Differences in the disposition and toxicity of 1-methyl-4-phenylpyridinium in cultured rat and mouse astrocytes

Species difference in the susceptibility to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was investigated in cultured rat and mouse astrocytes, where 1-methyl-4-phenylpyridinium (MPP⁺), the toxic mediator of MPTP, is formed. Type A monoamine oxidase (MAO) predominated in both rat and mouse astrocytes, while its activity was slightly higher in mouse cells compared to rat cells; MAO-B activity, on the other hand, was significantly lower in mouse astrocytes than in rat astrocytes. Because both types of MAO have been reported to make similar contributions to MPP⁺ production in astrocytes, their total activity was examined and results indicated that there was no significant difference between these two species. In additon, MPP^± caused a dose dependent loss of cell viability as judged by the amount of lactate dehydrogenase released into the incubation medium. The toxicity of MPP^± on astrocytes started to be seen after a 2 day incubation period. Mouse astrocytes were more vulnerable to MPP^± than rat astrocytes. The threshhold values for MPP^± toxicity in mouse and rat cultures were 10 ±M and 70 ±M, respectively. After addition of [³H] MPP^± to the medium, intracellular [³H] MPP^± was found to increase in both cultures. Mouse astrocytes accumulated more MPP^± than rat astrocytes (150 pmol/mg protein vs. 65 pmol/mg protein). When astrocytes were allowed to accumulate [³H] MPP^± and then incubated in fresh medium medium not containing [³H] MPP^±, intracellular levels of [³H] MPP^± in both cells rapidly declined (110 pmol/protein in mouse vs. 40 pmol/mg protein in rat of MPP^± been released). These results indicated that (1) MPP^± could cross the plasma membrane of astrocytes despite of its charged chemical structure, (2) mouse astrocytes had a higher capacity for MPP^± accumulation (approximately 2-fold), as well as release (approximately 2.7-fold), than rat astrocytes, and (3) mouse astrocytes were more vulnerable to MPP^± than rat astrocytes.     

Pinocembrin Protects SH-SY5Y Cells Against MPP+-Induced Neurotoxicity Through the Mitochondrial Apoptotic Pathway

Pinocembrin (PB), the most abundant flavonoid in propolis, has been proven to have neuroprotective property against neurotoxicity in vivo and in vitro. Our recent study demonstrated the neuroprotective effect of PB against Aβ_25–35-induced SH-SY5Y neurotoxicity. However, the mechanism as how PB can induce neuroprotection is not known. In the present study, we demonstrate here that PB abrogates the effects of the neurotoxin 1-methyl-4-phenylpyridinium (MPP⁺) which mimics Parkinson’s disease (PD) with elevation of intracellular reactive oxygen species (ROS) level and apoptotic death. We found that pretreatment of SH-SY5Y cells with PB significantly reduced the MPP⁺-induced loss of cell viability, the generation of intracellular ROS, apoptotic rate, and the cleavage of caspase-3. PB strikingly inhibited MPP⁺-induced mitochondrial dysfunctions, including lowered membrane potential, decreased Bcl-2/Bax ratio, and the release of cytochrome c. Overall, these results suggest that PB is intimately involved in inhibiting MPP⁺-induced loss of mitochondrial function and induction of apoptosis that contributes toward neuronal survival. These data indicated that PB might provide a valuable therapeutic strategy for the treatment of PD.     

l-Deprenyl fails to protect mesencephalic dopamine neurons and PC12 cells from the neurotoxic effect of 1-methyl-4-phenylpyridinium ion

