The Protective Role of <Emphasis Type="SmallCaps">d</Emphasis>-Glucose Against 1-Methyl-4-Phenylpyridinium Ion (MPP<Superscript>+</Superscript>): Induced Mitochondrial Dysfunction in C6 Astroglial Cells

Impaired mitochondrial function in glial and neuronal cells in the substantia nigra is one of the most likely causes of Parkinson’s disease. In this study, we investigated the protective role of glucose on early key events associated with MPP⁺-induced changes in rat C6 astroglial cells. Studies were carried out to examine alterations in mitochondrial respiratory status, membrane potential, glutathione levels, and cell cycle phase inhibition at 48 h in 2 and 10 mM glucose in media. The results obtained suggest that MPP⁺ caused significant cell death in 2 mM glucose with LC₅₀ 0.14 ± 0.005 mM, while 10 mM glucose showed highly significant protection against MPP⁺ toxicity with LC₅₀ 0.835 ± 0.03 mM. This protection was not observed with cocaine, demonstrating its compound specificity. MPP⁺ in 2 mM glucose decreased significantly mitochondrial respiration, membrane potential and glutathione levels in a dose dependent manner, while 10 mM glucose significantly restored them. MPP⁺ in 2 mM glucose arrested the cells at G0/G1 and G2/M phases, demonstrating its dual inhibitory effects. However, in 10 mM glucose, MPP⁺ caused G0/G1 arrest only. In summary, the results suggest that loss of cell viability in 2 mM glucose group with MPP⁺ treatments was due to mitochondrial dysfunction caused by multilevel mechanism, involving significant decrease in mitochondrial respiration, membrane potential, glutathione levels, and dual arrest of cell phases, while 10 mM glucose rescued astroglial cells from MPP⁺ toxicity by significant maintenance of these factors.     

Anaerobic Glycolysis Protection against 1-Methy-4-Phenylpyridinium (MPP<Superscript>+</Superscript>) Toxicity in C6 Glioma Cells

The neurotoxin 1-methy-4-phenylpyridinium (MPP⁺) is used for its’ capacity to induce Parkinsonism through its inhibitory effects on mitochondrial complex I. This inhibition disrupts cellular energy formation and aerobic glycolysis. The objective of this study was to demonstrate that the toxic effect of mitochondrial aerobic pathway inhibition with MPP⁺ can be reduced by stimulating anaerobic glycolysis using glucose supplementation. In this study, C6 Glioma cell viability was examined in the presence of different concentrations of MPP alone and with the addition of glucose. The results obtained indicate that there was a significant increase (P < 0.001) in cell viability in cells treated with glucose and MPP⁺ verses cells treated with MPP⁺ alone. Fluorometric analysis using 100 uM Rhodamine 123 indicated mitochondrial membrane potential was not restored in MPP⁺ treated cells with glucose; however, normal cell viability was confirmed using 2 ug/ml Fluorescein diacetate. This dual fluorescence indicated mitochondrial damage from MPP⁺ while glucose augmented cell survival. Further confirmation of cell survival upon damage to the mitochondria was evident in TUNEL staining. Positive staining was prominent only in MPP⁺ treatment groups alone, while control and co-treated groups exhibited little to no TUNEL staining. ATP measurements of all MPP⁺ treated groups exhibited a significant (P < 0.001) decrease verses control. Groups co-treated with MPP⁺ and glucose revealed a significant increase (250 μM group: P < 0.001) in ATP. It was concluded from this study that glucose supplementation was able to sustain cellular viability and ATP production through anaerobic glycolysis despite the inhibitory effect of MPP⁺ on aerobic glycolysis.     

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.     

