PI3-K/Akt and ERK pathways activated by VEGF play opposite roles in MPP+-induced neuronal apoptosis

Vascular endothelial growth factor (VEGF), a specific pro-angiogenic peptide, has shown neuroprotective effects in the Parkinson’s disease (PD) models, but the underlying mechanisms remain elusive. In this study, the neuroprotective properties of VEGF on 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced neurotoxicity in primary cerebellar granule neurons were investigated. Pretreatment of VEGF prevented MPP⁺-induced neuronal apoptosis in a concentration- and time-dependent manner. And this prevention was blocked by PTK787/ZK222584, a VEGF receptor-2 specific inhibitor. Both inhibition of the Akt pathway and activation of the extracellular signal-regulated kinase (ERK) pathway contribute to MPP⁺-induced neuronal apoptosis. VEGF reversed the inhibition of phosphoinositide 3-kinase (PI3-K)/Akt pathway caused by MPP⁺, but further enhanced the activation of ERK induced by MPP⁺. Interestingly, VEGF and PD98059 (an ERK kinase inhibitor) play a synergistic role in protecting neurons from MPP⁺-induced toxicity. Collectively, these findings suggest that the PI3-K/Akt and ERK pathways activated by VEGF play opposite roles in MPP⁺-induced neuronal apoptosis. This finding offers not only a new and clinically significant modality as to how VEGF exerts its neuroprotective effects but also a novel therapeutic strategy for PD by differentially regulating PD-associated signaling pathways.     

MPP+-induced cytotoxicity in neuroblastoma cells: Antagonism and reversal by guanosine

Guanosine exerts neuroprotective effects in the central nervous system. Apoptosis, a morphological form of programmed cell death, is implicated in the pathophysiology of Parkinson’s disease (PD). MPP⁺, a dopaminergic neurotoxin, produces in vivo and in vitro cellular changes characteristic of PD, such as cytotoxicity, resulting in apoptosis. Undifferentiated human SH-SY5Y neuroblastoma cells had been used as an in vitro model of Parkinson’s disease. We investigated if extracellular guanosine affected MPP⁺-induced cytotoxicity and examined the molecular mechanisms mediating its effects. Exposure of neuroblastoma cells to MPP⁺ (10 μM–5 mM for 24–72 h) induced DNA fragmentation in a time-dependent manner (p < 0.05). Administration of guanosine (100 μM) before, concomitantly with or, importantly, after the addition of MPP⁺ abolished MPP⁺-induced DNA fragmentation. Addition of MPP⁺ (500 μM) to cells increased caspase-3 activity over 72 h (p < 0.05), and this was abolished by pre- or co-treatment with guanosine. Exposure of cells to pertussis toxin prior to MPP⁺ eliminated the anti-apoptotic effect of guanosine, indicating that this effect is dependent on a Gi protein-coupled receptor, most likely the putative guanosine receptor. The protection by guanosine was also abolished by the selective inhibitor of the enzyme PI-3-K/Akt/PKB (LY294002), confirming that this pathway plays a decisive role in this effect of guanosine. Neither MPP⁺ nor guanosine had any significant effect on α-synuclein expression. Thus, guanosine antagonizes and reverses MPP⁺-induced cytotoxicity of neuroblastoma cells via activation of the cell survival pathway, PI-3-K/Akt/PKB. Our results suggest that guanosine may be an effective pharmacological intervention in PD.     

Insulin-like growth factor 1 protects human neuroblastoma cells SH-EP1 against MPP<Superscript>+</Superscript>-induced apoptosis by AKT/GSK-3β/JNK signaling

