MPP+-Induced Changes in Cellular Impedance as a Measure for Organic Cation Transporter (SLC22A1-3) Activity and Inhibition.

The organic cation transporters OCT1-3 (SLC22A1-3) facilitate the transport of cationic endo- and xenobiotics and are important mediators of drug distribution and elimination. Their polyspecific nature makes OCTs highly susceptible to drug-drug interactions (DDIs). Currently, screening of OCT inhibitors depends on uptake assays that require labeled substrates to detect transport activity. However, these uptake assays have several limitations. Hence, there is a need to develop novel assays to study OCT activity in a physiological relevant environment without the need to label the substrate. Here, a label-free impedance-based transport assay is established that detects OCT-mediated transport activity and inhibition utilizing the neurotoxin MPP+. Uptake of MPP+ by OCTs induced concentration-dependent changes in cellular impedance that were inhibited by decynium-22, corticosterone, and Tyrosine Kinase inhibitors. OCT-mediated MPP+ transport activity and inhibition were quantified on both OCT1-3 overexpressing cells and HeLa cells endogenously expressing OCT3. Moreover, the method presented here is a valuable tool to identify novel inhibitors and potential DDI partners for MPP+ transporting solute carrier proteins (SLCs) in general.

Long non-coding RNA myocardial infarction-associated transcript promotes 1-Methyl-4-phenylpyridinium ion-induced neuronal inflammation and oxidative stress in Parkinson's disease through regulating microRNA-221-3p/ transforming growth factor /nuclear factor E2-related factor 2 axis.

This study attempted to evaluate the role of long non-coding RNA myocardial infarction-associated transcript (LncRNA MIAT) in Parkinson's disease (PD). The mouse model was established through intraperitoneal injection with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and in vitro model was induced by administrating cell with 1-Methyl-4-phenylpyridinium ion (MPP+). Rotarod test was conducted to evaluate the motor coordination of PD mice. In order to investigate the roles of LncRNA MIAT in neuronal inflammation and oxidative stress, MIAT shRNA (shMIAT) was transfected into MPP+-treated cells, and cell viability, cell apoptosis and oxidative stress response were evaluated. To evaluate the interactions between LncRNA MIAT and microRNA-221-3p (miR-221-3p)/TGF-ß1/Nrf2, miR-221-3p mimic, miR-221-3p inhibitor, NC-inhibitor and transforming growth factor-ß1 shRNA (shTGF-ß1) were subsequently transfected into MPP+-treated cells. Dual-luciferase reporter gene assays were performed to determine the interaction of miR-221-3p with MIAT or TGFB receptor 1 (TGFBR1). The expressions of LncRNA MIAT, miR-221-3p, TGFBR1, transforming growth factor (TGF-ß1) and nuclear factor E2-related factor 2 (Nrf2) were measured by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and immunoblotting. As a result, LncRNA MIAT was abundantly expressed in PD mice and cells, while downregulation of LncRNA MIAT promoted the survival of neurons, inhibited apoptosis and oxidative stress in neurons. LncRNA MIAT bound to miR-221-3p, and there was a negative correlation between miR-221-3p and LncRNA MIAT expression. In addition, miR-221-3p targeted TGFBR1 and suppressed TGF-ß1 expression but increased Nrf2 expression. LncRNA MIAT promoted MPP+-induced neuronal injury in PD via regulating TGF-ß1/Nrf2 axis through binding with miR-221-3p.

Thioredoxin-1 mediates neuroprotection of Schisanhenol against MPP+-induced apoptosis via suppression of ASK1-P38-NF-κB pathway in SH-SY5Y cells.