Differential Involvement of Intracellular Ca2+ in 1-Methyl-4-phenylpyridinium- or 6-Hydroxydopamine-Induced Cell Viability Loss in PC12 Cells

1-Methyl-4-phenylpyridinium (MPP⁺) or 6-hydroxydopamine (6-OHDA) caused a nuclear damage, the mitochondrial membrane permeability changes, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in PC12 cells. Nicardipine (a calcium channel blocker), EGTA (an extracellular calcium chelator), BAPTA-AM (a cell permeable calcium chelator) and calmodulin antagonists (W-7 and calmidazolium) attenuated the MPP⁺-induced mitochondrial damage and cell death. In contrast, the compounds did not reduce the toxicity of 6-OHDA. Treatment with MPP⁺ or 6-OHDA evoked the elevation of intracellular Ca²⁺ levels. Unlike cell injury, addition of nicardipine, BAPTA-AM and calmodulin antagonists prevented the elevation of intracellular Ca²⁺ levels due to both toxins. The results show that the MPP⁺-induced formation of the mitochondrial permeability transition seems to be mediated by elevation of intracellular Ca²⁺ levels and calmodulin action. In contrast, the 6-OHDA-induced cell death seems to be mediated by Ca²⁺-independent manner.     

Differential Involvement of Mitochondrial Permeability Transition in Cytotoxicity of 1-Methyl-4-Phenylpyridinium and 6-Hydroxydopamine

Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The aim of the present study was to assess the influence of the mitochondrial membrane permeability transition inhibition against the toxicity of 1-methyl-4-phenylpyridinium (MPP⁺) and 6-hydroxydopamine (6-OHDA) in relation to the mitochondria-mediated cell death process and role of oxidative stress. Both MPP⁺ and 6-OHDA induced the nuclear damage, the changes in the mitochondrial membrane permeability, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in differentiated PC12 cells. Cyclosporin A (CsA), trifluoperazine and aristolochic acid, inhibitors of mitochondrial permeability transition, significantly attenuated the MPP⁺-induced mitochondrial damage leading to caspase-3 activation, increased oxidative stress and cell death. In contrast to MPP⁺, the cytotoxicity of 6-OHDA was not reduced by the addition of the mitochondrial permeability transition inhibitors. The results show that the cytotoxicity of MPP⁺ may be mediated by the mitochondrial permeability transition formation, which is associated with formation of reactive oxygen species and the depletion of GSH. In contrast, the 6-OHDA-induced cell injury appears to be mediated by increased oxidative stress without intervention of the mitochondrial membrane permeability transition.     

Differential effect of platelet activating factor on 1-methyl-4-phenylpyridinium-induced cell death through regulation of apoptosis-related protein activation

Platelet activating factor (PAF) has been suggested to play a critical role in the pathogenesis of neurological disorders. We assessed the effect of PAF against the toxicity of 1-methyl-4-phenylpyridinium (MPP⁺), a parkinsonian toxin, in relation to apoptotic process. PAF exhibited differential effect against the MPP⁺ toxicity in differentiated PC12 cells depending on concentration. Treatment with 0.75 μM PAF significantly attenuated the MPP⁺-induced increase in Bax levels, decrease in Bid and Bcl-2 levels, and mitochondrial membrane potential loss that lead to the release of cytochrome c and subsequent caspase-3 activation. The inhibitory effect of PAF was not associated with nuclear factor-κB activation. In contrast, PAF at the concentrations greater than 2.5 μM exhibited a toxicity and additive effect on the MPP⁺ toxicity. The results show that PAF at low concentrations, which does not induce a significant toxicity, may prevent the MPP⁺ toxicity by suppressing the apoptosis-related protein activation and mitochondrial membrane permeability change that lead to the cytochrome c release and caspase-3 activation. The preventive effect seems to be associated with the inhibitory effect on the formation of reactive oxygen species and depletion of GSH. In contrast, PAF at higher concentrations may exhibit an additive toxic effect against the MPP⁺ toxicity by increasing apoptosis-related protein activation.     

α-Synuclein and mitochondrial bioenergetics regulate tetrahydrobiopterin levels in a human dopaminergic model of Parkinson disease