Parkinson’s disease (PD) is primarily caused by severe degeneration and loss of dopamine neurons in the substantia nigra pars compacta. Thus, preventing the death of dopaminergic neurons is thought to be a potential strategy to interfere with the development of PD. In the present work, we studied the effect of insulin-like growth factor-1 (IGF-1) on 1-methyl-4-phenylpyridinium (MPP⁺)-induced apoptosis in human neuroblastoma SH-EP1 cells. We found that the PI3K/AKT pathway plays a central role in IGF-mediated cell survival against MPP⁺ neurotoxicity. Furthermore, we demonstrated that the protective effect of AKT is largely dependent on the inactivation of GSK-3β, since inhibition of GSK-3β by its inhibitor, BIO, could mimic the protective effect of IGF-1 on MPP⁺-induced cell death in SH-EP1 cells. Interestingly, the IGF-1 potentiated PI3K/AKT activity is found to negatively regulate the JNK related apoptotic pathway and this negative regulation is further shown to be mediated by AKT-dependent GSK-3β inactivation. Thus, our results demonstrated that IGF-1 protects SH-EP1 cells from MPP⁺-induced apoptotic cell death via PI3K/AKT/GSK-3β pathway, which in turn inhibits MPP⁺-induced JNK activation.     

2′,3′-Dideoxycytidine Protects Dopaminergic Neurons in a Mouse Model of Parkinson’s Disease

DNA polymerase-β (DNA pol-β) plays a crucial role in the pathogenesis of Parkinson’s disease (PD). The aim of this study was to investigate the neuroprotective effects of a DNA polymerase-β inhibitor 2′,3′-dideoxycytidine (DDC) in PD models. In the in vitro studies, primary cultured neurons were challenged with 1-methyl-4-phenylpyridinium ion (MPP⁺). The expression of DNA pol-β was assessed using western blot. The neuroprotective effect of DNA pol-β knockdown and DNA pol-β inhibitor DDC was determined using cell viability assay and caspase-3 activity assay. We found that MPP⁺ induced neuronal death and the activation of caspase-3 in a dose-dependent manner. The expression of DNA pol-β increased after the neurons were exposed to MPP⁺. DNA pol-β siRNA or DNA pol-β inhibitor DDC attenuated neuronal death induced by MPP⁺. In the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD, MPTP treatment triggered behavioral deficits and nigrostriatal lesions. Pretreatment with DDC attenuated MPTP-induced behavioral deficits, dopaminergic neuronal death and striatal dopamine depletion in the MPTP mouse model. These results indicate that DNA pol-β inhibitors may present a novel promising therapeutic option for the neuroprotective treatment of PD.

     

AMP-activated protein kinase is activated in Parkinson’s disease models mediated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

The selective loss of dopaminergic neurons in the substantia nigra pars compacta is a feature of Parkinson’s disease (PD). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity is the most common experimental model used to investigate the pathogenesis of PD. Administration of MPTP in mice produces neuropathological defects as observed in PD and 1-methyl-4-pyridinium (MPP⁺) induces cell death when neuronal cell cultures are used. AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis. In the present study, we demonstrated that AMPK is activated by MPTP in mice and MPP⁺ in SH-SY5Y cells. The inhibition of AMPK by compound C resulted in an increase in MPP⁺-induced cell death. We further showed that overexpression of AMPK increased cell viability after exposure to MPP⁺ in SH-SY5Y cells. Based on these results, we suggest that activation of AMPK might prevent neuronal cell death and play a role as a survival factor in PD.     

Miltirone Attenuates Reactive Oxygen Species-Dependent Neuronal Apoptosis in MPP+-Induced Cell Model of Parkinson’s Disease Through Regulating the PI3K/Akt Pathway