l-Deprenyl, a monoamine oxidase (MAO)-B inhibitor, appears to slow down the progression of Parkinson's disease. While inhibition of MAO-B activity can account for some of the effects of this substance, the basis by whichl-deprenyl slows the progression of the disease remains controversial. In recent years, a new mechanism of action has emerged that may explain the ability ofl-deprenyl to increase neuronal survival.l-deprenyl has been reported to modify gene expression and protein synthesis in astrocytes and PC12 cells. In this study, we tested the ability ofl-deprenyl to protect mouse mesencephalic cells from the toxicity of the 1-methyl-4-phenyl pyridinium ion (MPP⁺). We exposed mouse mesencephalic cell cultures to L-deprenyl (10 μM) and, 24 h later, to MPP⁺ (2.5 μM). On the fifth day afterl-deprenyl and MPP⁺ exposition, cells were washed free of drugs, and the following day they were tested for dopamine uptake, intracellular dopamine content and tyrosine hydroxylase immunoreactivity. The experiments were performed either in the presence or in the absence of glia. It was found thatl-deprenyl pretreatment failed to achieve any protection against MPP⁺ toxicity. The fall in dopamine uptake and intracellular dopamine content, and the diminution of tyrosine hydroxylase immunoreactivity observed in cells pretreated withl-deprenyl and then given MPP⁺ were not significantly different from the values observed in cells treated with MPP⁺ alone. Additional experiments performed in PC12 cells, confirmed the failure ofl-deprenyl to abolish the toxicity of MPP⁺. Our data seem to be at variance with previous reports demonstrating that the MAO-B inhibitorl-deprenyl protects dopaminergic neurons against MPP⁺ toxicity [12,20]; furthermore they do not support alternative mechanisms of action ofl-deprenyl against MPP⁺ toxicity.     

MALDI Mass Spectrometry Imaging of 1-Methyl-4-phenylpyridinium (MPP+) in Mouse Brain

Parkinson’s disease (PD) is the second most common neurodegenerative disorder affecting ~1 % of the population older than 60 years. The administration of the proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice is one of the most widely used approach to elucidate the mechanisms of cell death involved in PD. Its toxicity is attributed to its active metabolite 1-methyl-4-phenylpyridinium (MPP⁺). However, the magnitude of the PD-like neurodegeneration induced by MPTP depends on many variables, including the route of administration. Different groups, including us, demonstrated that intranasal (i.n.) administration of MPTP constitutes a new route of toxin delivery to the brain that mimics environmental exposure to neurotoxins. In particular, our previous data showed that mice submitted to acute i.n. MPTP administration displayed a significant decrease of striatal dopamine (DA) and a loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. However, little is known about the timing and the anatomical distribution of MPP⁺ after i.n. MPTP administration in mice. In the present study, C57BL/6J mice received one dose of i.n. MPTP (1 mg/nostril) and were sacrificed at two different times after the administration. Using matrix-assisted laser desorption–ionization mass spectrometry imaging, a new technique for the detection of endogenous unlabeled molecules in tissue sections, we showed for the first time the MPP⁺ anatomical distribution in different brain regions. We demonstrated that the toxin first reached almost all the brain areas; however, in a second time MPP⁺ remained highly concentrated in the olfactory bulb, the basal ganglia, the ventral mesencephalon, and the locus coeruleus, regions differently affected in PD.     

Nicotine suppresses the neurotoxicity by MPP+/MPTP through activating α7nAChR/PI3K/Trx-1 and suppressing ER stress

Parkinson’s disease (PD) is a neurodegenerative disease. Nicotine has been reported to have the role in preventing Parkinson’s disease. However, its mechanism is still unclear. In present study we found that nicotine suppressed 1-methyl-4-phenylpyridinium ion(MPP⁺) toxicity in PC12 cells by MTT assay. The expression of thioredoxin-1(Trx-1) was decreased by MPP⁺, which was restored by nicotine. The nicotine suppressed expressions of Glucose-regulated protein 78(GRP78/Bip) and C/EBP homologous protein (CHOP) induced by MPP⁺. The methyllycaconitine (MLA), the inhibitor of α7nAChR and LY294002, the inhibitor of phosphatidylinositol 3-kinase (PI3K) blocked the suppressions of above molecules, respectively. Consistently, pretreatment with nicotine ameliorated the motor ability, restored the declines of Trx-1 and tyrosine hydroxylase (TH), and suppressed the expressions of Bip and CHOP induced by 1-Methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice. Our results suggest that nicotine plays role in resisting MPP⁺/MPTP neurotoxicity through activating the α7nAChR/PI3K/Trx-1 pathway and suppressing ER stress.     