Parkinson disease (PD) is a multifactorial disease resulting in preferential death of the dopaminergic neurons in the substantia nigra. Studies of PD-linked genes and toxin-induced models of PD have implicated mitochondrial dysfunction, oxidative stress, and the misfolding and aggregation of α-synuclein (α-syn) as key factors in disease initiation and progression. Many of these features of PD may be modeled in cells or animal models using the neurotoxin 1-methyl-4-phenylpyridinium (MPP⁺). Reducing oxidative stress and nitric oxide synthase (NOS) activity has been shown to be protective in cell or animal models of MPP⁺ toxicity. We have previously demonstrated that siRNA-mediated knockdown of α-syn lowers the activity of both dopamine transporter and NOS activity and protects dopaminergic neuron-like cells from MPP⁺ toxicity. Here, we demonstrate that α-syn knockdown and modulators of oxidative stress/NOS activation protect cells from MPP⁺-induced toxicity via postmitochondrial mechanisms rather than by a rescue of the decrease in mitochondrial oxidative phosphorylation caused by MPP⁺ exposure. We demonstrate that MPP⁺ significantly decreases the synthesis of the antioxidant and obligate cofactor of NOS and TH tetrahydrobiopterin (BH4) through decreased cellular GTP/ATP levels. Furthermore, we demonstrate that RNAi knockdown of α-syn results in a nearly twofold increase in GTP cyclohydrolase I activity and a concomitant increase in basal BH4 levels. Together, these results demonstrate that both mitochondrial activity and α-syn play roles in modulating cellular BH4 levels.     

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.     

Inhibition of NADH oxidation by 1-methyl-4-phenylpyridinium analogs as the basis for the prediction of the inhibitory potency of novel compounds

Inhibition of NADH dehydrogenase (Complex I) of the mitochondrial respiratory chain by 1-methyl-4-phenylpyridinium (MPP⁺) and its analogs results in dopaminergic cell death. In the present study, the inhibition of mitochondrial respiration and of NADH oxidation in inverted inner membrane preparations by the oxidation products of N-methyl-stilbazoles (N-methyl-styrylpyridiniums) are characterized. These nonflexible MPP⁺ analogs were found to be considerably more potent inhibitors than the corresponding MPP⁺ derivatives. The IC₅₀ values for these compounds and previously published figures for MPP⁺ analogs were then used to select a computer model based on structural parameters to predict the inhibitory potency of other compounds that react at the “rotenone site” in Complex I. A series of 12 novel inhibitors different in structure from the basic set were used to test the predictive capacity of the models selected. Despite major structural differences between the novel test compounds and the MPP⁺ and styrylpyridinium analogs on which the models were based, substantial agreement was found between the predicted and experimentally determined IC₅₀ values. The value of this technique lies in the potential for the prediction of the inhibitory potency of other drugs and toxins which block mitochondrial respiration by interacting at the rotenone sites. © 1996 John Wiley & Sons, Inc.     

Homer1 knockdown protects dopamine neurons through regulating calcium homeostasis in an in vitro model of Parkinson's disease

Homer1 protein is an important scaffold protein at postsynaptic density and has been demonstrated to play a central role in calcium signaling in the central nervous system. The aim of this study was to investigate the effects of Homer1 knockdown on MPP⁺ induced neuronal injury in cultured dopamine (DA) neurons. We found that down-regulating Homer1 expression with specific small interfering RNA (siRNA) significantly suppressed LDH release, reduced Propidium iodide (PI) or Hoechst staining, increased the number of tyrosine hydroxylase (TH) positive cells and DA uptake, and attenuated apoptotic and necrotic cell death after MPP⁺ injury. Homer1 knockdown decreased intracellular reactive oxygen species (ROS) generation through inhibition of intracellular calcium overload, but did not affect the endogenous antioxidant enzyme activities. Calcium imaging was used to examine the changes of intracellular Ca^2 + concentration ([Ca^2 +]_cyt) and Ca^2 + in endoplasmic reticulum (ER) ([Ca^2 +]_ER), and the results showed that Homer1 siRNA transfection attenuated ER Ca^2 + release up to 120 min after MPP⁺ injury. Furthermore, decrease of [Ca^2 +]_cyt induced by Homer1 knockdown in MPP⁺ treated neurons was further enhanced by NMDA receptor antagonists MK-801 and AP-5, but not canonical transient receptor potential (TRPC) channel antagonist SKF-96365. l-type calcium antagonist isradipine but not nimodipine further inhibited intracellular calcium overload after MPP⁺ insult in Homer1 down-regulated neurons. These results suggest that Homer1 knockdown has protective effects against neuronal injury in in vitro PD model by reducing calcium overload mediated ROS generation, and this protection may be dependent at least in part on the regulatory effects on the function of calcium channels in both plasma membrane and ER.     