Miltirone is a phenanthrene-quinone derived from Salvia miltiorrhiza Bunge with anti-inflammatory and anti-oxidant effects. Our study aimed to explore the protective effect of miltirone on 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell model of Parkinson’s disease (PD). PharmMapper database was employed to predict the targets of miltirone. PD-related genes were identified using GeneCards database. The overlapping genes between miltirone and PD were screened out using Venn diagram. KEGG analysis was performed using DAVID and KOBAS databases. Cell viability, reactive oxygen species (ROS) generation, apoptosis, and caspase-3 activity were detected by CCK-8 assay, a ROS assay kit, TUNEL, and caspase-3 activity assay, respectively. Effect of miltirone on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway was explored by western blot analysis. A total of 214 targets of miltirone and 372 targets related to PD were attained, including 29 overlapping targets. KEGG analysis demonstrated that the 29 overlapping targets were both significantly enriched in the PI3K/Akt pathway. MPP⁺ stimulation reduced the cell viability in SH-SY5Y cells and neuronal primary cultures derived from human brain. Miltirone or N-acetylcysteine (NAC) attenuated MPP⁺-induced reduction in cell viability, ROS production, SOD activity reduction, apoptosis, and increase of caspase-3 activity. Additionally, miltirone recuperated MPP⁺-induced inactivation of the PI3K/Akt pathway. Moreover, treatment with LY294002, an inhibitor of the PI3K/Akt pathway, reversed the inhibitory effect of miltirone on MPP⁺-induced ROS generation and apoptosis in SH-SY5Y cells and neuronal primary cultures. In conclusion, miltirone attenuated ROS-dependent apoptosis in MPP⁺-induced cellular model of PD through activating the PI3K/Akt pathway.

     

Calbindin-D28K prevents drug-induced dopaminergic neuronal death by inhibiting caspase and calpain activity

Calbindin-D28K protects against apoptotic and necrotic cell death; these effects have been attributed to its ability to buffer calcium. In this study, we investigated the mechanisms underlying the neuroprotective effects of calbindin-D28K in staurosporine (STS)-induced apoptosis and 1-methyl-4-phenylpyridinium (MPP⁺)-induced necrosis. Treatment of the dopaminergic neuronal cell line MN9D with STS or MPP⁺ induced cell death that was associated with increased levels of free intracellular calcium. However, only MPP⁺-induced death was inhibited by co-treatment of the cells with a calcium chelator or a sodium/calcium antiporter inhibitor. Overexpression of calbindin-D28K prevented MPP⁺-induced MN9D cell death, which occurs in the absence of any detectable caspase activation. These pro-survival effects of calbindin-D28K were associated with the inhibition of calcium-mediated calpain activation, as determined by processing of Bax. Overexpression of calbindin-D28K also blocked STS-induced MN9D death. However, this effect was accompanied by the inhibition of capase-3 cleavage, poly(ADP-ribose)polymerase cleavage, and caspase activity. These findings suggest that calbindin-D28K protects against both types of cell death by inhibiting caspase- or calcium-mediated death signaling pathway.     

α-Lipoic acid alleviates ferroptosis in the MPP+-induced PC12 cells via activating the PI3K/Akt/Nrf2 pathway

Parkinson's disease (PD) is a typical neurodegenerative disease. α-Lipoic acid (α-LA) can reduce the incidence of neuropathy. The present study explored the role and mechanism of α-LA in 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell model of PD. The PD model was induced via treating PC12 cells with MPP⁺ at different concentrations. MPP⁺ and α-LA effects on PC12 cells were assessed from cell viability and ferroptosis. Cell viability was detected using the cell counting kit-8 assay. Malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), iron, reactive xygen species (ROS), and glutathione (GSH) concentrations, and ferroptosis-related protein SLC7A11 and GPx4 expressions were used for ferroptosis evaluation. p-PI3K, p-Akt, and nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels were detected. The PI3K/Akt/Nrf2 pathway inhibitors were applied to verify the role of the PI3K/Akt/Nrf2 pathway in α-LA protection against MPP⁺-induced decreased cell viability and ferroptosis. MPP⁺-reduced cell viability and induced ferroptosis as presented by increased MDA, 4-HNE, iron, and ROS concentrations, and reduced levels of GSH and ferroptosis marker proteins (SLC7A11 and GPx4). α-LA attenuated MPP⁺-induced cell viability decline and ferroptosis. The PI3K/Akt/Nrf2 pathway was activated after α-LA treatment. Inhibiting the PI3K/Akt/Nrf2 pathway weakened the protection of α-LA against MPP⁺ treatment. We highlighted that α-LA alleviated MPP⁺-induced cell viability decrease and ferroptosis in PC12 cells via activating the PI3K/Akt/Nrf2 pathway.     

