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

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

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

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

Vasonatrin peptide,a novel protector of dopaminergic neurons against the injuries induced by n-methyl-4-phenylpyridiniums

Vasonatrin peptide (VNP), a novel manmade natriuretic peptide, is known as a cardiovascular active substance. However, its neuroeffects are largely unknown. Here, cultured dopaminergic neurons from ventral mesencephalon of mouse were exposed to N-methyl-4-phenylpyridinium (MPP⁺), and the effects of VNP on the neurotoxicity of MPP⁺ were investigated. As a result, MPP⁺ caused injuries in the dopaminergic neurons. VNP significantly reduced the cytotoxicity of MPP⁺ by increasing axon number and length of dopaminergic neurons, and by enhancing the cell viability. Also, the MPP⁺-induced depolymerization of β-Tubulin III was attenuated by the treatment of VNP. In addition, VNP significantly increased the intracellular levels of cGMP. These effects of VNP were mimicked by 8-br-cGMP (a cell-permeable analog of cGMP), whereas inhibited by HS-142-1 (the antagonist of the particulate guanylyl cyclase-coupled natriuretic peptide receptors), or KT-5823 (a cGMP-dependent protein kinase inhibitor). Taken together, VNP attenuates the neurotoxicity of MPP⁺ via guanylyl cyclase-coupled NPR/cGMP/PKG pathway, indicating that VNP might be a new effective reagent in the treatment of neuron degeneration of dopaminergic neurons in Parkinson's disease (PD).     

Environmental Estrogen-Like Chemicals and Hydroxyl Radicals Induced by MPTP in the Striatum: A Review

Oxygen free radical formation has been implicated in lesions caused by the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and iron. Although MPTP produces a parkinsonian syndrome after its conversion to 1-methyl-4-phenylpyridine (MPP⁺) by type B monoamine oxidase (MAO) in the brain, the etiology of this disease remains obscure. This review focuses on the role of an environmental neurotoxin chemically related to MPP⁺-induced free radical generation in the pathogenesis of Parkinson's disease. Environmental-like chemicals, such as para-nonylphenol or bisphenol A, significantly stimulated hydroxyl radical (OH) formation in the striatum. Allopurinol, a xanthine oxidase inhibitor, prevents para-nonylphenol and MPP⁺-induced OH generation. Tamoxifen, a synthetic nonsteroidal antiestrogen, suppressed the OH generation via dopamine efflux induced by MPP⁺. These results confirm that free radical production might make a major contribution at certain stages in the progression of the injury. Such findings may be useful in elucidating the actual mechanism of free radical formation in the pathogenesis of neurodegenerative brain disorders, including Parkinson's disease and traumatic brain injuries.     

Nitric oxide promotes in vitro organogenesis in <Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.

The effects of nitric oxide (NO) on caulogenesis, shoot organogenesis and rhizogenesis from hypocotyl explants of Linum usitatissimum were investigated. Exogenously supplied NO donors, 5 μM sodium nitroprusside (SNP), 2 μM S-nitroso-N-acetylpenicillamine (SNAP) and 2 μM 3-morpholinosydnonimine (SIN-1), significantly promoted shoot differentiation from the hypocotyl explants of L. usitatissimum excised from its in vitro raised seedlings. Potassium ferrocyanide, a structural analogue of SNP, lacking NO group, did not promote shoot organogenesis. Likewise, products of NO, \textNO₂ ^- {\text{NO}}_{2}^{ - } and \textNO₃ ^- {\text{NO}}_{3}^{ - } supplied as 5 μM NaNO₂ and 5 μM NaNO₃ did not enhance shoot differentiation. Another source of NO, a mixture of sodium nitrite (SN) provided along with ascorbic acid (AsA), also caused significant promotion in the average number of shoots per responding explant. SNP also augmented the rhizogenic response of the microshoots in terms of percentage of responding explants, number of roots per responding explant and average root length. The NO scavengers, 2-(4-carboxy-phenyl)-4, 4, 5, 5-tetramethylimideazoline-1-oxyl-3-oxide (cPTIO) or methylene blue (MB), provided along with SNP, SNAP, SIN-1 or SN + AsA, at concentrations equimolar to the optimum concentration of the donors, reversed the promotory influence, thereby, confirming the role of NO in promotion of in vitro morphogenesis. However, NO scavengers individually did not affect the observed morphogenic processes. Morphological and histological studies of hypocotyl segments cultured on BM or BM + SNP for 4, 8 and 12 days demonstrated that SNP enhanced shoot differentiation by inducing a higher number of shoot primordia, each of which develops into a single shoot.     

