The proto-oncogene Bcl-2 inhibits cellular toxicity of dopamine: possible implications for Parkinson's Disease

It is currently believed that excessive oxidant stress induced by metabolism of dopamine (DA), plays a major role in the pathogenesis of the selective nigrostriatal neuronal loss in Parkinson's disease. We recently showed that the neurotransmitter DA, in physiological concentrations, is capable of initiating apoptosis in cultured, post-mitotic sympathetic neurons. Bcl-2 is a proto-oncogene that blocks apoptosis. We now report that Bcl-2 is a powerful inhibitor of DA toxicity in PC-12 pheochromocytoma cells. We induced stable expression of Bcl-2 in PC-12 cells by transfection with recombinant pCMV5 expression vector, containing mouse bcl-2 (coding-sequence) cDNA. Cells expressing Bcl-2 manifested marked resistance to otherwise lethal (300 uM) in vitroconcentrations of DA. This protective effect was reflected in the trypan-blue test of cell survival, 3 H-thymidine incorporation and inhibition of the characteristic apoptotic morphologic alterations in scanning electron microscopic studies. Bcl-2 and associated control systems of apoptosis may have an important physiological role in restraining the apop-tosis-triggering potential of DA in nigrostriatal neurons. This novel field of research may yield insights into the pathogenesis of Parkinson's disease and lead to development of novel therapeutic approaches.     

Early events in Bcl-2-enhanced apoptosis

Transfection of PC12 pheochromocytoma cells with bcl-2 potentiates apoptosis induced by the antimitotic agent, neocarzinostatin (NCS). The mechanism of potentiation involves caspase 3-dependent cleavage of Bcl-2 to its pro-apoptotic counterpart, but the cellular events proximal to caspase 3 activation in this system are not known. Two min after initiation of NCS treatment, Bax begins to translocate from cytosol to the mitochondria; the mitochondrial localization of Bax persists for 30 min after NCS treatment. At the same time, cytochrome C is released from the mitochondria to cytosol. The mitochondrial membrane potential exhibits differential change in mock- and bcl-2-transfected PC12 cells. In mock-transfected PC12 cells, the mitochondrial membrane potential increases immediately, peaks at 15 min following initiation of NCS treatment, and drops thereafter. In contrast, in bcl-2-transfected PC12 cells, the membrane potential drops immediately following NCS treatment. Caspase 9 is activated and peaks at 10 min in both mock- and bcl-2 transfected PC12 cells, however, the peak activity of caspase 9 is higher and caspase 9 activation lasts longer (30 min) after the treatment in bcl-2 transfectants. Not until 30 min after initiation of a 1 h treatment with NCS is Bcl-2 protein cleaved in bcl-2-transfected cells. Thus, in bcl-2-transfected cells, the mitochondrial membrane potential drops and cytochrome C is released from the mitochondria despite the presence of large amounts of intact mitochondrial Bcl-2. This makes it unlikely that cleavage of Bcl-2 is the only factor involved in potentiation of NCS-induced apoptosis by Bcl-2.     

Bcl-2 protects against Abeta(25-35)-induced oxidative PC12 cell death by potentiation of antioxidant capacity

A substantial body of data indicates that reactive oxygen intermediates (ROIs) are implicated in pathogenesis of diverse human diseases. Oxidative stress induced by ROIs often causes cell death via apoptosis that is regulated by a plenty of functional genes and their protein products. Bcl-2 is one such protein that blocks apoptosis induced by various death stimuli. In spite of extensive research, the molecular mechanisms underlying antiapoptotic function of Bcl-2 are not fully clarified. In the present work, we have investigated the role of bcl-2 in protecting against beta-amyloid (Abeta)-induced oxidative death in rat pheochromocytoma (PC12) cells. Transfection with the antiapoptotic bcl-2 gene rescued PC12 cells from apoptotic death induced by Abeta. Addition of an NF-kappaB inhibitor, such as pyrrolidine dithiocarbamate or N-tosyl-l-phenylalanine chloromethyl ketone, to the media aggravated Abeta-induced PC12 cell death. PC12 cells overexpressing bcl-2 exhibited higher levels of constitutively activated NF-kappaB compared with vector-transfected controls, which appear to be mediated by the elevated activation of Akt/protein kinase B. The ectopic expression of bcl-2 enhanced both the expression and the activity of catalase, which were attenuated by NF-kappaB blockers. These results suggest that NF-kappaB plays a role in bcl-2-mediated protection against Abeta-induced apoptosis in PC12 cells through augmentation of cellular antioxidant capacity.     