Neuroprotective cytokines repress PUMA induction in the 1-methyl-4-phenylpyridinium (MPP(+)) model of Parkinson's disease

The hematopoietic cytokines erythropoietin (Epo) and granulocyte-colony stimulating factor (G-CSF) provide neuroprotection in several in vitro and in vivo models of Parkinson’s disease (PD). The molecular mechanism by which Epo and G-CSF signals reduce the neuronal death in PD is not clear. Here, we show that in rat pheochromocytoma PC12 cells, Epo and G-CSF efficiently repressed the 1-methyl-4-phenylpyridinium (MPP⁺)-induced expression of the proapoptotic protein PUMA (p53 up-regulated modulator of apoptosis). Accordingly, Epo and G-CSF treatment reduced the PC12 cell fraction that underwent apoptosis by MPP⁺ treatment and thus improved cell viability. Downregulation of PUMA expression by Epo and G-CSF in MPP⁺-treated PC12 cells seems to be mediated by repression of p53, as the expression of p53 was increased by MPP⁺-treatment and reduced by Epo and G-CSF. Together, these results suggest that the neuroprotective activities of Epo and G-CSF in an experimental model of PD involve the repression of the apoptosis-inducing action of PUMA.     

Discovery of a benzofuran derivative (MBPTA) as a novel ROCK inhibitor that protects against MPP+-induced oxidative stress and cell death in SH-SY5Y cells

Parkinson disease (PD) is a neurodegenerative disease with multifactorial etiopathogenesis. The discovery of drug candidates that act on new targets of PD is required to address the varied pathological aspects and modify the disease process. In this study, a small compound, 2-(5-methyl-1-benzofuran-3-yl)-N-(5-propylsulfanyl-1,3,4-thiadiazol-2-yl) acetamide (MBPTA) was identified as a novel Rho-associated protein kinase inhibitor with significant protective effects against 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced damage in SH-SY5Y neuroblastoma cells. Further investigation showed that pretreatment of SH-SY5Y cells with MBPTA significantly suppressed MPP⁺-induced cell death by restoring abnormal changes in nuclear morphology, mitochondrial membrane potential, and numerous apoptotic regulators. MBPTA was able to inhibit MPP⁺-induced reactive oxygen species (ROS)/NO generation, overexpression of inducible NO synthase, and activation of NF-κB, indicating the critical role of MBPTA in regulating ROS/NO-mediated cell death. Furthermore, MBPTA was shown to activate PI3K/Akt survival signaling, and its cytoprotective effect was abolished by PI3K and Akt inhibitors. The structural comparison of a series of MBPTA analogs revealed that the benzofuran moiety probably plays a crucial role in the anti-oxidative stress action. Taken together, these results suggest that MBPTA protects against MPP⁺-induced apoptosis in a neuronal cell line through inhibition of ROS/NO generation and activation of PI3K/Akt signaling.     

SIRT1 mediates salidroside‐elicited protective effects against MPP+‐induced apoptosis and oxidative stress in SH‐SY5Y cells: involvement in suppressing MAPK pathways

Parkinson's disease (PD) is a progressive neurodegenerative disease, leading to tremor, rigidity, bradykinesia, and gait impairment. Salidroside has been reported to exhibit antioxidative and neuroprotective properties in PD. However, the underlying neuroprotective mechanisms effects of salidroside are poorly understood. Recently, a growing body of evidences suggest that silent information regulator 1 (SIRT1) plays important roles in the pathophysiology of PD. Hence, the present study investigated the roles of SIRT1 in neuroprotective effect of salidroside against N‐methyl‐4‐phenylpyridinium (MPP⁺)‐induced SH‐SY5Y cell injury. Our findings revealed that salidroside attenuates MPP⁺‐induced neurotoxicity as evidenced by the increase in cell viability, and the decreases in the caspase‐3 activity and apoptosis ratio. Simultaneously, salidroside pretreatment remarkably increased SIRT1 activity, SIRT1 mRNA and protein levels in MPP⁺‐treated SH‐SY5Y cell. However, sirtinol, a SIRT1 activation inhibitor, significantly blocked the inhibitory effects of salidroside on MPP⁺‐induced cytotoxicity and apoptosis. In addition, salidroside abolished MPP⁺‐induced the production of reactive oxygen species (ROS), the up‐regulation of NADPH oxidase 2 (NOX2) expression, the down‐regulations of superoxide dismutase (SOD) activity and glutathione (GSH) level in SH‐SY5Y cells, while these effects were also blocked by sirtinol. Finally, we found that the inhibition of salidroside on MPP⁺‐induced phosphorylation of p38, extracellular signal‐regulated kinase (ERK) and c‐Jun NH2‐terminal kinase (JNK) were also reversed by sirtinol in SH‐SY5Y cells. Taken together, these results indicated that SIRT1 contributes to the neuroprotection of salidroside against MPP⁺‐induced apoptosis and oxidative stress, in part through suppressing of mitogen‐activated protein kinase (MAPK) pathways.     