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

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

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.     

Involvement of nitric oxide/reactive oxygen species signaling via 8-nitro-cGMP formation in 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in PC12 cells and rat cerebellar granule neurons

To investigate the role of nitric oxide (NO)/reactive oxygen species (ROS) redox signaling in Parkinson's disease-like neurotoxicity, we used 1-methyl-4-phenylpyridinium (MPP⁺) treatment (a model of Parkinson's disease). We show that MPP⁺-induced neurotoxicity was dependent on ROS from neuronal NO synthase (nNOS) in nNOS-expressing PC12?cells (NPC12?cells) and rat cerebellar granule neurons (CGNs). Following MPP⁺ treatment, we found production of 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), a second messenger in the NO/ROS redox signaling pathway, in NPC12?cells and rat CGNs, that subsequently induced S-guanylation and activation of H-Ras. Additionally, following MPP⁺ treatment, extracellular signal-related kinase (ERK) phosphorylation was enhanced. Treatment with a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor attenuated MPP⁺-induced ERK phosphorylation and neurotoxicity. In conclusion, we demonstrate for the first time that NO/ROS redox signaling via 8-nitro-cGMP is involved in MPP⁺-induced neurotoxicity and that 8-nitro-cGMP activates H-Ras/ERK signaling. Our results indicate a novel mechanism underlying MPP⁺-induced neurotoxicity, and therefore contribute novel insights to the mechanisms underlying Parkinson's disease.     

MPP+ toxicity in rat striatal slices: Relationship between non-selective effects and free radical production

Incubations of rat striatal slices have been used to assay MPP⁺ neurotoxicity. MPP⁺, at concentrations of 1 mM or higher, caused a marked increase in hydroxyl radicals, measured as malondialdehyde (MDA) accumulation, but not in nitric oxide production. At these doses, MPP⁺ showed an effect on dopamine terminals, causing a massive dopamine decrease, and on non-neuronal glial cells, where a marked reduction in glutamine synthetase activity was detected. At lower concentrations (25 μM), the toxic effect on dopaminergic endings was maintained without increasing malondialdehyde concentrations or inhibiting glutamine synthetase activity. The effect on glutamine synthetase was prevented by the addition to the medium of 0.5% dimethyl sulfoxide, a hydroxyl-radical scavenger, but this did not protect the effect of dopamine depletion. We propose that non-selective effects of MPP⁺, at doses of 1 mM or higher, are mediated by extracellular overproduction of hydroxyl radicals. The main factor responsible for this overproduction would not be the released dopamine but rather the MPP⁺ itself, through non selective inhibition of the mitochondrial respiratory chain or through a redox cycling that can trigger oxygen radical production.     

Naringin protects the nigrostriatal dopaminergic projection through induction of GDNF in a neurotoxin model of Parkinson's disease

This study investigated the effect of naringin, a major flavonoid in grapefruit and citrus fruits, on the degeneration of the nigrostriatal dopaminergic (DA) projection in a neurotoxin model of Parkinson's disease (PD) in vivo and the potential underlying mechanisms focusing on the induction of glia-derived neurotrophic factor (GDNF), well known as an important neurotrophic factor involved in the survival of adult DA neurons. 1-Methyl-4-phenylpyridinium (MPP⁺) was unilaterally injected into the medial forebrain bundle of rat brains for a neurotoxin model of PD in the presence or absence of naringin by daily intraperitoneal injection. To ascertain whether naringin-induced GDNF contributes to neuroprotection, we further investigated the effects of intranigral injection of neutralizing antibodies against GDNF in the MPP⁺ rat model of PD. Our observations demonstrate that naringin could increase the level of GDNF in DA neurons, contributing to neuroprotection in the MPP⁺ rat model of PD, with activation of mammalian target of rapamycin complex 1. Moreover, naringin could attenuate the level of tumor necrosis factor-α in microglia increased by MPP⁺-induced neurotoxicity in the substantia nigra. These results indicate that naringin could impart to DA neurons the important ability to produce GDNF as a therapeutic agent against PD with anti-inflammatory effects, suggesting that naringin is a beneficial natural product for the prevention of DA degeneration in the adult brain.     