Protection of intracellular dopamine cytotoxicity by dopamine disposition and metabolism factors

Dopamine has been hypothesized as a contributing factor for the selective degeneration of dopaminergic neurons in Parkinson's disease. However, the cytotoxic mechanisms of dopamine and its metabolites remain poorly understood. Using a stable aromatic amino acid decarboxylase (AADC) expressing a fibroblast cell line, we previously demonstrated a novel, non-oxidative cytotoxicity of intracellular dopamine. In this study, we further investigate the roles of dopamine metabolism and disposition proteins against intracellular dopamine cytotoxicity by co-expressing these factors in AADC-expressing cells. Our results indicate that overexpression of the vesicular monoamine transporter and monoamine oxidase A-induced protection against intracellular dopamine toxicity, and conversely that pharmacological inhibition of these pathways potentiated L-DOPA toxicity in catecholaminergic PC12 cells. Macrophage migration inhibitory factor and glutathione S-transferase (GST), factors that have recently been shown to be involved in dopamine metabolism, also exhibited a strong protective role against intracellular dopamine cytotoxicity. Our results support a potential role for non-oxidative cytoplasmic dopamine toxicity, and imply that disruption in dopamine disposition and/or metabolism could underlie the progressive degeneration of dopaminergic neurons in Parkinson's disease.     

Peroxisome proliferator-activated receptor gamma up-regulates the Bcl-2 anti-apoptotic protein in neurons and induces mitochondrial stabilization and protection against oxidative stress and apoptosis

Peroxisome proliferator-activated receptor gamma (PPARgamma) has been proposed as a therapeutic target for neurodegenerative diseases because of its anti-inflammatory action in glial cells. However, PPARgamma agonists preventbeta-amyloid (Abeta)-induced neurodegeneration in hippocampal neurons, and PPARgamma is activated by the nerve growth factor (NGF) survival pathway, suggesting a neuroprotective anti-inflammatory independent action. Here we show that the PPARgamma agonist rosiglitazone (RGZ) protects hippocampal and dorsal root ganglion neurons against Abeta-induced mitochondrial damage and NGF deprivation-induced apoptosis, respectively, and promotes PC12 cell survival. In neurons and in PC12 cells RGZ protective effects are associated with increased expression of the Bcl-2 anti-apoptotic protein. NGF-differentiated PC12 neuronal cells constitutively overexpressing PPARgamma are resistant to Abeta-induced apoptosis and morphological changes and show functionally intact mitochondria and no increase in reactive oxygen species when challenged with up to 50 microM H2O2. Conversely, cells expressing a dominant negative mutant of PPARgamma show increased Abeta-induced apoptosis and disruption of neuronal-like morphology and are highly sensitive to oxidative stress-induced impairment of mitochondrial function. Cells overexpressing PPARgamma present a 4- to 5-fold increase in Bcl-2 protein content, whereas in dominant negative PPARgamma-expressing cells, Bcl-2 is barely detected. Bcl-2 knockdown by small interfering RNA in cells overexpressing PPARgamma results in increased sensitivity to Abeta and oxidative stress, further suggesting that Bcl-2 up-regulation mediates PPARgamma protective effects. PPARgamma prosurvival action is independent of the signal-regulated MAPK or the Akt prosurvival pathways. Altogether, these data suggest that PPARgamma supports survival in neurons in part through a mechanism involving increased expression of Bcl-2.     

Bcl-2 protects against FCCP-induced apoptosis and mitochondrial membrane potential depolarization in PC12 cells.

