppressed 6-hydroxydopamine (6-OHDA)-induced apoptosis. Our results showed that 6-OHDA remarkably decreased phosphorylation of glycogen synthase kinase 3β (GSK3β) as well as enhanced the level of Bax in the mitochondria. Besides, 6-OHDA decreased the mitochondrial expression of Bcl-2 without altering the cytoplasmic expression of Bcl-2. In line with these results, 6-OHDA treatment enhanced the apoptosis and caspase 3 activity in PC12 cells. These findings indicated that mitochondrial dysfunction was involved in the neurotoxicity of 6-OHDA and GSK3β might act upstream of Bax/Bcl-2 and the caspase 3 pathways in 6-OHDA-treated PC12 cells. Furthermore, EPO reduced 6-OHDA-induced growth inhibition. Western blot exhibited that GSK3β inhibitor 4-benzyl-2-methyl-1, 2,4-thiadiazolidine-3, 5-dione (TDZD8) and EPO not only increased the phosphorylation of GSK3β but also inhibited the mitochondrial translocation of Bax. In agreement with these results, EPO and TDZD8 obviously increased the mitochondrial expression of Bcl-2. Finally, TDZD-8 and EPO significantly suppressed the enhanced apoptosis and activity of caspase 3 induced by 6-OHDA. Taken together, GSK3β-mediated mitochondrial cell death pathway is involved in the neuroprotective effect of EPO against 6-OHDA-induced apoptosis.     

Caspase activation pathways induced by staurosporine and low potassium: role of caspase-2

Apoptotic death is a physiological process with regulatory mechanisms that are under the control of different molecules such as caspases. These are classified as initiators, such as caspases-8 and -9, and effectors, such as caspases-3 and -7. The participation of caspase-2 in the effector phase of apoptosis has been commonly observed in many cell types; however, it is able to act as an initiator caspase, depending on the apoptotic stimulus. Cerebellar granule cells (CGCs) undergo apoptosis when they are transferred from high potassium (K25) to low potassium (K5); this process seems to be mediated by caspase-3 activation. Staurosporine (STS), a full strength inhibitor of kinase proteins, also induces apoptosis in these cells. To characterize the caspase cascade induced by two stimuli in the same cell type we studied the activation of different caspases in CGCs treated with STS or K5. We found that both K5 and STS induce the activation of caspase-3. This result was confirmed by the proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), an endogenous caspase-3 substrate. Caspase-2 was activated preferentially by STS, which showed a temporal course suggesting that this caspase was induced before caspase-3. The initiator caspase-9 was also activated by both K5 and STS, as well as cytochrome-c release. The results obtained in this study suggest that STS and K5 induced different activation caspase pathways for apoptotic cell death of CGCs.     

Effects of CDNF on 6-OHDA-induced apoptosis in PC12 cells via modulation of Bcl-2/Bax and caspase-3 activation

Progressive dopamine neuron degeneration in the substantia nigra pars compacta is considered the most prominent pathological characteristic of Parkinson’s disease (PD). Currently, there is no cure, but only the capability to relieve the symptoms of PD. The conserved dopamine neurotrophic factor (CDNF) protects and rescues dopamine neurons in vivo. However, the molecular function of CDNF in PD remains unclear. In present study, we investigated the role and intrinsic mechanism of CDNF in preventing and reversing rat pheochromocytoma (PC12) cells from apoptosis induced by 6-hydroxydopamine (6-OHDA). We demonstrate that 6-OHDA induces cell death in PC12 cells, but that CDNF attenuates this effect in a dose-dependent manner. Further study shows that upregulation of the Bcl-2/Bax ratio and downregulation of caspase-3 activity are observed in a dose-dependent manner upon pre-treatment or post-treatment with CDNF, suggesting a pathway of regulation of apoptosis by CDNF. These data demonstrate that CDNF prevents the apoptosis of PC12 cells induced by 6-OHDA by modulating Bcl-2/Bax and caspase-3 activation.     

Baculovirus p35 gene greatly enhances PC12 cell's resistance against oxidative stress

Oxidative stress is thought to be a major contributor to the progress of the Parkinson's Disease (PD) because of the high vulnerability of dopaminergic cells against oxidative stress. The present work demonstrates that with the expression of the baculovirus p35 gene, PC12 cells could gain a high resistance against oxidative toxicants, hydrogen peroxide (H(2)O(2)) and 6-hydroxydopamine (6-OHDA). The DNA fragmentation analysis showed that PC12 cells underwent apoptosis after exposure to H(2)O(2) or 6-OHDA, while PP35 cells, a p35-expressing PC12 cell line, did not. Flow cytometric analysis showed that treatment with 150 microM H(2)O(2) or 120 microM 6-OHDA for 24 h caused 52.86% or 66.36% apoptotic cell, respectively, in PC 12 cells, but only 4.26% or 5.80% in PP35 cells. The cell viability measured by 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay indicated that H(2)O(2) and 6-OHDA induced a dose-dependent cell death on PC12 cells that were greatly remitted on PP35 cells. The viability of PP35 cells was even stronger than that of PC12 cells protected by glial cell line deprived neurotrophic factor (GDNF). The surviving PP35 cells remained normal cell morphology and showed positive with tyrosine hydroxylase (TH) immunocytochemical staining. These results indicate that baculovirus p35 gene possesses remarkable ability to rescue PC12 cells from death in experimental paradigms associated with oxidative stress.