MPP<Superscript>+</Superscript>-induced degeneration is potentiated by dicoumarol in cultures of the RCSN-3 dopaminergic cell line. Implications of neuromelanin in oxidative metabolism of dopamine neurotoxicity

We have tested the idea that oxidative metabolism of dopamine may be involved in MPTP toxicity using the RCSN-3 cell line derived from the substantia nigra of an adult rat. Treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (10 μM), MPTP combined with 40 μM dicoumarol (an inhibitor of DT-diaphorase) and dicoumarol alone, did not induce toxicity in RCSN-3 cells after 72 h incubation. The lack of toxicity MPTP-treated RCSN-3 cells may be explained by the fact that they are unable to metabolize MPTP to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium ion (MPP⁺) as determined by HPLC. Incubation for 72 h with 100 μM MPP⁺ induced 6.6±1.4% cell death of RCSN-3 cells compared to 3.5±0.4 observed in control cells. However, when the cells were treated with 100 μM MPP⁺ and 40 μM dicoumarol, cell death increased 4-fold compared to that of cells treated solely with MPP⁺ (27±2%;P<0.001). Underthese conditions, a significant increase in DNA fragmentation (3-fold compared to MPP⁺ alone;P<0.01) and in calpain activation (P <0.05 compared to control) was evident. The inhibition of DT-diaphorase by dicoumarol supports the idea that oxidative metabolism of dopamine is involved in MPP⁺ toxicity in RCSN-3 cells.     

SR-A Regulates the Inflammatory Activation of Astrocytes

Scavenger receptor Class A (SR-A) participates in the regulation of inflammatory processes against pathogens and in inflammatory stimulation. We have recently demonstrated the presence of SR-A in astrocytes, but its participation in their inflammatory response is unknown. Astrocytes regulate neuroinflammation through the regulation of microglial cell activation and the production of cytokines, neurotrophic factors, and reactive species. Using astrocytes from SR-A^?/? mice in culture, we assessed the participation of SR-A in their inflammatory activation, evaluating the activation of IκB/NF-κB and MAPK signaling pathways and the production of nitric oxide (NO) and IL-1β in response to SR-A ligands. In SR-A^?/? astrocytes, lipopolysaccharide (LPS) induced higher levels of NO and reduced levels of IL-1β compared to SR-A^+/+ cells. In addition, SR-A^?/? astrocytes had a reduced basal and LPS-stimulated JNK phosphorylation, and a delayed activation on IκB/NF-κB signaling pathway in response to LPS. Moreover, inhibition of the ERK pathway reduced NO production by SR-A^?/? cells, suggesting that this signaling pathway modulated LPS-induced NO production, an effect that depended on the presence of SR-A. Our results suggest that SR-A participates in the modulation of signaling pathways involved in the production of soluble molecules implicated in the neuroinflammatory response.     

Downregulation of miR‐7116‐5p in microglia by MPP+ sensitizes TNF‐α production to induce dopaminergic neuron damage

Activation of microglia resulting in exacerbated inflammation expression plays an important role in degeneration of dopaminergic (DA) neurons in the pathogenesis of Parkinson's disease (PD). However, how this enhanced inflammation is induced in microglia remains largely unclear. Here, in the mouse PD model induced by 1‐methyl‐4‐phenyl‐1,2,3,6‐tetra hydropyridine (MPTP), we found that miR‐7116‐5p in microglia has a crucial role in this inflammation. 1‐methyl‐4‐phenylpyridinium (MPP⁺) is uptaken by microglia through organic cation transporter 3 (OCT3) to downregulate miR‐7116‐5p, an miRNA found to target tumor necrosis factor alpha (TNF‐α). Production of TNF‐α in microglia is specifically potentiated by MPP⁺ via downregulation of miR‐7116‐5p to elicit subsequent inflammatory responses. Furthermore, enhancement of miR‐7116‐5p expression in microglia in mice inhibits the production of TNF‐α and the activation of glia, and further prevents loss of DA neurons. Together, our studies suggest that MPP⁺ suppresses miR‐7116‐5p level in microglia and potentiates TNF‐α production and inflammatory responses to contribute to DA neuron damage.     