Changes in Neuronal Dopamine Homeostasis following 1-Methyl-4-phenylpyridinium (MPP+) Exposure

1-Methyl-4-phenylpyridinium (MPP⁺), the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, selectively kills dopaminergic neurons in vivo and in vitro via a variety of toxic mechanisms, including mitochondrial dysfunction, generation of peroxynitrite, induction of apoptosis, and oxidative stress due to disruption of vesicular dopamine (DA) storage. To investigate the effects of acute MPP⁺ exposure on neuronal DA homeostasis, we measured stimulation-dependent DA release and non-exocytotic DA efflux from mouse striatal slices and extracellular, intracellular, and cytosolic DA (DA_cyt) levels in cultured mouse ventral midbrain neurons. In acute striatal slices, MPP⁺ exposure gradually decreased stimulation-dependent DA release, followed by massive DA efflux that was dependent on MPP⁺ concentration, temperature, and DA uptake transporter activity. Similarly, in mouse midbrain neuronal cultures, MPP⁺ depleted vesicular DA storage accompanied by an elevation of cytosolic and extracellular DA levels. In neuronal cell bodies, increased DA_cyt was not due to transmitter leakage from synaptic vesicles but rather to competitive MPP⁺-dependent inhibition of monoamine oxidase activity. Accordingly, monoamine oxidase blockers pargyline and l-deprenyl had no effect on DA_cyt levels in MPP⁺-treated cells and produced only a moderate effect on the survival of dopaminergic neurons treated with the toxin. In contrast, depletion of intracellular DA by blocking neurotransmitter synthesis resulted in ∼30% reduction of MPP⁺-mediated toxicity, whereas overexpression of VMAT2 completely rescued dopaminergic neurons. These results demonstrate the utility of comprehensive analysis of DA metabolism using various electrochemical methods and reveal the complexity of the effects of MPP⁺ on neuronal DA homeostasis and neurotoxicity.     

MicroRNA-7 Promotes Glycolysis to Protect against 1-Methyl-4-phenylpyridinium-induced Cell Death

Parkinson disease is associated with decreased activity of the mitochondrial electron transport chain. This defect can be recapitulated in vitro by challenging dopaminergic cells with 1-methyl-4-phenylpyridinium (MPP⁺), a neurotoxin that inhibits complex I of electron transport chain. Consequently, oxidative phosphorylation is blocked, and cells become dependent on glycolysis for ATP production. Therefore, increasing the rate of glycolysis might help cells to produce more ATP to meet their energy demands. In the present study, we show that microRNA-7, a non-coding RNA that protects dopaminergic neuronal cells against MPP⁺-induced cell death, promotes glycolysis in dopaminergic SH-SY5Y and differentiated human neural progenitor ReNcell VM cells, as evidenced by increased ATP production, glucose consumption, and lactic acid production. Through a series of experiments, we demonstrate that targeted repression of RelA by microRNA-7, as well as subsequent increase in the neuronal glucose transporter 3 (Glut3), underlies this glycolysis-promoting effect. Consistently, silencing Glut3 expression diminishes the protective effect of microRNA-7 against MPP⁺. Further, microRNA-7 fails to prevent MPP⁺-induced cell death when SH-SY5Y cells are cultured in a low glucose medium, as well as when differentiated ReNcell VM cells or primary mouse neurons are treated with the hexokinase inhibitor, 2-deoxy-d-glucose, indicating that a functional glycolytic pathway is required for this protective effect. In conclusion, microRNA-7, by down-regulating RelA, augments Glut3 expression, promotes glycolysis, and subsequently prevents MPP⁺-induced cell death. This protective effect of microRNA-7 could be exploited to correct the defects in oxidative phosphorylation in Parkinson disease.     

A microarray study of MPP<Superscript>+</Superscript>-treated PC12 Cells: Mechanisms of toxicity (MOT) analysis using bioinformatics tools

Background

This paper describes a microarray study including data quality control, data analysis and the analysis of the mechanism of toxicity (MOT) induced by 1-methyl-4-phenylpyridinium (MPP⁺) in a rat adrenal pheochromocytoma cell line (PC12 cells) using bioinformatics tools. MPP⁺ depletes dopamine content and elicits cell death in PC12 cells. However, the mechanism of MPP⁺-induced neurotoxicity is still unclear.

