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.     

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.     

SIRT1 deficiency attenuates MPP+-induced apoptosis in dopaminergic cells

One of the functions mediated by sirtuin 1 (SIRT1), the NAD⁺-dependent protein deacetylase, has been suggested to be neuroprotective since resveratrol, a SIRT1 activator, inhibits 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced cytotoxicity. In this study, we show that SIRT1 siRNA transfection blocks MPP⁺-induced apoptosis in SH-SY5Y cells. The ratio of potential pro-apoptotic BNIP2 to antiapoptotic BCL-xL was attenuated in SIRT1-deficient cells following MPP⁺ treatment. In addition, BNIP2 shRNA-transfected cells showed reduced cleavage of PARP-1, while BNIP2 overexpression intensified the cleavage in MPP⁺-treated SH-SY5Y cells, suggesting that BNIP2 participates in the MPP⁺-induced apoptosis. Overall, these data imply that SIRT1 may mediate MPP⁺-induced cytotoxicity, possibly through the regulation of BNIP2.     

Regulation of Histone Acetylation by Autophagy in Parkinson Disease

Parkinson disease (PD) is the most common age-dependent neurodegenerative movement disorder. Accumulated evidence indicates both environmental and genetic factors play important roles in PD pathogenesis, but the potential interaction between environment and genetics in PD etiology remains largely elusive. Here, we report that PD-related neurotoxins induce both expression and acetylation of multiple sites of histones in cultured human cells and mouse midbrain dopaminergic (DA) neurons. Consistently, levels of histone acetylation are markedly higher in midbrain DA neurons of PD patients compared to those of their matched control individuals. Further analysis reveals that multiple histone deacetylases (HDACs) are concurrently decreased in 1-methyl-4-phenylpyridinium (MPP⁺)-treated cells and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mouse brains, as well as midbrain tissues of human PD patients. Finally, inhibition of histone acetyltransferase (HAT) protects, whereas inhibition of HDAC1 and HDAC2 potentiates, MPP⁺-induced cell death. Pharmacological and genetic inhibition of autophagy suppresses MPP⁺-induced HDACs degradation. The study reveals that PD environmental factors induce HDACs degradation and histone acetylation increase in DA neurons via autophagy and identifies an epigenetic mechanism in PD pathogenesis.     

Embelin averts MPTP-induced dysfunction in mitochondrial bioenergetics and biogenesis via activation of SIRT1

Parkinson's disease (PD) is a chronic neurodegenerative disease characterized by the death of dopamine neurons of Substantia nigra pars compacta (SNpc) leading to motor deficits. Amongst the mechanisms proposed, mitochondrial dysfunction, reduced complex-I and PGC1α levels were found to correlate with the pathology of PD. As embelin is a natural product with structural resemblance to ubiquinone, exhibits mitochondrial uncoupling and antioxidant effects, in the present study, we sought to examine its role in the mechanisms mediating PD. Results indicate that embelin protects from MPP⁺-induced oxidative stress and apoptosis in a time and dose-dependent manner in N27 dopaminergic cells. Cells treated with embelin exhibited increased levels of pAMPK, SIRT1 and PGC1α leading to enhanced mitochondrial biogenesis. Though treatment of cells with MPP⁺ also increased pAMPK levels, but, SIRT1 and PGC1α levels decreased substantially, possibly due to the block in the mitochondrial electron transport chain and reduced NAD/NADH levels. The mitochondrial uncoupling effects of embelin leading to increased NAD/NADH levels followed by enhanced SIRT1, PGC1α and mitochondrial biogenesis were found to confer embelin mediated protection as treatment of cells with SIRT1 inhibitor or siRNA nullified this effect. Embelin (10 mg/kg) also conferred protection in vivo in MPTP mouse model of PD, wherein, MPTP-induced loss of TH staining, reduced striatal dopamine and markers of mitochondrial biogenesis pathway were averted by embelin.     

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.     

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.     