Purinergic receptor P2Y6 contributes to 1‐methyl‐4‐phenylpyridinium‐induced oxidative stress and cell death in neuronal SH‐SY5Y cells

Oxidative stress and neural degeneration have been shown to be involved in the pathogenesis of Parkinson's disease (PD). The P2Y6 purinergic receptor (P2Y6R) has been shown to participate in the activation of microglia and the production of pro‐inflammatory factors induced by lipopolysaccharide to cause neuronal loss. However, the function of P2Y6R during oxidative stress in neurons is unclear. In the present study, 1‐methyl‐4‐phenylpyridinium (MPP⁺) treatment increased the level of UDP/P2Y6R on neuronal SH‐SY5Y cells. Importantly, pharmacological inhibition of P2Y6R or knockdown of P2Y6R using a siRNA exerted an increased protective effect by preventing MPP⁺‐induced increases in the levels of reactive oxygen species (ROS), superoxide anion, inducible nitric oxide synthase (iNOS), and malondialdehyde (MDA) and down‐regulation of superoxide dismutase 1 (SOD1) expression. UDP, an agonist of P2Y6R, enhanced the effects of MPP⁺, which was also inhibited by apyrase or MRS2578. Additionally, P2Y6R knockdown also significantly reversed both the loss of cell viability and the increase in the levels of phosphorylated extracellular signal‐regulated protein kinase (p‐ERK1/2) and p38 (p‐p38) caused by MPP⁺ stimulation. However, the inhibition of the ERK1/2 and p38 kinase signaling pathways had no effect on P2Y6R expression. Taken together, these results support the hypothesis that P2Y6R expressed on neuronal SH‐SY5Y cell is associated with the progression of oxidative stress and cell death induced by MPP⁺, suggesting that P2Y6R may play an important role in the pathogenesis of PD.     

Mitochondrial Uncoupling Protein-2 (UCP2) Mediates Leptin Protection Against MPP+ Toxicity in Neuronal Cells

Mitochondrial dysfunction is involved in the pathogenesis of neurodegenerative diseases, including Parkinson’s disease (PD). Uncoupling proteins (UCPs) delink ATP production from biofuel oxidation in mitochondria to reduce oxidative stress. UCP2 is expressed in brain, and has neuroprotective effects under various toxic insults. We observed induction of UCP2 expression by leptin in neuronal cultures, and hypothesize that leptin may preserve neuronal survival via UCP2. We showed that leptin preserved cell survival in neuronal SH-SY5Y cells against MPP⁺ toxicity (widely used in experimental Parkinsonian models) by maintaining ATP levels and mitochondrial membrane potential (MMP); these effects were accompanied by increased UCP2 expression. Leptin had no effect in modulating reactive oxygen species levels. Stable knockdown of UCP2 expression reduced ATP levels, and abolished leptin protection against MPP⁺-induced mitochondrial depolarization, ATP deficiency, and cell death, indicating that UCP2 is critical in mediating these neuroprotective effects of leptin against MPP⁺ toxicity. Interestingly, UCP2 knockdown increased UCP4 expression, but not of UCP5. Our findings show that leptin preserves cell survival by maintaining MMP and ATP levels mediated through UCP2 in MPP⁺-induced toxicity.     

KATP channel openers protect mesencephalic neurons against MPP+‐induced cytotoxicity via inhibition of ROS production

Opening of ATP‐sensitive potassium (K_ATP) channels has been demonstrated to exert significant neuroprotection in in vivo and in vitro models of Parkinson's disease (PD), but the exact mechanism remains unclear. In the present study, various K_ATP channel openers (KCOs) sensitive to diverse K_ATP subunits were used to clarify the protective role of K_ATP channel opening in 1‐methyl‐4‐phenylpyridinium (MPP⁺)‐induced oxidative stress injury in mouse primary cultured mesencephalic neurons. The results showed that pretreatment with nonselective KCO pinacidil (Pin) or diazoxide (Dia), a KCO sensitive to Kir6.2/SUR1 K_ATP channels, protected mesencephalic neurons, especially dopaminergic neurons, against MPP⁺‐induced injury in a concentration‐dependent manner. However, cromakalim (Cro), an opener of Kir6.1/SUR2 but not Kir6.2/SUR1 K_ATP channels, failed to protect against MPP⁺‐induced cytotoxicity. Furthermore, Pin and Dia but not Cro significantly suppressed the elevation of reactive oxygen species (ROS) triggered by MPP⁺ and prevented the loss of mitochondrial member potential (ΔΨm) and the release of mitochondrial cyotchrome c. Consequently, opening of K_ATP channels expressed in neurons could protect primary mesencephalic neurons against MPP⁺‐induced cytotoxicity via inhibiting ROS overproduction and subsequently ameliorating mitochondrial function. © 2009 Wiley‐Liss, Inc.     