This report addresses the relation between Bcl-2 and mitochondrial membrane potential (DeltaPsi(m)) in apoptotic cell death. Rat pheochromocytoma (PC12) cells are differentiated into neuron-like cells with nerve growth factor (NGF). It is known that Bcl-2 can attenuate apoptosis induced by deprivation of neurotrophic factor. The protective effect of Bcl-2 has been correlated with preservation of DeltaPsi(m). Protonophores, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), collapse the proton gradient across the mitochondrial inner membrane, resulting in a complete abolition of the mitochondrial membrane potential. Based on the analysis of morphology, of phosphatidylserine exposure and of nuclear fragmentation we conclude that FCCP induces apoptosis in PC12 cells, which can be prevented by overexpression of Bcl-2. To determine whether the cytoprotective effect of Bcl-2 is due to stabilization of DeltaPsi(m), we investigated the effect of Bcl-2 on changes in DeltaPsi(m), induced by FCCP in PC12 cells. We showed that treatment with FCCP induced a reduction in DeltaPsi(m), as assessed with the lipophilic cationic membrane potential-sensitive dye JC-1, and that Bcl-2 protects against FCCP-induced changes in NGF differentiated PC12 cells. Our data indicate that Bcl-2 protects against FCCP-induced cell death by stabilizing DeltaPsi(m).     

Neuroprotective effects of bcl-2 overexpression in hippocampal cultures: interactions with pathways of oxidative damage

Overexpression of bcl-2protects neurons from numerous necrotic insults, both in vitro and in vivo. While the bulk of such protection is thought to arise from Bcl-2 blocking cytochrome c release from mitochondria, thereby blocking apoptosis, the protein can target other steps in apoptosis, and can protect against necrotic cell death as well. There is evidence that these additional actions may be antioxidant in nature, in that Bcl-2 has been reported to protect against generators of reactive oxygen species (ROS), to increase antioxidant defenses and to decrease levels of ROS and of oxidative damage. Despite this, there are also reports arguing against either the occurrence, or the importance of these antioxidant actions. We have examined these issues in neuron-enriched primary hippocampal cultures, with overexpression of bcl-2 driven by a herpes simplex virus amplicon: (i) Bcl-2 protected strongly against glutamate, whose toxicity is at least partially ROS-dependent. Such protection involved reduction in mitochondrially derived superoxide. Despite that, Bcl-2 had no effect on levels of lipid peroxidation, which is thought to be the primary locus of glutamate-induced oxidative damage; (ii) Bcl-2 was also mildly protective against the pro-oxidant adriamycin. However, it did so without reducing levels of superoxide, hydrogen peroxide or lipid peroxidation; (iii) Bcl-2 protected against permanent anoxia, an insult likely to involve little to no ROS generation. These findings suggest that Bcl-2 can have antioxidant actions that may nonetheless not be central to its protective effects, can protect against an ROS generator without targeting steps specific to oxidative biochemistry, and can protect in the absence of ROS generation. Thus, the antioxidant actions of Bcl-2 are neither necessary nor sufficient to explain its protective actions against these insults in hippocampal neurons.     

Low concentrations of methamphetamine can protect dopaminergic cells against a larger oxidative stress injury: mechanistic study

Mild stress can protect against a larger insult, a phenomenon termed preconditioning or tolerance. To determine if a low intensity stressor could also protect cells against intense oxidative stress in a model of dopamine deficiency associated with Parkinson disease, we used methamphetamine to provide a mild, preconditioning stress, 6-hydroxydopamine (6-OHDA) as a source of potentially toxic oxidative stress, and MN9D cells as a model of dopamine neurons. We observed that prior exposure to subtoxic concentrations of methamphetamine protected these cells against 6-OHDA toxicity, whereas higher concentrations of methamphetamine exacerbated it. The protection by methamphetamine was accompanied by decreased uptake of both [(3)H] dopamine and 6-OHDA into the cells, which may have accounted for some of the apparent protection. However, a number of other effects of methamphetamine exposure suggest that the drug also affected basic cellular survival mechanisms. First, although methamphetamine preconditioning decreased basal pERK1/2 and pAkt levels, it enhanced the 6-OHDA-induced increase in these phosphokinases. Second, the apparent increase in pERK1/2 activity was accompanied by increased pMEK1/2 levels and decreased activity of protein phosphatase 2. Third, methamphetamine upregulated the pro-survival protein Bcl-2. Our results suggest that exposure to low concentrations of methamphetamine cause a number of changes in dopamine cells, some of which result in a decrease in their vulnerability to subsequent oxidative stress. These observations may provide insights into the development of new therapies for prevention or treatment of PD.     