DNA polymerase-β is required for 1-methyl-4-phenylpyridinium-induced apoptotic death in neurons

The biochemical pathways that mediate the degeneration of dopaminergic neurons in the substantia nigra of patients with Parkinson’s disease are largely unknown. Recently, aberrant cell cycle events have been shown to be associated with neuronal death in several neurodegenerative diseases. In the present study, we investigated the role of DNA polymerases (DNA pols) in 1-methyl-4-phenylpyridinium (MPP⁺)-induced neuronal apoptosis in cerebellar granule cells. After exposure to MPP⁺, the neurons entered S phase of the cell cycle. Neuronal cell cycle re-entry and apoptosis were attenuated by flavopiridol, which is a broad inhibitor of cyclin-dependent kinases (CDKs). MPP⁺ exposure significantly increased the expression of DNA pol-β and primase but did not affect the expression of the canonical replicative DNA pols, including DNA pol-δ and pol-ε. Dideoxycytidine, which is a pharmacological inhibitor of DNA pol-β, attenuated the neuronal apoptosis mediated by MPP⁺. In a similar manner, the expression of a dominant negative form of DNA pol-β was also neuroprotective. These results suggest that DNA pol-β may have a causal role in MPP⁺-induced neuronal apoptosis.     

Protective effects of activated signaling pathways by insulin on C6 glial cell model of MPP+-induced Parkinson’s disease

Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease (AD) associated with mitochondrial dysfunction mediated by oxidative stress. Astrocytes regulate neuronal function via the modulation of synaptic transmission and plasticity, secretion of growth factors, uptake of neurotransmitters, and regulation of extracellular ion concentrations and metabolic support of neurons. Therefore, this study was undertaken to investigate the mechanism of action of insulin on a 1-methyl-4-phenylpyridinium (MPP⁺)-induced toxicity of events associated in cell viability and toxicity to the expression profile of cell signaling pathway proteins and genes in rat C6 glial cells. The various concentrations of MPP⁺ alone inhibited cell viability in a dose-dependent manner. Pretreatment of insulin prevented the cell death and lowered the intracellular reactive oxygen species and calcium ion influx by MPP⁺. Insulin also suppressed the α-synuclein and elevated the insulin signaling pathway molecules IR, IGF-1R, IRS-1 and IRS-2 in C6 cells through phosphorylation of Akt/ERK survival pathways. Moreover, insulin inhibits MPP⁺-induced Bax to Bcl-2 ratio. These results suggest that insulin has a protective effect on the MPP⁺-toxicity in C6 glial cells.     

Commitment of 1-Methyl-4-phenylpyrinidinium Ion-induced Neuronal Cell Death by Proteasome-mediated Degradation of p35 Cyclin-dependent Kinase 5 Activator