Methylmercury chloride exposure aggravates proinflammatory mediators and Notch-1 signaling in CD14+ and CD40+ cells and is associated with imbalance of neuroimmune function in BTBR T+ Itpr3tf/J mice

Autism spectrum disorder (ASD) is a severe neurodevelopmental disorder characterized by deficits in social interaction, communication, and repetitive behaviors. A key role for immune dysfunction has been suggested in ASD. Recent studies have indicated that inflammatory mediators and Notch-1 signaling may contribute to the development of ASD. Methylmercury chloride (MeHgCl) is an environmental pollutant that primarily affects the central nervous system, causing neurological alterations. Its effects on immunological responses have not been fully investigated in ASD. In this study, we examined the influence of MeHgCl exposure on inflammatory mediators and Notch-1 signaling in BTBR T⁺ Itpr3tf/J (BTBR) mice, a model of ASD. We examined the effects of MeHgCl on the IL-6-, GM-CSF-, NF-κB p65-, Notch-1-, and IL-27-producing CD14⁺ and CD40⁺ cells in the spleen. We assessed the effect of MeHgCl on IL-6, GM-CSF, NF-κB p65, Notch-1, and IL-27 mRNA levels in brain tissue. We also measured IL-6, GM-CSF, and NF-κB p65 protein expression levels in brain tissue. MeHgCl exposure of BTBR mice significantly increased IL-6-, GM-CSF-, NF-κB p65-, and Notch-1-, and decreased IL-27-producing CD14⁺, and CD40⁺ cells in the spleen. MeHgCl exposure of BTBR mice upregulated IL-6, GM-CSF, NF-κB p65, and Notch-1, and decreased IL-27 mRNA expression levels in brain tissue. Moreover, MeHgCl resulted in elevated expression of the IL-6, GM-CSF, and NF-κB p65 proteins in brain tissue. Taken together, these results indicate that MeHgCl exposure aggravates proinflammatory mediators and Notch-1 signaling which are associated with imbalance of neuroimmune function in BTBR mice.     

Protective effects of heme oxygenase-1 against MPP+-induced cytotoxicity in PC-12 cells

Heme oxygenase-1 (HO-1) catalyses the rate-limiting step of heme degradation to biliverdin, which is in turn reduced to bilirubin, CO and free iron. HO-1 can be induced by several harmful stimuli including oxidative stress, and it has a protective role against the cytotoxicity in different cells. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridinium (MPP⁺) is a neurotoxic substance that induces the degeneration of dopaminergic neurons. This study examined whether HO-1 can be induced by MPP⁺ and whether HO-1 has a protective role against the MPP⁺-induced cytotoxicity in PC-12 cells. MPP⁺ triggered a relatively rapid induction of HO-1. The MPP⁺-induced cytotoxicity and reactive oxygen species (ROS) production markedly increased by HO-1 inhibitor, zinc protoporphyrin-IX (ZnPP-IX). The increase of ROS production by ZnPP-IX was completely abrogated by either two products of HO (biliverdin or bilirubin) while the increase of cytotoxicity by ZnPP-IX was attenuated partially. These suggest that HO-1 expression might have some cytoprotective effect against MPP⁺-induced cytotoxicity.     