Oxidative stress-induced dopaminergic neuronal loss and apoptosis play a crucial role in the pathogenesis of Parkinson's disease (PD), and as a vital antioxidant protein, thioredoxin (Trx) exerts neuroprotection against PD. In this study, we investigated the effect of Schisanhenol (Sal), an active component from a traditional Chinese herb Schisandra rubriflora (Franch.), on MPP+-induced apoptosis and its association with thioredoxin-1 (Trx1) in SH-SY5Y cells. The protein levels of Trx1 and apoptosis-related proteins were detected by Western blot, the expression of Trx1 mRNA by real time qPCR, and apoptosis was detected by fluorescence microscopy and flow cytometry. Pretreatment with Sal (1 µM, 10 µM, and 50 µM) dose-dependently ameliorated MPP+-induced neuronal injury, confirmed by the improvement of the viability and morphological changes. Sal decreased the apoptosis rate of cells, suppressed the production of DNA ladder and sub-G1 peak, inhibited the Caspase-3 activity and the expression of apoptosis-related proteins. Sal enhanced the expression of Trx1 both in the protein and mRNA levels. However, the Trx1 inhibitor PX-12 suppressed the protective effects of Sal. In addition, Sal inhibited NF-κB translocation and activation. These results suggest that Sal has a protective effect against MPP+-induced apoptosis in SH-SY5Y cells via up-regulation of Trx1 expression and suppression of ASK1-P38-NF-κB pathway.

PEP-1-GLRX1 Reduces Dopaminergic Neuronal Cell Loss by Modulating MAPK and Apoptosis Signaling in Parkinson's Disease.

Parkinson's disease (PD) is characterized mainly by the loss of dopaminergic neurons in the substantia nigra (SN) mediated via oxidative stress. Although glutaredoxin-1 (GLRX1) is known as one of the antioxidants involved in cell survival, the effects of GLRX1 on PD are still unclear. In this study, we investigated whether cell-permeable PEP-1-GLRX1 inhibits dopaminergic neuronal cell death induced by 1-methyl-4-phenylpyridinium (MPP+) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We showed that PEP-1-GLRX1 protects cell death and DNA damage in MPP+-exposed SH-SY5Y cells via the inhibition of MAPK, Akt, and NF-κB activation and the regulation of apoptosis-related protein expression. Furthermore, we found that PEP-1-GLRX1 was delivered to the SN via the blood-brain barrier (BBB) and reduced the loss of dopaminergic neurons in the MPTP-induced PD model. These results indicate that PEP-1-GLRX1 markedly inhibited the loss of dopaminergic neurons in MPP+- and MPTP-induced cytotoxicity, suggesting that this fusion protein may represent a novel therapeutic agent against PD.

Protective effects of irigenin against 1-methyl-4-phenylpyridinium-induced neurotoxicity through regulating the Keap1/Nrf2 pathway.

The rhizome of Belamcanda chinensis possesses antiinflammatory and antioxidant activities. However, the effect of irigenin, isolated from the rhizome of B. chinensis, on 1-methyl-4-phenylpyridinium (MPP+ )-induced neurotoxicity is unknown. MTT assay showed that MPP+ exposure dose dependently inhibited the viability of mouse microglia BV-2 cells, whereas irigenin suppressed MPP+ -induced viability reduction. The production of nitric oxide, prostaglandin E2, tumor necrosis factor-α and interleukin-6 were increased by MPP+ treatment, which were abolished by irigenin treatment. Irigenin-attenuated MPP+ -induced increase of malondialdehyde content and activities of superoxide dismutase, catalase and glutathione peroxidase in BV-2 cells. Irigenin treatment also repressed apoptosis, caspase-3/7 activity and Cytochrome C expression in MPP+ -challenged BV-2 cells. Interestingly, irigenin activated the Keap1/Nrf2 pathway in MPP+ -induced BV-2 cells. Nrf2 knockdown attenuated the effects of irigenin on MPP+ -induced viability reduction, inflammation, oxidative stress and apoptosis in BV-2 cells. In conclusion, irigenin alleviated MPP+ -induced neurotoxicity in BV-2 cells through regulating the Keap1/Nrf2 pathway.

Hydrogen sulfide alleviates oxidative stress injury and reduces apoptosis induced by MPP+ in Parkinson's disease cell model.