Omega‐3 fatty acid eicospentaenoic acid attenuates MPP+‐induced neurodegeneration in fully differentiated human SH‐SY5Y and primary mesencephalic cells

Eicosapentaenoic acid (EPA), a neuroactive omega‐3 fatty acid, has been demonstrated to exert neuroprotective effects in experimental models of Parkinson's disease (PD), but the cellular mechanisms of protection are unknown. Here, we studied the effects of EPA in fully differentiated human SH‐SY5Y cells and primary mesencephalic neurons treated with MPP⁺. In both in‐vitro models of PD, EPA attenuated an MPP⁺‐induced reduction in cell viability. EPA also prevented the presence of electron‐dense cytoplasmic inclusions in SH‐SY5Y cells. Then, possible mechanisms of the neuroprotection were studied. In primary neurons, EPA attenuated an MPP⁺‐induced increase in Tyrosine‐related kinase B (TrkB) receptors. In SH‐SY5Y cells, EPA down‐regulated reactive oxygen species and nitric oxide. This antioxidant effect of EPA may have been mediated by its inhibition of neuronal NADPH oxidase and cyclo‐oxygenase‐2 (COX‐2), as MPP⁺ increased the expression of these enzymes. Furthermore, EPA prevented an increase in cytosolic phospholipase A2 (cPLA2), an enzyme linked with COX‐2 in the potentially pro‐inflammatory arachidonic acid cascade. Lastly, EPA attenuated an increase in the bax:bcl‐2 ratio, and cytochrome c release. However, EPA did not prevent mitochondrial enlargement or a decrease in mitochondrial membrane potential. This study demonstrated cellular mechanisms by which EPA provided neuroprotective effects in experimental PD.     

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.     

Curcumin exerts anti-inflammatory and antioxidative properties in 1-methyl-4-phenylpyridinium ion (MPP<Superscript>+</Superscript>)-stimulated mesencephalic astrocytes by interference with TLR4 and downstream signaling pathway

Neuroinflammation is closely associated with the pathophysiology of neurodegenerative diseases including Parkinson’s disease (PD). Recent evidence indicates that astrocytes also play pro-inflammatory roles in the central nervous system (CNS) by activation with toll-like receptor (TLR) ligands. Therefore, targeting anti-inflammation may provide a promising therapeutic strategy for PD. Curcumin, a polyphenolic compound isolated from Curcuma longa root, has been commonly used for the treatment of neurodegenerative diseases. However, the details of how curcumin exerts neuroprotection remain uncertain. Here, we investigated the protective effect of curcumin on 1-methyl-4-phenylpyridinium ion-(MPP⁺-) stimulated primary astrocytes. Our results showed that MPP⁺ stimulation resulted in significant production of tumor necrosis factor (TNF)-α, interleukin (IL-6), and reactive oxygen species (ROS) in primary mesencephalic astrocytes. Curcumin pretreatment decreased the levels of these pro-inflammatory cytokines while increased IL-10 expression in MPP⁺-stimulated astrocytes. In addition, curcumin increased the levels of antioxidant glutathione (GSH) and reduced ROS production. Our results further showed that curcumin decreased the levels of TLR4 and its downstream effectors including NF-κB, IRF3, MyD88, and TIRF that are induced by MPP⁺ as well as inhibited the immunoreactivity of TLR4 and morphological activation in MPP⁺-stimulated astrocytes. Together, data suggest that curcumin might exert a beneficial effect on neuroinflammation in the pathophysiology of PD.     

Protective Effects of SKF-96365, a Non-Specific Inhibitor of SOCE,against MPP+-Induced Cytotoxicity in PC12 Cells: Potential Role of Homer1

Parkinson''s disease (PD) is the most common neurodegenerative movement disorder, characterized by loss of dopominergic (DA) neurons in substantia nigra pars compacta (SNpc), and can be experimentally mimicked by the neurotoxin MPP⁺ in vitro models. In this study, we investigated the potential protective effect of SKF-96365, a non-specific inhibitor of SOCE (store-operated calcium entry), on MPP⁺ induced cytotoxicity in PC12 cells. We found that pretreatment with SKF-96365 (10 µM and 50 µM) 30 min before injury significantly increased cell viability, decreased LDH release, prevented nuclear damage, and inhibited apoptotic cell death in MPP⁺ stressed PC12 cells. The results of calcium image using the ratiometric calcium indicator Fura-2-AM also showed that SKF-96365 reduced the intracellular calcium overload induced by MPP⁺ in PC12 cells. In addition, SKF-96365 decreased the expression of Homer1, a more recently discovered postsynaptic scaffolding protein with calcium modulating function, following MPP⁺ administration in PC12 cells, while had no statistically significant effects on endoplasmic reticulum (ER) calcium concentration. Furthermore, overexpression of Homer1 by using recombinant lentivirus partly reversed protective effects of SKF-96365 against MPP⁺ injury. The ER Ca²⁺ release was further amplified and ER calcium recovery was delayed by Homer1 upregulation in PC12 cells following MPP⁺ insult. Taken together, these data suggest that SKF-96365 protects PC12 cells against MPP⁺ induced cytotoxicity, and this protection may be at least in part on the inhibition of intracellular calcium overload and suppression of Homer1-mediated ER Ca²⁺ release.     