Inhibition of Hydrogen Sulfide Generation Contributes to 1-Methy-4-Phenylpyridinium Ion-Induced Neurotoxicity

Reactive oxygen species (ROS) overproduction contributes to the neurotoxicity of 1-methy-4-phenylpyridinium ion (MPP⁺). Increasing studies have shown that hydrogen sulfide (H₂S) is an endogenous antioxidant gas. We have hypothesized that MPP⁺-caused neurotoxicity may involve the imbalance of proportion to this endogenous protective antioxidant gas. The aim of this study is to evaluate whether MPP⁺ disturbs H₂S synthesis in PC12 cells, a clonal rat pheochromocytoma cell line, and whether disturbance of H₂S generation induced by MPP⁺ is an underlying mechanism of MPP⁺-induced neurotoxicity. We show that exposure of PC12 cells to MPP⁺ causes a significant decrease in H₂S generation and results in remarkable cell damage. We find that cystathionine-β-synthetase (CBS) is catalyzed in PC12 cells to generate H₂S, and that both expression and activity of CBS are inhibited by MPP⁺ treatment. Exposure of sodium hydrosulfide (NaHS), a donor of H₂S, extenuates MPP⁺-induced cytotoxicity and ROS accumulation in PC12 cells, while inhibition of CBS by amino-oxyacetate (AOAA) exacerbates the effects of MPP⁺. These results indicate that MPP⁺ neurotoxicity involves reduction of H₂S production, which is caused by inhibition of CBS. This study provides novel insights into cell death observed in neurodegenerative disease such as Parkinson’s disease.     

Baicalein protects against MPP+/MPTP-induced neurotoxicity by ameliorating oxidative stress in SH-SY5Y cells and mouse model of Parkinson’s disease

Baicalein, a major bioactive flavone constituent isolated from Scutellaria baicalensis Georgi, has neuroprotective properties in several neurological disorders. Many studies suggest that oxidative stress plays a central role in the pathogenesis of Parkinson’s disease (PD). Baicalein has also been shown to have antioxidant effects. Therefore, the current study was designed to investigate whether baicalein could protect against MPP⁺/MPTP-induced neurotoxicity via suppressing oxidative stress in vitro and in vivo. In vitro, our results showed that baicalein increased cell viability in MPP⁺-treated SH-SY5Y cells. Treatment with baicalein could reversed the increased MDA and ROS levels, and the decreased GSH levels in MPP⁺-treated SH-SY5Y cells. In MPTP-treated mice, baicalein ameliorated MPTP-induced motor impairment and suppressed the MPTP-induced accumulation of iron and lipid peroxides. Besides, baicalein improved the neurotoxicity induced by MPTP as seen by a significant raise of tyrosine hydroxylase (TH) and simultaneous decrease of monoamine-oxidase-B (MAO-B). The inhibitory effect of baicalein on oxidative stress probably was partially governed by inhibition of ERK activation. In conclusion, our results suggest that baicalein could prevent MPP+/MPTP-induced neurotoxicity via suppressing oxidative stress.     

Glutamate is not involved in the MPP+-induced dopamine overflow in the striatum of freely moving C57BL/6 mice

Summary. The role of glutamate in the N-methyl-4-phenyl-dihydropyridinium (MPP⁺) toxicity has been argued in the past decade. However, the effects of glutamate efflux and NMDA antagonist on MPP⁺-induced dopamine overflow have not been documented. To clarify this, we perfused MPP⁺ through a microdialysis probe in the striatum of freely moving mature C57BL/6 mice. The 60-min perfusion of 10 and 100 μM MPP⁺ strikingly increased dopamine levels to 28- and 93-fold of the basal values, respectively. In contrast, an administration of MPP⁺ did not induce marked glutamate release: the MPP⁺-perfusion slightly increased the glutamate level at 100 μM, but not at 10 μM. The addition of 100 μM (+)-MK-801 or 200 μM (±)-AP-7 to the perfusate did not attenuate MPP⁺-induced dopamine overflow. The extent of dopamine release only depended on the amount of MPP⁺ accumulation into the cells. These results indicated that, at least in the striatum, neither glutamate release nor the NMDA antagonist, including (+)-MK-801, could regulate MPP⁺-evoked dopamine overflow. Received November 7, 2000; accepted February 28, 2001