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Mitochondrial dysfunction and subsequent energy failure is a contributing factor to degeneration of the substantia nigra pars compacta associated with Parkinson's disease (PD). In this study, we investigate molecular events triggered by cell exposure to the mitochondrial toxin 1-methyl-4-phenylpyridine (MPP+) using whole genome-expression microarray, Western Blot and metabolic studies. The data show that MPP+ (500 μM) obstructs mitochondrial respiration/oxidative phosphorylation (OXPHOS) in mouse neuroblastoma Neuro-2a cells, juxtaposing accelerated glucose consumption and production of lactic acid. While additional glucose concentrations restored viability in the presence of MPP+ (500 μM), the loss of OXPHOS was sustained, suggesting that compensatory anaerobic metabolic systems were fulfilling required energy needs. Under these conditions, MPP+ initiated significant changes to the transcription of 439 genes of which 287 DAVID IDs were identified and subsequent functional annotation clusters identified. Prominent changes were as follows; MPP+ initiated loss of mRNA for mitochondrial encoded 3-hydroxybutyratedehydrogenase, type 2(Bdh2), tv1, NADH dehydrogenase 4,5 genes, cytochrome b and NADH dehydrogenase (ubiquinone) flavoprotein 3, concomitant to rise in a mitochondrial fission gene; ganglioside-induced differentiation-associated-protein 1 (GDAP1). The negative changes to OXPHOS components were accompanied by protective forces within the mitochondria espousing elevated ratio of anti/pro-apoptotic processes. These included a loss of apoptotic Bcl-2/adenovirus E1B 19-kDa-interacting protein (BNIP3) and family with sequence similarity 162, member A (FAM162a) and rise of heat shock protein 1 and Lon peptidase 1. There were no changes indicative of free radical damage (e.g. SOD, GSH-Px), rather MPP+ initiated significant elevation in G protein signaling components (which trigger catabolic processes) and anaerobic metabolic systems involving carboxylic acid/transamination reactions (e.g. glutamate oxaloacetate transaminase 1 (GOT1), glutamic pyruvate-alanine transaminase 2 (GPT2), cystathionase and redox proteins such as cytochrome b5 reductase 1 and ferredoxin reductase. Counter-intuitively, the data show reduction of mRNA in glycolytic processes [DAVID enrichment score 9.96 p value 1.90E-19], some corroborated by Western Blot, bringing in to question the sources of lactate observed in the presence of MPP+. Examining this aspect, the data show that diverse carboxylic acids (succinate, oxaloacetate and a-ketoglutarate) are capable of contributing to the lactate pool in addition to phosph(enolpyruvate) or pyruvate in the absence of glucose by this cell line. In conclusion, these findings show that MPP+ negatively affects the transcriptome involved with complex I, but initiated an elevation of G protein signaling and anaerobic metabolic systems involved with nitrogen/carboxylic acid metabolism. Future research will be required to elucidate the survival pathways that drive anaerobic substrate level phosphorylation, and define functional ramification to the loss of mitochondrial FAM162a and BNIP3 proteins., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Environmental or occupational exposure to high levels of manganese (Mn) can lead to manganism, a symptomatic neuro-degenerative disorder similar to idiopathic Parkinson's disease. The underlying mechanism of Mn neurotoxicity remains unclear. In this study, we evaluate the primary toxicological events associated with MnCl(2) toxicity in rat PC12 cells using whole genome cDNA microarray, RT-PCR, Western blot and functional studies. The results show that a sub-lethal dose range (38-300 μM MnCl(2)) initiated slight metabolic stress evidenced by heightened glycolytic rate and induction of enolase/aldolase - gene expression. The largest shift observed in the transcriptome was MnCl(2) induction of heme-oxygenase 1 (HO-1) [7.7 fold, p<0.001], which was further corroborated by RT-PCR and Western blot studies. Concentrations in excess of 300 μM corresponded to dose dependent loss of cell viability which was associated with enhanced production of H(2)O(2) concomitant to elevation of gene expression for diverse antioxidant enzymes; biliverdin reductase, arsenite inducible RNA associated protein, dithiolethione-inducible gene-1 (DIG-1) and thioredoxin reductase 1. Moreover, Mn initiated significant reduction of gene expression of mitochondrial glutaryl-coenzyme A dehydrogenase (GCDH), an enzyme involved with glutaric acidemia, oxidative stress, lipid peroxidation and striatal degeneration observed in association with severe dystonic-dyskinetic movement disorder. Future research will be required to elucidate a defined role for HO-1 and GCDH in Mn toxicity., (Published by Elsevier B.V.)