Pharmacological study of the novel compound FLZ against experimental parkinson’s models and its active mechanism

FLZ is a synthetic new derivative of squamosamide. Pharmacological study found that FLZ given orally improved the abnormal behavior caused by the functional disturbance of dopaminergic and cholinergic neurons in mice. FLZ significantly increased the content of dopamine and its metabolites in striatum in MPTP model mice. FLZ also remarkably protected dopaminergic PC-12 cells against dopamine and MPP⁺ induced injury and apoptosis in vitro. The compound inhibited the formation of dopamine-melanin and protein polymers. Additionally, FLZ inhibited cytochrome-c release from mitochondria and caspase-3 activation by dopamine in PC-12 cells. The above results suggest that compound FLZ possesses anti-PD activity through neuroprotection.     

Adenovirus-mediated transfer of Bcl-X(L) protects neuronal cells from Bax-induced apoptosis

Bax-mediated apoptosis in neurons is involved in many pathologic conditions affecting the central nervous system, including degenerative diseases, stroke, and trauma. Two molecules belonging to the Bcl-2 family, Bcl-2 and Bcl-X(L), protect cells from Bax-induced apoptosis and show distinct expression patterns in adult neurons, with downregulated Bcl-2 and highly upregulated Bcl-X(L) expression. To investigate the biological functions of these two molecules in Bax-mediated apoptosis in neurons, we transduced various levels of Bcl-X(L) or Bcl-2 via adenoviral vectors into nerve growth factor (NGF)-treated PC12 cells. Overexpression of Bax induced drastic apoptosis in NGF-treated PC12 cells. Bcl-X(L) expressed at a wide range of levels conferred a high level of protection against Bax-mediated apoptosis. In contrast, Bcl-2 at various levels conferred far less protection against apoptosis. Moreover, Bcl-X(L) protected PC12 cells from apoptosis induced by NGF withdrawal. These data indicate that Bcl-X(L)-mediated protection is the major pathway that suppresses apoptosis in NGF-treated PC12 cells and that Bcl-X(L) would be a more relevant target of manipulation in future treatment strategies, including gene therapies.     

Shift of the Cellular Oxidation-Reduction Potential in Neural Cells Expressing Bcl-2

Abstract: Expression of the protooncogene bcl-2 inhibits both apoptotic and in some cases necrotic cell death in many cell types, including neural cells, and in response to a wide variety of inducers. The mechanism by which the Bcl-2 protein acts to prevent cell death remains elusive. One mechanism by which Bcl-2 has been proposed to act is by decreasing the net cellular generation of reactive oxygen species. To evaluate this proposal, we measured activities of antioxidant enzymes as well as levels of glutathione and pyridine nucleotides in control and bcl-2 transfectants in two different neural cell lines—rat pheochromocytoma PC12 and the hypothalamic GnRH cell line GT1-7. Both neural cell lines overexpressing bcl-2 had elevated total glutathione levels when compared with control transfectants. The ratios of oxidized glutathione to total glutathione in PC12 and GT1-7 cells overexpressing bcl-2 were significantly reduced. In addition, the NAD⁺/NADH ratio of bcl-2 -expressing PC12 and GT1-7 cells was two- to threefold less than that of control cell lines. GT1-7 cells overexpressing bcl-2 had the same level of glutathione peroxidase, catalase, superoxide dismutase, and glutathione reductase activities as control cells. PC12 cells overexpressing bcl-2 had a twofold increase in superoxide dismutase and catalase activity when compared with matched control transfected cells. The levels of glutathione peroxidase and glutathione reductase in PC12 cells overexpressing bcl-2 were similar to those of control cells. These results indicate that the overexpression of bcl-2 shifts the cellular redox potential to a more reduced state, without consistently affecting the major cellular antioxidant enzymes.     

Bcl-2 protects neuronal cells against taxol-induced apoptosis by inducing multi-nucleation

Taxol-induced peripheral neuropathy is a commonly-occurring side-effect in the treatment of cancer patients with taxoteres or taxanes. Taxol is known to induce apoptosis in a number of tumor cells. This report documents that, similar to proliferating cells, taxol induces apoptosis in NGF-differentiated PC12 cells, as assessed by exogenous FITC-annexin-V binding and nuclear fragmentation. It is shown that PC12 cells that stably overexpress Bcl-2 are protected against the toxic effect of taxol, as evidenced by the XTT assay and by a decreased fraction of propididum iodide positive cells in a dye exclusion test. Also the number of annexin-V-positive cells and the number of fragmented nuclei are lower in the Bcl-2 transfected cells. The effect is similar to the protective effect of Bcl-2 against NGF deprivation in differentiated PC12 cells. Although taxol forced both wild-type and Bcl-2-overexpressing cells into a mitotic state, only in Bcl-2-overexpressing cells did this lead to the appearance of metabolically active, multi-nucleated cells. This suggests that Bcl-2 is able to induce an alternative escape pathway, downstream of the G2/M block, in taxol-treated differentiated PC12 cells.     