Results

In this study, Agilent rat oligo 22K microarrays were used to examine alterations in gene expression of PC12 cells after 500 μM MPP⁺ treatment. Relative gene expression of control and treated cells represented by spot intensities on the array chips was analyzed using bioinformatics tools. Raw data from each array were input into the NCTR ArrayTrack database, and normalized using a Lowess normalization method. Data quality was monitored in ArrayTrack. The means of the averaged log ratio of the paired samples were used to identify the fold changes of gene expression in PC12 cells after MPP⁺ treatment. Our data showed that 106 genes and ESTs (Expressed Sequence Tags) were changed 2-fold and above with MPP⁺ treatment; among these, 75 genes had gene symbols and 59 genes had known functions according to the Agilent gene Refguide and ArrayTrack-linked gene library. The mechanism of MPP⁺-induced toxicity in PC12 cells was analyzed based on their genes functions, biological process, pathways and previous published literatures.

Conclusion

Multiple pathways were suggested to be involved in the mechanism of MPP⁺-induced toxicity, including oxidative stress, DNA and protein damage, cell cycling arrest, and apoptosis.

     

Age-Dependent Sensitivity of Cultured Peripheral Sympathetic Neurons to 1-Methyl-4-Phenylpyridinium: Role of Glutathione

Abstract: We demonstrate that 1-methyl-4-phenylpyridinium (MPP⁺) is toxic to chick peripheral sympathetic neurons maintained in culture in the presence of nerve growth factor (NGF). When MPP⁺ was added to the culture medium at the time the neurons were plated, cell loss after 3 days in culture was evident at concentrations as low as 3 nM, and near maximal at 1 µM. Toxicity was blocked by brief preincubation with the norepinephrine (NE)-reuptake blocker desipramine (DMI; 10 µM for 30 min). MPP⁺ blocked the uptake of [³H]NE by sympathetic neurons in a dose-dependent manner with a potency roughly equal to DMI. At concentrations up to 10 µM, MPP⁺ had no neurotoxic effect on the survival of sensory neurons maintained in the presence of NGF. The sensitivity of sympathetic neurons to the toxic effects of MPP⁺ diminished gradually with increasing lengths of time in culture. When MPP⁺ was added to the culture medium 48 h after plating, concentrations up to 100 µM did not cause neuronal death. This increasing resistance of sympathetic neurons to MPP⁺-induced cell death could not be explained by an increasing capacity for sequestration of MPP⁺ within synaptic vesicles. The loss of sensitivity with time in culture was, however, accompanied by a threefold increase in the levels of glutathione (GSH). Furthermore, addition of MPP⁺ (1 µM) to cultures previously maintained for 2 days in the presence of the GSH-synthesis inhibitor l -buthionine-[S,R]-sulfoximine (1 µM) caused the same degree of cell death as when added to freshly plated neurons. These results suggest that the observed toxicity of MPP⁺ in freshly plated chick sympathetic neurons may involve the formation of free radicals and that GSH plays a role in protecting sympathetic neurons in vivo from the toxicity of MPP⁺.     

The parkinsonian neurotoxin MPP+ opens the mitochondrial permeability transition pore and releases cytochrome c in isolated mitochondria via an oxidative mechanism