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

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

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

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

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

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

Unexpected Neuronal Protection of SU5416 against 1-Methyl-4-Phenylpyridinium Ion-Induced Toxicity via Inhibiting Neuronal Nitric Oxide Synthase

SU5416 was originally designed as a potent and selective inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) for cancer therapy. In this study, we have found for the first time that SU5416 unexpectedly prevented 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced neuronal apoptosis in cerebellar granule neurons, and decreased 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced loss of dopaminergic neurons and impairment of swimming behavior in zebrafish in a concentration-dependent manner. However, VEGFR-2 kinase inhibitor II, another specific VEGFR-2 inhibitor, failed to reverse neurotoxicity at the concentration exhibiting anti-angiogenic activity, strongly suggesting that the neuroprotective effect of SU5416 is independent from its anti-angiogenic action. SU5416 potently reversed MPP⁺-increased intracellular nitric oxide level with an efficacy similar to 7-nitroindazole, a specific neuronal nitric oxide synthase (nNOS) inhibitor. Western blotting analysis showed that SU5416 reduced the elevation of nNOS protein expression induced by MPP⁺. Furthermore, SU5416 directly inhibited the enzyme activity of rat cerebellum nNOS with an IC₅₀ value of 22.7 µM. In addition, knock-down of nNOS expression using short hairpin RNA (shRNA) abolished the neuroprotective effects of SU5416 against MPP⁺-induced neuronal loss. Our results strongly demonstrate that SU5416 might exert its unexpected neuroprotective effects by concurrently reducing nNOS protein expression and directly inhibiting nNOS enzyme activity. In view of the capability of SU5416 to cross the blood-brain barrier and the safety for human use, our findings further indicate that SU5416 might be a novel drug candidate for neurodegenerative disorders, particularly those associated with NO-mediated neurotoxicity.     

Involvement of tubulin in MPP+ neurotoxicity on NGF-differentiated PC12 cells.

In vivo, the neurotoxin MPTP is oxidated to MPP⁺, which is toxic to dopaminergic neurons. In this paper, we have used MPP⁺ as a tool to evoke neurotoxicity in the PC12 cell line and investigate the intracellular events that are involved. A cytotoxicity test, performed on undifferentiated and NGF-differentiated PC12 cells, showed that MPP⁺ is much more toxic on differentiated cells and indicated the suitable range of concentrations for studying the starting events evoked by the neurotoxin. By indirect immunofluorescence we have shown that the localisation of α - and β -tubulin in NGF-differentiated cells was modified by a 24 h treatment with 15 μmol/l MPP⁺. A biochemical analysis was performed on cell extracts and the results showed that MPP⁺ treatment induced an increase in α -tubulin levels and a decrease in β -tubulin levels. These results suggest the involvement of the two microtubule proteins in MPP⁺ neurotoxicity on NGF-differentiated PC12 cells.     

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.     

The Peptidyl-prolyl Isomerase Pin1 Up-regulation and Proapoptotic Function in Dopaminergic Neurons: RELEVANCE TO THE PATHOGENESIS OF PARKINSON DISEASE*