Phencyclidine (PCP) is a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist and exposing the developing brain to PCP has been shown to cause deficits in neurobehavioral functions. In the present study we tested the effects of PCP, as an NMDA receptor inhibitor, on the neuronal differentiation and biogenic amines levels including norepinephrine (NE), epinephrine, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5-HIAA) in the rat pheochromocytoma (PC12) cells. After PC12 cells were differentiated with nerve growth factor (NGF) in the presence of PCP, NMDA binding kinetics, biogenic amines analysis and NMDA receptor protein expression assay were conducted. The results showed that NMDA receptor binding activities were significantly increased after differentiated with NGF in PC12 cells. B(max) values were increased in differentiated cells by four-folds, whereas K(d) values were not changed. All of biogenic amines were significantly increased in differentiated cells. On the other hand, PCP at 50 and 100 microM inhibited neuronal differentiation in a dose-dependent manner in NGF-stimulated PC12 cells without affecting cell viability. PCP treatment during differentiation significantly reduced NMDA binding activity and biogenic amine levels. Western blotting analysis revealed that NMDA receptor protein expression was significantly higher in NGF-differentiated cells and PCP treatment decreased the expression of NMDA receptor proteins. These results indicate that NMDA receptor functions and monoaminergic nervous systems are significantly stimulated during NGF-induced differentiation. PCP suppresses neuronal outgrowth and hampers neuronal functions possibly by inhibiting NMDA receptor functions and biogenic amine production, implying the suppressive effects of PCP exposure on neuronal developments.

Excessive methylation has been proposed to be involved in the pathogenesis of Parkinson's disease (PD), via mechanisms that involve phospholipid methylation. Meanwhile, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was found to stimulate phospholipid methylation via the oxidized metabolite, 1-methyl-4-phenyl-pyridinium (MPP+), in the rat brain and liver tissues. In the present study, we investigated the effect of MPP+ on phosphatidylethanolamine N-methyltransferases (PENMT) and the potential role of this pathway in MPP(+)-induced neurotoxicity using PC12 cells. The results obtained indicate that MPP+ stimulated phosphatidylethanolamine (PTE) methylation to phosphatidylcholine (PTC) and correspondingly increased the formation of lysophosphatidylcholine (lyso-PTC). Moreover, the addition of S-adenosylmethionine (SAM) to the cell culture medium increases MPP(+)-induced cytotoxicity. The incubation of 1mM MPP+ and various concentrations of SAM (0-4 mM) decreased the viability of PC12 cells from 80% with MPP+ alone to 38% viability with 4 mM SAM for 4 days incubation. The data also revealed that the addition of S-adenosylhomocysteine (SAH), a methylation inhibitor, offered significant protection against MPP(+)-induced cytotoxicity, indicating that methylation plays a role in MPP(+)-induced cytotoxicity. Interestingly, lyso-PTC showed similar actions to MPP+ in causing many cytotoxic changes with at least 10 times higher potency. Lyso-PTC induced dopamine release and inhibited dopamine uptake in PC12 cells. Lyso-PTC also caused the inhibition of mitochondrial potential and increased the formation of reactive oxygen species in PC12 cells. These results indicate that phospholipid methylation pathway might be involved in MPP+ neurotoxicity and lyso-PTC might play a role in MPP(+)-induced neurotoxicity.

Long-term treatment of levodopa for Parkinson's disease (PD) patients is known to elevate homocysteine level in their plasma. The present study was designed to examine the possible neurotoxic effects of the increased homocysteine level on the dopaminergic system. Homocysteine was administered into Sprague-Dawley male rats intracerebroventricularly or C57BL/6 mice intraperitoneally. Following homocysteine injection the locomotor activities, the levels of dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and immunohistochemistry of dopaminergic neurons were examined. The results obtained indicate that homocysteine administration (1 or 2 micromol, i.c.v.) into the rat brains for 5 days significantly decreased the locomotor activities and dopamine as well as its metabolites, DOPAC and HVA, in the rat striatal regions. Two different doses of homocysteine (50 and 100mg/100g, i.p. daily) were administered into mice for 36 days to evaluate the effect of systemic treatment of homocysteine on the dopaminergic neurons of the brain. The intraperitoneal injections of two doses of homocysteine significantly increased homocysteine levels in the striatal regions of mouse brains by 21.5 and 39.2%, while reducing dopamine turnover rates in the striatal regions by decreasing (DOPAC+HVA)/DA, 23.7 and 51.6%, respectively. Accordingly, homocysteine decreased locomotor activities significantly by decreasing movement time by 29 and 38%, total distance by 32 and 42%, and numbers of movement by 28 and 41%, respectively. Moreover, homocysteine decreased tyrosine hydroxylase immunoreactivity in substantia nigra of mouse brain. The data obtained indicate that the potential of homocysteine to be toxic to the dopaminergic system. Consequently, long-term levodopa therapy for PD may accelerate the progression of PD, at least in part by elevated homocysteine.