Protective effects of PEP-1-Catalase on stress-induced cellular toxicity and MPTP-induced Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disability caused by a decrease of dopaminergic neurons in the substantia nigra (SN). Although the etiology of PD is not clear, oxidative stress is believed to lead to PD. Catalase is antioxidant enzyme which plays an active role in cells as a reactive oxygen species (ROS) scavenger. Thus, we investigated whether PEP-1-Catalase protects against 1-methyl-4-phenylpyridinium (MPP⁺) induced SH-SY5Y neuronal cell death and in a 1-methyl-4-phenyl-1,2,3,6-trtrahydropyridine (MPTP) induced PD animal model. PEP-1-Catalase transduced into SH-SY5Y cells significantly protecting them against MPP⁺-induced death by decreasing ROS and regulating cellular survival signals including Akt, Bax, Bcl-2, and p38. Immunohistochemical analysis showed that transduced PEP-1-Catalase markedly protected against neuronal cell death in the SN in the PD animal model. Our results indicate that PEP-1-Catalase may have potential as a therapeutic agent for PD and other oxidative stress related diseases. [BMB Reports 2015; 48(7): 395-400]     

Theacrine,a purine alkaloid from kucha,protects against Parkinson's disease through SIRT3 activation

BackgroundOxidative damage of dopaminergic neurons is the fundamental causes of Parkinson's disease (PD) that has no standard cure at present. Theacrine, a purine alkaloid from Chinese tea Kucha, has been speculated to benefit the neurodegeneration in PD, through similar actions to its chemical analogue caffeine, albeit excluding side effects. Theacrine has nowadays gained a lot of interest for its multiple benefits, while the investigations are weak and insufficient.Hypothesis/PurposeIt is well-known that tea has a wide range of functions, especially in the prevention and treatment of neurodegenerative diseases. Theacrine is an active monomer compound in Camellia assamica var. kucha Hung T. Chang & H.S.Wang (Kucha), which appears to be effective and safe in PD therapy. The aim of this study is to examine its actions in diverse PD models and explore the mechanisms.Study DesignFor determination of theacrine's effects, we employed diverse oxidative damage-associated PD models, including 6-OHDA-treated rats, MPTP-treated mice/zebrafish and MPP⁺-treated SH-SY5Y cells, and using caffeine, selegiline and depranyl as positve control. For investigation and verification of the mechanisms, we utilized approaches testing mitochondrial function-related parameters and enzyme activity as well as applied gene knockdown and overexpression.MethodsWe employed behavioral tests including spontaneous activity, pole, swimming, rotarod and gait, immunohistochemistry, HPLC, flow cytometry, immunohistochemistry, Western blot, gene knockdown by siRNA and overexpression by plasmid in this study.ResultsTheacrine is demonstrated to retrieve the loss of dopaminergic neurons and the damages of behavioral performance in multiple animal models of PD (6-OHDA-treated rats and in MPTP-treated mice and zebrafish). The followed data of MPP⁺-treated SH-SY5Y cells indicate that theacrine relieves apoptosis resulted from oxidative damage and mitochondrial dysfunction. Further investigations illustrate that theacrine activates SIRT3 directly. It is of advantage to prevent apoptosis through SIRT3-mediated SOD2 deacetylation that reduces ROS accumulation and restores mitochondrial function. This concept is elaborated by 3TYP that inhibits SIRT3 enzyme activity and knockdown/overexpression of SIRT3 gene, demonstrating a crucial role of SIRT3 in theacrine-benefited dopaminergic neurons.ConclusionTheacrine prevents apoptosis of dopaminergic neurons through directly activating SIRT3 which deacetylating SOD2 and restoring mitochondrial functions.     