The dysfunction of proteasomes and mitochondria has been implicated in the pathogenesis of Parkinson disease. However, the mechanism by which this dysfunction causes neuronal cell death is unknown. We studied the role of cyclin-dependent kinase 5 (Cdk5)-p35 in the neuronal cell death induced by 1-methyl-4-phenylpyrinidinium ion (MPP⁺), which has been used as an in vitro model of Parkinson disease. When cultured neurons were treated with 100 μm MPP⁺, p35 was degraded by proteasomes at 3 h, much earlier than the neurons underwent cell death at 12–24 h. The degradation of p35 was accompanied by the down-regulation of Cdk5 activity. We looked for the primary target of MPP⁺ that triggered the proteasome-mediated degradation of p35. MPP⁺ treatment for 3 h induced the fragmentation of the mitochondria, reduced complex I activity of the respiratory chain without affecting ATP levels, and impaired the mitochondrial import system. The dysfunction of the mitochondrial import system is suggested to up-regulate proteasome activity, leading to the ubiquitin-independent degradation of p35. The overexpression of p35 attenuated MPP⁺-induced neuronal cell death. In contrast, depletion of p35 with short hairpin RNA not only induced cell death but also sensitized to MPP⁺ treatment. These results indicate that a brief MPP⁺ treatment triggers the delayed neuronal cell death by the down-regulation of Cdk5 activity via mitochondrial dysfunction-induced up-regulation of proteasome activity. We propose a role for Cdk5-p35 as a survival factor in countering MPP⁺-induced neuronal cell death.Parkinson disease (PD)³ is the second most common neurodegenerative disease, characterized pathologically by degenerated dopaminergic neurons and ubiquitin-positive aggregates known as Lewy bodies (1). Most cases of PD are sporadic, but a small proportion of patients with PD have the familial form. Several causative genes have been identified for familial PDs, including α-synuclein (2), ubiquitin C-terminal hydrolase L1 (UCH-L1) (3), and parkin, an ubiquitin ligase E3 of the ubiquitin-proteasome system (4), implicating the impairment of the ubiquitin-proteasome pathway in the pathogenesis of PD. However, the mechanisms underlying the involvement of the ubiquitin-proteasome system in the development of PD are not yet understood.The 1-methyl-4-phenylpyrinidinium ion (MPP⁺), a toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a neurotoxin used widely to induce dopaminergic neuronal cell death in in vitro models of PD (5). Previous studies have indicated that MPP⁺ induces neuronal cell death via several pathways, including the inhibition of complex I activity of the respiratory chain in mitochondria, leading to energy depletion, protein peroxidation, and DNA damage by producing reactive oxygen species and the induction of cytotoxic glutamate secretion (6, 7). However, the precise molecular pathway resulting in neuronal cell death remains to be identified.Cyclin-dependent kinase 5 (Cdk5) is a member of the Cdk serine/threonine kinase family. Cdk5 plays a role in a variety of neuronal activities including neuronal migration during central nervous system development (8, 9), synaptic activity in matured neurons (10), and neuronal cell death in neurodegenerative diseases (11, 12). Generally, when Cdk5 are activated by their respective activator cyclins, they function in cell cycle progression. However, unlike those cell cycle Cdk5, the kinase activity of Cdk5 is detected mainly in post mitotic neurons. This is because Cdk5 activators p35 and p39 are expressed predominantly in neurons (13, 14). The amount of p35 is the major determinant of Cdk5 activity, and it is normally a short-lived protein degraded by the ubiquitin-proteasome pathway (15, 16). However, in stressed neurons, the calcium-activated protease calpain cleaves p35 to the more stable and active form, p25 (17–21). Hyperactivated or mislocalized Cdk5-p25 has been implicated in the pathogenesis of numerous neurodegenerative disorders including PD and Alzheimer disease. In the case of PD, Cdk5 and p35 are found in the Lewy bodies of the dopaminergic neurons of the brain (22, 23). Cdk5 is activated by p25 and is required for cell death in mouse models of PD induced with MPTP (24) or 6-hydroxydopamine (25). It has been shown that Cdk5-p25 in MPTP-treated neurons phosphorylates the survival factor, myocyte enhancer factor 2 (MEF2), to inactivate it, leading to cell death (26, 27). However, further studies are required to clarify the involvement of p35 metabolism in the PD pathway.Contrary to its role in cell death progression, recent studies have also suggested a survival function for Cdk5 in maintaining survival signals or counteracting apoptotic signals. For example, Cdk5 inhibits c-Jun phosphorylation by c-Jun-N-terminal protein kinase 3, which is activated by UV irradiation (28). Cdk5 also promotes the survival of neurons by activating Akt through the well known neuregulin/phosphatidylinositol 3-kinase (PI3K) survival pathway, which leads to the down-regulation of proapoptotic factors (29). Cdk5 attenuates cell death either by up-regulating Bcl-2 through the phosphorylation of ERK (30) or by phosphorylating Bcl-2 to maintain its neuroprotective effect (31). However, whether Cdk5 acts as the anti-apoptotic factor in the PD model of neuronal cell death has not been determined.Here, we studied the role of Cdk5-p35 in the cell death of neurons treated with MPP⁺. We found that p35 was proteolysed in cultured neurons by either calpain or proteasomes depending on the concentration of MPP⁺ used. The proteasomal MPP⁺-induced degradation of p35 occurred earlier and at lower MPP⁺ concentrations than did its cleavage by calpain. MPP⁺ up-regulated the overall proteasome activity in the neurons by impairing the mitochondrial protein import system. A brief MPP⁺ treatment for up to ∼3 h was sufficient to induce delayed cell death at 24 h. The overexpression of p35 suppressed this MPP⁺-induced cell death, and depletion of p35 increased cell death. Together, these results implicate a role for Cdk5-p35 as a survival factor in MPP⁺-treated neurons.     