Epidemiological studies consistently report an inverse correlation between cigarette smoking and associated risk for Parkinson's disease (PD). The degeneration of dopaminergic neurons may involve the toxic metabolic products of glial cell monoamine oxidase (MAO) and inducible nitric oxide synthase (iNOS). This study evaluates the direct protective effects of cigarette smoke (CS) against potential neurotoxic products of MAO, such as 1-methyl-4-phenylpyridinium (MPP+), 6-hydroxydopamine (6-OHDA) and hydrogen peroxide (H2O2) in brain neuroblastoma. Moreover, the effects of CS were also evaluated on endotoxin/cytokine activated glioma iNOS protein expression and MAO enzyme activity. Cigarette smoke condensates (CSCs) were acquired from Marlboro 20 Class A and Kentucky 2R4F reference research (2R4F) cigarettes. The CSCs did not protect against 6-OHDA or H2O2 toxicity in neuroblastoma, and exhibited a very mild protective effect [approximately 10%] against MPP+. Neither CSC demonstrated antioxidant capability, but conversely contained high concentration of NO2-. Paradoxically, in glioma cells, iNOS protein expression and endogenous enzymatic NO2- production were significantly blocked by both CSCs. Both CSCs also inhibited glioma MAO-A and MAO-B [1.4.3.4]. Kinetic analysis indicated that 2R4F-CSC displayed competitive inhibition and the Marlboro-CSC exerted potent competitive and non-competitive inhibition. In conclusion, these data suggest that cigarette smoke does not appear to directly protect against the toxicity of the selected neurotoxins. In contrast, CS exerts pronounced effects on glia, whereby its presence can simultaneously attenuate cytokine induction of iNOS and MAO.

Lysophosphatidylcholine (lyso-PTC), a secondary product of S-adenosylmethionine (SAM)-dependent phosphatidylethanolamine (PTE) methylation, is a potent cytotoxin and might be involved in the pathogenesis of Parkinson's disease (PD). Our previous studies showed that the injection of SAM into the brain caused PD-like changes in rodents. Moreover, 1-methyl-4-phenylpyridinium (MPP+), a Parkinsonism-inducing agent, increased lyso-PTC formation via the stimulation of PTE methylation pathway. These results indicate a possible role of lyso-PTC in the PD-like changes seen following the injection of SAM or MPP+. In the present study, lyso-PTC was injected into the lateral ventricle of rats and locomotor activities and the biogenic amine levels were measured to evaluate the effects of lyso-PTC on the dopaminergic system. Quinacrine, a phospholipase A2 (PLA2) inhibitor, was employed to determine its protective effect on SAM-induced PD-like changes by the inhibition of lyso-PTC formation. The results showed that 1 h after the injection, 0.4 and 0.8 micromol of lyso-PTC increased striatal dopamine (DA) by 20 and 24%, decreased 3,4-dihydroxyphenylacetic acid (DOPAC) by 37 and 45% and decreased homovanilic acid (HVA) by 24 and 13%, respectively. Consequently, dopamine turnover rate, (DOPAC + HVA)/DA, was significantly reduced by 44 and 48% in the rat striatum. Meanwhile, the administration of 0.4 or 0.8 micromol of lyso-PTC decreased movement time by 52 and 63%, total distance by 44 and 48% and the number of movements by 43 and 64%, respectively. Quinacrine attenuated SAM-induced hypokinesia without affecting SAM metabolism prior to its action on rat brain. The results obtained indicate that the hypokinesia observed following the administration of lyso-PTC might be related to the decline in DA turnover in the striatum in response to lyso-PTC exposure. The present study suggests that inhibitory effects of lyso-PTC on dopaminergic neurotransmission is one of the contributing factors in SAM and MPP+-induced PD-like changes.