Mast Cells Release Chemokine CCL2 in Response to Parkinsonian Toxin 1-Methyl-4-Phenyl-Pyridinium (MPP<Superscript>+</Superscript>)

Microglial activation and release of inflammatory cytokines and chemokines are crucial events in neuroinflammation. Microglial cells interact and respond to other inflammatory cells such as T cells and mast cells as well as inflammatory mediators secreted from these cells. Recent studies have shown that neuroinflammation causes and accelerates neurodegenerative disease such as Parkinson’s disease (PD) pathogenesis. 1-methyl-4-phenyl-pyridinium ion (MPP⁺), the active metabolite of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine activates glial cells and mediate neurodegeneration through release of inflammatory mediators. We have shown that glia maturation factor (GMF) activates glia and induces neuroinflammation and neurodegeneration and that MPP⁺ activates mast cells and release proinflammatory cytokines and chemokines. The chemokine (C-C motif) ligand 2 (CCL2) levels have been shown to be elevated and play a role in PD pathogenesis. In the present study, we analyzed if MPP⁺ activates mouse and human mast cells to release chemokine CCL2. Mouse bone marrow-derived mast cells (BMMCs) and human umbilical cord blood-derived cultured mast cells (hCBMCs) were incubated with MPP⁺ (10 µM) for 24 h and CCL2 levels were measured in the supernatant media by ELISA. MPP⁺-significantly induced CCL2 release from BMMCs and hCBMCs. Additionally, GMF overexpression in BMMCs obtained from wild-type mice released significantly more CCL2, while BMMCs obtained from GMF-deficient mice showed less CCL2 release. Further, we show that MPP⁺-induced CCL2 release was greater in BMMCs–astrocyte co-culture conditions. Uncoupling protein 4 (UCP4) which is implicated in neurodegenerative diseases including PD was detected in BMMCs by immunocytochemistry. Our results suggest that mast cells may play role in PD pathogenesis.     

DLP1-dependent mitochondrial fragmentation mediates 1-methyl-4-phenylpyridinium toxicity in neurons: implications for Parkinson's disease

Selective degeneration of nigrostriatal dopaminergic neurons in Parkinson’s disease (PD) can be modeled by the administration of the neurotoxin 1‐methyl‐4‐phenylpyridinium (MPP⁺). Because abnormal mitochondrial dynamics are increasingly implicated in the pathogenesis of PD, in this study, we investigated the effect of MPP⁺ on mitochondrial dynamics and assessed temporal and causal relationship with other toxic effects induced by MPP⁺ in neuronal cells. In SH‐SY5Y cells, MPP⁺ causes a rapid increase in mitochondrial fragmentation followed by a second wave of increase in mitochondrial fragmentation, along with increased DLP1 expression and mitochondrial translocation. Genetic inactivation of DLP1 completely blocks MPP⁺‐induced mitochondrial fragmentation. Notably, this approach partially rescues MPP⁺‐induced decline in ATP levels and ATP/ADP ratio and increased [Ca²⁺]_i and almost completely prevents increased reactive oxygen species production, loss of mitochondrial membrane potential, enhanced autophagy and cell death, suggesting that mitochondria fragmentation is an upstream event that mediates MPP⁺‐induced toxicity. On the other hand, thiol antioxidant N‐acetylcysteine or glutamate receptor antagonist D‐AP5 also partially alleviates MPP⁺‐induced mitochondrial fragmentation, suggesting a vicious spiral of events contributes to MPP⁺‐induced toxicity. We further validated our findings in primary rat midbrain dopaminergic neurons that 0.5 μm MPP⁺ induced mitochondrial fragmentation only in tyrosine hydroxylase (TH)‐positive dopaminergic neurons in a similar pattern to that in SH‐SY5Y cells but had no effects on these mitochondrial parameters in TH‐negative neurons. Overall, these findings suggest that DLP1‐dependent mitochondrial fragmentation plays a crucial role in mediating MPP⁺‐induced mitochondria abnormalities and cellular dysfunction and may represent a novel therapeutic target for PD.     

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.