Fasudil Mesylate Protects PC12 Cells from Oxidative Stress Injury via the Bax-Mediated Pathway

We previously reported that fasudil mesylate (FM) improves neurological deficit and neuronal damage in rats with ischemia following middle cerebral artery occlusion and reperfusion in vivo. In this study, the properties of FM on hydrogen peroxide (H₂O₂)-induced oxidative stress insult in cultured PC12 cells as well as the underlying mechanisms were investigated in vitro. Pretreatment with FM (5, 10 μM) prior to H₂O₂ exposure significantly elevated cell viability, reduced cell apoptosis by MTT assay, LDH assay, Hoechst 33258 dye staining, and FM also decreased the accumulation of reactive oxygen species (ROS) by DCFH-DA staining and NBT test. Furthermore, FM also reversed the upregulation of Bax/Bcl-2 ratio, the downstream cascade following ROS. FM protected PC12 cells from oxidative stress insult via down-regulating the Bax/Bcl-2 ratio. These findings indicate that a direct effect of fasudil mesylate on PC12 cells may be partly responsible for its protective effect against oxidative stress injury.     

Glutathione depletion resulting in selective mitochondrial complex I inhibition in dopaminergic cells is via an NO-mediated pathway not involving peroxynitrite: implications for Parkinson's disease

An early biochemical change in the Parkinsonian substantia nigra (SN) is reduction in total glutathione (GSH + GSSG) levels in affected dopaminergic neurons prior to depletion in mitochondrial complex I activity, dopamine loss, and cell death. We have demonstrated using dopaminergic PC12 cell lines genetically engineered to inducibly down-regulate glutathione synthesis that total glutathione depletion in these cells results in selective complex I inhibition via a reversible thiol oxidation event. Here, we demonstrate that inhibition of complex I may occur either by direct nitric oxide (NO) but not peroxinitrite-mediated inhibition of complex I or through H2O2-mediated inhibition of the tricarboxylic acid (TCA) cycle enzyme alpha-ketoglutarate dehydrogenase (KGDH) which supplies NADH as substrate to the complex; activity of both enzymes are reduced in PD. While glutathione depletion causes a reduction in spare KGDH enzymatic capacity, it produces a complete collapse of complex I reserves and significant effects on mitochondrial function. Our data suggest that NO is likely the primary agent involved in preferential complex I inhibition following acute glutathione depletion in dopaminergic cells. This may have major implications in terms of understanding mechanisms of dopamine cell death associated with PD especially as they relate to complex I inhibition.     

Bcl-2 Protects Isolated Plasma and Mitochondrial Membranes Against Lipid Peroxidation Induced by Hydrogen Peroxide and Amyloid β-Peptide

Abstract: The bcl-2 protooncogene product possesses antiapoptotic properties in neuronal and nonneuronal cells. Recent data suggest that Bcl-2's potency as a survival factor hinges on its ability to suppress oxidative stress, but neither the subcellular site(s) nor the mechanism of its action is known. In this report electron paramagnetic resonance (EPR) spectroscopy analyses were used to investigate the local effects of Bcl-2 on membrane lipid peroxidation. Using hydrogen peroxide (H₂O₂) and amyloid β-peptide (Aβ) as lipoperoxidation initiators, we determined the loss of EPR-detectable paramagnetism of nitroxyl stearate (NS) spin labels 5-NS and 12-NS. In intact cell preparations and postnuclear membrane fractions, Aβ and H₂O₂ induced significant loss of 5-NS and 12-NS signal amplitude in control PC12 cells, but not PC12 cells expressing Bcl-2. Cells were subjected to differential subcellular fractionation, yielding preparations of plasma membrane and mitochondria. In preparations derived from Bcl-2-expressing cells, both fractions contained Bcl-2 protein. 5-NS and 12-NS signals were significantly decreased following Aβ and H₂O₂ exposure in control PC12 mitochondrial membranes, and Bcl-2 largely prevented these effects. Plasma membrane preparations containing Bcl-2 were also resistant to radical-induced loss of spin label. Collectively, our data suggest that Bcl-2 is localized to mitochondrial and plasma membranes where it can act locally to suppress oxidative damage induced by Aβ and H₂O₂, further highlighting the important role of lipid peroxidation in apoptosis.     