The mitochondrial transition pore (MTP) is implicated as a mediator of cell injury and death in many situations. The MTP opens in response to stimuli including reactive oxygen species and inhibition of the electron transport chain. Sporadic Parkinson’s disease (PD) is characterized by oxidative stress and specifically involves a defect in complex I of the electron transport chain. To explore the possible involvement of the MTP in PD models, we tested the effects of the complex I inhibitor and apoptosis-inducing toxin N-methyl-4-phenylpyridinium (MPP⁺) on cyclosporin A (CsA)-sensitive mitochondrial swelling and release of cytochrome c. In the presence of Ca²⁺ and P_i, MPP⁺ induced a permeability transition in both liver and brain mitochondria. MPP⁺ also caused release of cytochrome c from liver mitochondria. Rotenone, a classic non-competitive complex I inhibitor, completely inhibited MPP⁺-induced swelling and release of cytochrome c. The MPP⁺-induced permeability transition was synergistic with nitric oxide and the adenine nucleotide translocator inhibitor atractyloside, and additive with phenyl arsine oxide cross-linking of dithiol residues. MPP⁺-induced pore opening and cytochrome c release were blocked by CsA, the Ca²⁺ uniporter inhibitor ruthenium red, the hydrophobic disulfide reagent N-ethylmaleimide, butacaine, and the free radical scavenging enzymes catalase and superoxide dismutase. MPP⁺ neurotoxicity may derive from not only its inhibition of complex I and consequent ATP depletion, but also from its ability to open the MTP and to release mitochondrial factors including Ca²⁺ and cytochrome c known to be involved in apoptosis.     

Inhibition of Store-Operated Calcium Entry Attenuates MPP+-Induced Oxidative Stress via Preservation of Mitochondrial Function in PC12 Cells: Involvement of Homer1a

The process of store-operated calcium entry (SOCE), whereby the release of intracellular Ca²⁺ from endoplasmic reticulum (ER) activates Ca²⁺ influx channels in the plasma membrane, has been demonstrated to impact a diverse range of cell functions. In the present study, we investigated the potential protective effect of SOCE inhibition against 1-methyl-4-phenylpyridinium (MPP⁺) injury by using pharmacological antagonists or specific small interfering RNA (siRNA) in PC12 cells. The results showed that both antagonists (15 μM MRS-1845 and 50 μM ML-9) and stromal interacting molecule-1 (STIM1) targeted siRNA (Si-STIM1) significantly increased cell viability, decreased apoptotic cell death and reduced intracellular reactive oxygen species (ROS) production and lipid peroxidation in MPP⁺ injured PC12 cells. SOCE inhibition also prevented MPP⁺ induced mitochondrial dysfunction and activation of mitochondrial related apoptotic factors, while had no effect on mitochondrial biogenesis. Moreover, inhibition of SOCE by antagonists and siRNA increased the expression levels of Homer1a mRNA and protein, and knockdown of Homer1a expression by specific siRNA partly reversed the protective effects induced by SOCE inhibition in PC12 cells. All these results indicated that SOCE inhibition protected PC12 cells against MPP⁺ insult through upregulation of Homer1a expression, and SOCE might be an ideal target for investigating therapeutic strategy against neuronal injury in PD patients.     

Endogenous Hydrogen Sulfide is Involved in Asymmetric Dimethylarginine-induced Protection Against Neurotoxicity of 1-Methyl-4-phenyl-pyridinium Ion

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, is profoundly protective against 1-methy-4-phenylpyridinium ion (MPP⁺)-induced neurotoxicity. Reactive oxygen species (ROS) overproduction contributes to the neurotoxicity of MPP⁺; while hydrogen sulfide (H₂S) is a pivotal endogenous antioxidant. This study is to assess the potential role of endogenous H₂S in the neuroprotection of ADMA against MPP⁺-induced toxicity in PC12 cells. We showed that ADMA prevented MPP⁺-induced inhibition of endogenous H₂S generation through inhibiting the down-regulation of cystathionine-β-synthetase (CBS, the major enzyme responsible for endogenous H₂S generation in PC12 cells) expression and activity elicited by MPP⁺. ADMA obviously attenuated MPP⁺-triggered accumulation of intracellular ROS, dissipation of mitochondrial membrane potential (MMP), release of cytochrome c (Cyt-c), and downregulation of Bcl-2 protein expression in PC12 cells. Inhibition of CBS activity by amino-oxyacetate and CBS silencing with a short hairpin RNA vector targeting rat CBS gene reversed the protective action of ADMA against MPP⁺-caused cytotoxicity, ROS overproduction, and MMP loss in PC12 cells. These results indicate that the protection of ADMA against MPP⁺-mediated neurotoxicity involves the melioration of MPP⁺-induced inhibition of endogenous H₂S generation. Our findings suggest that modulation of H₂S production provide new therapeutic targets for the treatment of neurodegenerative disease, such as Parkinson’s disease.