Hydrogen sulfide (H2S), an endogenously produced gas, is a cardioprotective agent against neurotoxin-induced neurodegeneration in Parkinson's disease (PD). However, the roles of H2S in 1-methyl-4-phenylpyridinium ion (MPP+)-treated SH-SY5Y cells with the involvement of reactive oxygen species-nitric oxide (ROS-NO) signaling pathway in PD remain unclear. For this study, a MPP+-treated SH-SY5Y cell model was established to explore the regulatory role of H2S in oxidative stress injury and cell apoptosis. With the cell viability, apoptosis, cytotoxicity, levels of reactive oxygen species (ROS) and nitric oxide (NO), mitochondrial transmembrane potential (Δψm), contents of oxidative stress injury-related markers (glutathione, superoxide dismutase, malondialdehyde), levels of apoptosis-related proteins (Caspase 3, Bax, Bcl-2) and inducible nitric oxide synthase (iNOS) determined, this study demonstrated that NaHS (an H2S donor) treatment could alleviated the reduction of cell viability and cytotoxicity, cell apoptosis, Δψm loss, contents of ROS and NO, and oxidative stress injury induced by MPP+. The present study showed that H2S may protect SH-SY5Y cells from MPP+-induced injury in PD cell model via the inhibition of ROS-NO signaling pathway and provide insight into the potential of H2S for PD therapy.

2,3,5,4'-Tetrahydroxystilbene-2-O-ß-d-glucoside attenuates MPP+/MPTP-induced neurotoxicity in vitro and in vivo by restoring the BDNF-TrkB and FGF2-Akt signaling axis and inhibition of apoptosis.

The major bioactive ingredient THSG of Polygonum multiflorum is well established for its anti-oxidation, anti-aging and anti-inflammation properties. Increasing evidence supports the capacity of THSG to ameliorate the biochemistry of neurotrophins and their downstream signaling axis in mouse models to attenuate neurodegenerative diseases such as Alzheimer's and Parkinson's disease. In this study, the neuroprotective effects of THSG were studied in vitro and in vivo. In cultured mesencephalic dopamine neurons and SH-SY5Y cell line, it was found that THSG protected the integrity of the cell body and neurite branching from MPP+-induced toxicity by restoring the expression of FGF2 and BDNF and their downstream signaling pathways to inhibit apoptosis and promote cell survival. The inhibition of Akt signaling by LY294002 or TrkB activity by K252a eliminated the neuroprotective effects of THSG. In the MPTP-induced mouse models of Parkinson's disease, THSG ameliorated the animal behaviors against MPTP-induced neurotoxicity, which was demonstrated by the pole test and the tail suspension test. Biochemical and immunohistochemical analysis verified the THSG-mediated restoration of the FGF2-Akt and BDNF-TrkB signaling axis in the substantia nigra and corpus striatum and the recovery of dopaminergic neurons. These results establish the neuroprotective effects of THSG in vitro and in vivo and unravel the underlying mechanism against toxin-induced neural atrophy, providing a new avenue for the use and pharmacological research of edible medicine for anti-neurodegenerative diseases.

Oridonin, A natural diterpenoid, protected NGF-differentiated PC12 cells against MPP+- and kainic acid-induced injury.

Oridonin (ORI) is a natural diterpenoid presented in some medicinal plants. The effects of pre-treatments from ORI against MPP+- or kainic acid (KA)-induced damage in nerve growth factor (NGF)-differentiated PC12 cells were investigated. Results showed that pre-treatments of ORI at 0.25-2 µM enhanced the viability and plasma membrane integrity of NGF-differentiated PC12 cells. MPP+ or KA exposure down-regulated Bcl-2 mRNA expression, up-regulated Bax mRNA expression, increased caspase-3 activity and decreased Na+-K+ ATPase activity. ORI pre-treatments at test concentrations reversed these changes. ORI pre-treatments decreased reactive oxygen species production, raised glutathione level, and increased glutathione peroxidase, glutathione reductase and catalase activities in MPP+ or KA treated cells. ORI pre-treatments lowered tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6 and prostaglandin E2 levels in MPP+ or KA treated cells. ORI also diminished MPP+ or KA induced increase in nuclear factor-κB binding activity. MPP+ exposure suppressed tyrosine hydroxylase (TH) mRNA expression and decreased dopamine content. KA exposure reduced glutamine synthetase (GS) mRNA expression, raised glutamate level and lowered glutamine level. ORI pre-treatments at 0.5-2 µM up-regulated mRNA expression of TH and GS, restored DA and glutamine content. These findings suggested that oridonin was a potent neuro-protective agent against Parkinson's disease and seizure.