Distinct mechanisms account for β-amyloid toxicity in PC12 and differentiated PC12 neuronal cells

Whether reactive oxygen species (ROS) mediate beta-amyloid (A beta) neurotoxicity remains controversial. Naive PC12 cells (PC12) and nerve growth factor-differentiated PC12 cells (dPC12) were used to study the role of ROS in cell death induced by A beta(25-35). The viability of PC12 and dPC12 cells decreased by 30-40% after a 48-hour exposure to 20 microM A beta(25-35). Microscopic examination showed that A beta(25-35) induced necrosis in PC12 cells and apoptosis in dPC12 cells. Vitamin E (100 microM) and other antioxidants protected PC12 cells, but not dPC12 cells, against the cytotoxic effect of A beta(25-35). Since H(2)O(2) has been proposed to be involved in A beta toxicity, the effects of H(2)O(2) on PC12 and dPC12 cells were studied. Differentiated PC12 cells appeared to be significantly more resistant to H(2)O(2) than naive PC12 cells. These data suggest that ROS may mediate A beta(25-35) toxicity in PC12 cells but not in dPC12 cells. Because the intracellular levels of ROS were elevated during the differentiation of PC12 cells, the baseline levels of ROS in these two model cell types may determine the intracellular mediators for A beta(25-35) toxicity. Therefore, the protective effects of antioxidants against A beta may depend upon the redox state of the cells.     

Simvastatin Inhibited the Apoptosis of PC12 Cells Induced by 1-Methyl-4-Phenylpyridinium Ion via Inhibiting Reactive Oxygen Species Production

In the present study, we investigated the neuroprotection of simvastatin in PC12 cells following 1-methyl-4-phenylpyridinium ion (MPP+) neurotoxicity. Simvastatin inhibited the decrease of cell viability induced by MPP+ in PC12 cells. The damage of PC12 cells in morphology was alleviated and the apoptotic rates were decreased due to simvatatin pretreatment against MPP+ cytotoxicity. The reactive oxygen species production exposure to MPP+ was inhibited by simvatatin in PC12 cells. So simvastatin may be of therapeutic benefit for PD patients.     

Human albumin prevents 6-hydroxydopamine-induced loss of tyrosine hydroxylase in in vitro and in vivo

Human albumin has recently been demonstrated to protect brain neurons from injury in rat ischemic brain. However, there is no information available about whether human albumin can prevent loss of tyrosine hydroxylase (TH) expression of dopaminergic (DA) neurons induced by 6-hydroxydopamine (6-OHDA) toxicity that is most commonly used to create a rat model of Parkinson's disease (PD). In the present study, two microliters of 1.25% human albumin were stereotaxically injected into the right striatum of rats one day before or 7 days after the 6-OHDA lesion in the same side. D-Amphetamine-induced rotational asymmetry was measured 7 days, 3 and 10 weeks after 6-OHDA lesion. We observed that intrastriatal administration of human albumin significantly reduced the degree of rotational asymmetry. The number of TH-immunoreactive neurons present in the substantia nigra was greater in 6-OHDA lesioned rats following human albumin-treatment than non-human albumin treatment. TH-immunoreactivity in the 6-OHDA-lesioned striatum was also significantly increased in the human albumin-treated rats. To examine the mechanisms underlying the effects of human albumin, we challenged PC12 cells with 6-OHDA as an in vitro model of PD. Incubation with human albumin prevented 6-OHDA-induced reduction of cell viability in PC12 cell cultures, as measured by MTT assay. Furthermore, human albumin reduced 6-OHDA-induced formation of reactive oxygen species (ROS) and apoptosis in cultured PC12 cells, as assessed by flow cytometry. Western blot analysis showed that human albumin inhibited 6-OHDA-induced activation of JNK, c-Jun, ERK, and p38 mitogen-activated protein kinases (MAPK) signaling in PC12 cultures challenged with 6-OHDA. Human albumin may protect against 6-OHDA toxicity by influencing MAPK pathway followed by anti-ROS formation and anti-apoptosis.