Integrated insight into the molecular mechanisms of selenium-modulated,MPP+-induced cytotoxicity in a Parkinson’s disease model

ObjectiveParkinson’s disease (PD) is a neurodegenerative disease that is associated with oxidative stress. Due to the anti-inflammatory and antioxidant functions of Selenium (Se), this molecule may have neuroprotective functions in PD; however, the involvement of Se in such a protective function is unclear.Methods1-methyl-4-phenylpyridinium (MPP⁺), which inhibits mitochondrial respiration, is generally used to produce a reliable cellular model of PD. In this study, a MPP⁺-induced PD model was used to test if Se could modulate cytotoxicity, and we further capture gene expression profiles following PC12 cell treatment with MPP⁺ with or without Se by genome wide high-throughput sequencing.ResultsWe identified 351 differentially expressed genes (DEGs) and 14 differentially expressed long non-coding RNAs (DELs) in MPP⁺-treated cells when compared to controls. We further document 244 DEGs and 27 DELs in cells treated with MPP⁺ and Se vs. cells treated with MPP⁺ only. Functional annotation analysis of DEGs and DELs revealed that these groups were enriched in genes that respond to reactive oxygen species (ROS), metabolic processes, and mitochondrial control of apoptosis. Thioredoxin reductase 1 (Txnrd1) was also identified as a biomarker of Se treatment.ConclusionsOur data suggests that the DEGs Txnrd1, Siglec1 and Klf2, and the DEL AABR07044454.1 which we hypothesize to function in cis on the target gene Cdkn1a, may modulate the underlying neurodegenerative process, and act a protective function in the PC12 cell PD model. This study further systematically demonstrated that mRNAs and lncRNAs induced by Se are involved in neuroprotection in PD, and provides novel insight into how Se modulates cytotoxicity in the MPP⁺-induced PD model.     

S-Allylcysteine, a garlic compound, protects against oxidative stress in 1-methyl-4-phenylpyridinium-induced parkinsonism in mice