1-Methyl-4-phenylpyridinium (MPP+) is a mitochondrial Complex I inhibitor and is frequently used to investigate the pathological degeneration of neurons associated with Parkinson's disease (PD). In vitro, extracellular concentration of glucose is one of the most critical factors in establishing the vulnerability of neurons to MPP+ toxicity. While glucose is the primary energy fuel for the brain, central nervous system (CNS) neurons can also take up and utilize other metabolic intermediates for energy. In this study, we compared various monosaccharides, disaccharides, nutritive/non-nutritive sugar alcohols, glycolytic and gluconeogenic metabolic intermediates for their cytoprotection against MPP+ in murine brain neuroblastoma cells. Several monosaccharides were effective against MMP+ (500 microM) including glucose, fructose and mannose, which restored cell viability to 109 +/- 5%, 70 +/- 5%, 99 +/- 3% of live controls, respectively. Slight protective effects were observed in the presence of 3-phosphoglyceric acid and glucose-6-phosphate; however, no protective effects were exhibited by galactose, sucrose, sorbitol, mannitol, glycerol or various gluconeogenic and ketogenic amino acids. On the other hand, fructose 1,6 bisphosphate and gluconeogenic energy intermediates [pyruvic acid, malic acid and phospho(enol)pyruvate (PEP)] were neuroprotective against MPP+. The gluconeogenic intermediates elevated intracellular levels of ATP and reduced propidium iodide (PI) nucleic acid staining to live controls, but did not alter the MPP(+)-induced loss of mitochondrial O2 consumption. These data indicate that malic acid, pyruvic acid and PEP contribute to anaerobic substrate level phosphorylation. The use of hydrazine sulfate to impede gluconeogenesis through PEP carboxykinase (PEPCK) inhibition heightened the protective effects of energy substrates possibly due to attenuated ATP demands from pyruvate carboxylase (PC) activity and pyruvate mitochondrial transport. It was concluded from these studies that several metabolic intermediates are effective in fueling anaerobic glycolysis during mitochondrial inhibition by MPP+.

The neuropathology associated with Parkinson's disease within and around the substantia nigra is thought to involve excessive production of free radicals, dopamine autoxidation, defects in the expression of glutathione peroxidase, attenuated levels of reduced glutathione, altered calcium homeostasis, excitotoxicity and genetic defects in mitochondrial complex I activity. While the neurotoxic mechanisms are vastly different for excitotoxins and N-methyl-4-phenylpyridinium ion (MPP+), both are thought to involve free radical production, compromised mitochondrial activity and excessive lipid peroxidation. In the present study, several dietary antioxidant compounds, monoamine oxidase inhibitors and ergogenic compounds were examined for protective action against neurotoxicity induced by L-glutamate (15 mM) or MPP+-HCl (5 mM) in a plastic adhering variant of murine pheochromocytoma cells. The results show no significant protective effects exhibited by azulene, (+)-catechin, curcrumin, (-)-epigallocatechin gallate, green tea, morin, pygnogenol, silymarin, clove oil, garlic oil or rosemary, extract. Compounds, which were effective in providing protection against L-glutamate-induced cell death, were coenzyme Q-0, coenzyme Q-10, L-deprenyl and N-acetyl-L-cysteine. Compounds, which provided protection against MPP+-HCl toxicity, were allopurinol, coenzyme Q-10, L-deprenyl, N-acetyl-L-cysteine and sesame oil. In both models, significant protection was achieved in the presence of coenzyme Q-10, L-deprenyl and N-acetyl-L-cysteine. These results indicate that the mechanism of cell death in both of these toxicity models is most likely not related to the destructive effects of free radicals.

The effects of triadimefon (TDF) were examined in male Sprague-Dawley rats. In this study, the acute administration of TDF (100 mg/kg) was found to significantly increase locomotor activity and induce stereotyped behavior. Acute administration of TDF was also found to significantly increase dopamine (DA) and homovanillic acid (HVA) levels while the dihydroxyphenylacetic acid (DOPAC) level remained unchanged in both the nucleus accumbens (NA) and striatal (ST) tissues when compared to control. Furthermore, DOPAC:DA ratios were significantly reduced in both brain regions suggesting an increase in DA turn overrate. On the other hand, in animals receiving repeated TDF administration, only the HVA level was significantly increased in both the ST and NA. TDF neither competed for binding to D2, D3 or D4 DA receptors nor altered the Kd or the Bmax of [3H] SCH 23390 and [3H] spiperone recognition sites associated with striatal D1 and D2 receptors, respectively. Meanwhile, TDF competed with [3H] GBR 12935 for binding to DA transporter sites with strong affinity, but repeated treatment with TDF had no sustained or cumulative effect on the DA transporter system. These results clearly show that acute TDF-induced behavioral effects may not be via binding to DA receptors, but through the interaction with DA transporter binding sites. Also, TDF does not appear to produce cumulative effects in the parameters evaluated.