Parkinson disease (PD) is a chronic neurodegenerative disease characterized by a slow and progressive degeneration of dopaminergic neurons in substantia nigra. The pathophysiological mechanisms underlying PD remain unclear. Pin1, a major peptidyl-prolyl isomerase, has recently been associated with certain diseases. Notably, Ryo et al. (Ryo, A., Togo, T., Nakai, T., Hirai, A., Nishi, M., Yamaguchi, A., Suzuki, K., Hirayasu, Y., Kobayashi, H., Perrem, K., Liou, Y. C., and Aoki, I. (2006) J. Biol. Chem. 281, 4117–4125) implicated Pin1 in PD pathology. Therefore, we sought to systematically characterize the role of Pin1 in PD using cell culture and animal models. To our surprise we observed a dramatic up-regulation of Pin1 mRNA and protein levels in dopaminergic MN9D neuronal cells treated with the parkinsonian toxicant 1-methyl-4-phenylpyridinium (MPP⁺) as well as in the substantia nigra of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. Notably, a marked expression of Pin1 was also observed in the substantia nigra of human PD brains along with a high co-localization of Pin1 within dopaminergic neurons. In functional studies, siRNA-mediated knockdown of Pin1 almost completely prevented MPP⁺-induced caspase-3 activation and DNA fragmentation, indicating that Pin1 plays a proapoptotic role. Interestingly, multiple pharmacological Pin1 inhibitors, including juglone, attenuated MPP⁺-induced Pin1 up-regulation, α-synuclein aggregation, caspase-3 activation, and cell death. Furthermore, juglone treatment in the MPTP mouse model of PD suppressed Pin1 levels and improved locomotor deficits, dopamine depletion, and nigral dopaminergic neuronal loss. Collectively, our findings demonstrate for the first time that Pin1 is up-regulated in PD and has a pathophysiological role in the nigrostriatal dopaminergic system and suggest that modulation of Pin1 levels may be a useful translational therapeutic strategy in PD.     

Protective effect of histidine on para-nonylphenol-enhanced hydroxyl free radical generation induced by 1-methyl-4-phenylpyridinium ion (MPP+) in rat striatum

The present study examined the antioxidant effect of histidine, a singlet oxygen (¹O₂) scavenger, on para-nonylphenol (an environmental estrogen-like chemical)-enhanced hydroxyl radical (·OH) generation induced by 1-methyl-4-phenylpyridinium ion (MPP⁺) in extracellular fluid of rat striatum. Rats were anesthetized, and sodium salicylate in Ringer’s solution (0.5 nmol/μl/min) was infused through a microdialysis probe to detect the generation of ·OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. Introduction of para-nonylphenol (10 μM) significantly enhanced MPP⁺-induced ·OH generation. Histidine (25 mM) decreased the para-nonylphenol-enhanced ·OH formation. Although the level of MPP⁺-induced ·OH formation trapped as DHBA after para-nonylphenol treatment increased, para-nonylphenol failed to increase either the level of dopamine and DHBA formation in the reserpinized animals. These results indicate that para-nonylphenol and MPP⁺-enhanced ·OH generation was based on ¹O₂ production, and histidine may have a preventive effect on para-nonylphenol and MPP⁺-induced ·OH generation in rat striatum.     

Lithospermic acid attenuates 1-methyl-4-phenylpyridine-induced neurotoxicity by blocking neuronal apoptotic and neuroinflammatory pathways

Background

Parkinson’s disease is the second most common neurodegenerative disorders after Alzheimer’s disease. The main cause of the disease is the massive degeneration of dopaminergic neurons in the substantia nigra. Neuronal apoptosis and neuroinflammation are thought to be the key contributors to the neuronal degeneration.

Results

Both CATH.a cells and ICR mice were treated with 1-methyl-4-phenylpyridin (MPP⁺) to induce neurotoxicity in vitro and in vivo. Western blotting and immunohistochemistry were also used to analyse neurotoxicity, neuroinflammation and aberrant neurogenesis in vivo. The experiment in CATH.a cells showed that the treatment of MPP⁺ impaired intake of cell membrane and activated caspase system, suggesting that the neurotoxic mechanisms of MPP⁺ might include both necrosis and apoptosis. Pretreatment of lithospermic acid might prevent these toxicities. Lithospermic acid possesses specific inhibitory effect on caspase 3. In mitochondria, MPP⁺ caused mitochondrial depolarization and induced endoplasmic reticulum stress via increasing expression of chaperone protein, GRP-78. All the effects mentioned above were reduced by lithospermic acid. In animal model, the immunohistochemistry of mice brain sections revealed that MPP⁺ decreased the amount of dopaminergic neurons, enhanced microglia activation, promoted astrogliosis in both substantia nigra and hippocampus, and MPP⁺ provoked the aberrant neurogenesis in hippocampus. Lithospermic acid significantly attenuates all of these effects induced by MPP⁺.

Conclusions

Lithospermic acid is a potential candidate drug for the novel therapeutic intervention on Parkinson’s disease.