S-Allylcysteine (SAC), the most abundant organosulfur compound in aged garlic extract, has multifunctional activity via different mechanisms and neuroprotective effects that are exerted probably via its antioxidant or free radical scavenger action. The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mouse has been the most widely used model for assessing neuroprotective agents for Parkinson's disease. 1-Methyl-4-phenylpyridinium (MPP⁺) is the stable metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and it causes nigrostriatal dopaminergic neurotoxicity. Previous studies suggest that oxidative stress, via free radical production, is involved in MPP⁺-induced neurotoxicity. Here, we report on the neuroprotective effect of SAC against oxidative stress induced by MPP⁺ in the striatum of C57BL/6J mice. Mice were pretreated with SAC (125 mg/kg ip) daily for 17 days, followed by administration of MPP⁺ (0.72 mg/kg icv), and were sacrificed 24 h later to evaluate lipid peroxidation, different antioxidant enzyme activities, spontaneous locomotor activity and dopamine (DA) content. MPP⁺ administration resulted in a significant decrease in DA levels in the striatum. Mice receiving SAC (125 mg/kg ip) had significantly attenuated MPP⁺-induced loss of striatal DA levels (32%). The neuroprotective effect of SAC against MPP⁺ neurotoxicity was associated with blocked (100% of protection) of lipid peroxidation and reduction of superoxide radical production — indicated by an up-regulation of Cu-Zn-superoxide dismutase activity — both of which are indices of oxidative stress. Behavioral analyses showed that SAC improved MPP⁺-induced impairment of locomotion (35%). These findings suggest that in mice, SAC attenuates MPP⁺-induced neurotoxicity in the striatum and that an antioxidant effect against oxidative stress may be partly responsible for its observed neuroprotective effects.     

Paeoniflorin, a Natural Neuroprotective Agent, Modulates Multiple Anti-Apoptotic and Pro-apoptotic Pathways in Differentiated PC12 Cells

Numerous studies have shown robust neuroprotective effects of paeoniflorin (PF), a natural compound derived from the herbal medicine Paeony radix. In the present study, we determined associations of PF neuroprotection with its modulation of various apoptotic and anti-apoptotic pathways. PF (50–400 μM) pretreatment significantly improved viability of differentiated PC12 cells exposed to methyl-4-phenylpyridine ion (MPP⁺), a neurotoxin, and inhibited over-release of lactate dehydrogenase, a biomarker of neuronal cell death. PF also ameliorated MPP⁺-induced nuclear and mitochondrial apoptotic alteration and intracellular calcium overload. PF treatment reversed MPP⁺ suppression of activity of B cell lymphoma-extra large, which is a mitochondrial membrane molecule that protects cells from DNA damage-induced apoptosis, and strikingly inhibited the enhanced level of cleaved poly(ADP-ribose)polymerase, which is involved in the process of apoptosis. PF alone and coadministration with MPP⁺ enhanced phospho activation of extracellular signal-regulated kinases, Akt, and its downstream element glycogen synthase kinase-3, but the effects were completely abolished in the presence of their blockers PD98059 and LY294002. The presence of the blockers also diminished the potency of PF in improving viability of MPP⁺-exposed cells. These results indicate that neuroprotective effects of PF are related to its modulation of multiple anti-apoptotic and pro-apoptotic pathways, including blockade of intracellular calcium overload, prevention of mitochondrial membrane integrity, inhibition of pro-apoptotic molecules, and up-regulation of anti-apoptotic proteins associated with cell survival and proliferation. The study provides evidence supporting PF as a potential therapeutic agent used for the treatment of neurodegenerative diseases and neural injury.     

Curcumin protects PC12 cells against 1-methyl-4-phenylpyridinium ion-induced apoptosis by bcl-2-mitochondria-ROS-iNOS pathway

The aim of present study is to explore the cytoprotection of curcumin against 1-methyl-4-phenylpridinium ions (MPP⁺)-induced apoptosis and the molecular mechanisms underlying in PC12 cells. Our findings indicated that MPP⁺ significantly reduced the cell viability and induced apoptosis of PC12 cells. Curcumin protected PC12 cells against MPP⁺-induced cytotoxicity and apoptosis not only by inducing overexpression of Bcl-2, but also reducing the loss of mitochondrial membrane potential (MMP), an increase in intracellular reactive oxygen species (ROS) and overexpression of inducible nitric oxide synthase (iNOS). The selective iNOS inhibitor AG partly blocked MPP⁺-induced apoptosis of PC12 cells. The results of present study suggested that the cytoprotective effects of curcumin might be mediated, at least in part, by the Bcl-2-mitochondria-ROS-iNOS pathway. Because of its non-toxic property, curcumin could be further developed to treat the neurodegenerative diseases which are associated with oxidative stress, such as Parkinson’s disease (PD). J. Chen and X. Q. Tang are contributed equally to this work.