Regulation of the Nrf2 Pathway by Glycogen Synthase Kinase-3ß in MPP⁺-Induced Cell Damage.

Recently, nuclear translocation and stability of nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) have gained increasing attention in the prevention of oxidative stress. The present study was aimed to evaluate the regulatory role of glycogen synthase kinase-3ß (GSK-3ß) inhibition by tideglusib through the Nrf2 pathway in a cellular damage model. Gene silencing (siRNA-mediated) was performed to examine the responses of Nrf2-target genes (i.e., heme oxygenase-1, NAD(P)H:quinone oxidoreductase1) to siRNA depletion of Nrf2 in MPP⁺-induced dopaminergic cell death. Nrf2 and its downstream regulated genes/proteins were analyzed using Real-time PCR and Western Blotting techniques, respectively. Moreover, free radical production, the changes in mitochondrial membrane potential, total glutathione, and glutathione-S-transferase were examined. The possible contribution of peroxisome proliferator-activated receptor gamma (PPARγ) to tideglusib-mediated neuroprotection was evaluated. The number of viable cells and mitochondrial membrane potential were increased following GSK-3ß enzyme inhibition against MPP⁺. HO-1, NQO1 mRNA/protein expressions and Nrf2 nuclear translocation significantly triggered by tideglusib. Moreover, the neuroprotection by tideglusib was not observed in the presence of siRNA Nrf2. Our study supports the idea that GSK-3ß enzyme inhibition may modulate the Nrf2/ARE pathway in cellular damage and the inhibitory role of tideglusib on GSK-3ß along with PPARγ activation may be responsible for neuroprotection.

Apelin-36 exerts the cytoprotective effect against MPP+-induced cytotoxicity in SH-SY5Y cells through PI3K/Akt/mTOR autophagy pathway.

AIMS: Parkinson's disease (PD) is a common neurodegenerative disease typically associated with the accumulation of α-synuclein. Autophagy impairment is thought to be involved in the dopaminergic neurodegeneration in PD. We investigate the effect of Apelin-36 on the activated phosphatidylinositol 3-kinase (PI3K)/protein kinase B(Akt)/the mammalian target of rapamycin (mTOR) autophagy pathway in 1-methyl-4-phenylpyridinium (MPP+)-treated SH-SY5Y cells, which is involved in the cytoprotective effect of Apelin-36. MAIN METHODS: SH-SY5Y cells were treated with 1-Methyl-4-phenylpyridine (MPP+) with or without Apelin-36. The cell viability, apoptotic ratio, the form of autophagic vacuoles, the expression of tyrosine hydroxylase (TH), α-synuclein, phosphorylation of PI3K, AKT, mTOR, microtubule-associated protein 1 Light Chain 3 II/I (LC3II/I) and p62 were detected to investigate the neuroprotective effect of Apelin-36. KEY FINDINGS: The results indicate that Apelin-36 significantly improved the cell viability and decreased the apoptosis in MPP+-treated SH-SY5Y cells. The decreased expression of tyrosine hydroxylase (TH) induced by MPP+ was significantly increased by Apelin36 pretreatment. Moreover, Apelin36 significantly increased the autophagic vacuoles. The ratio of LC3II/I was significantly increased by Apelin36, as well as the decreased p62 expression. In addition, the activated PI3K/AKT/mTOR pathway induced by MPP+ was significantly inhibited by Apelin36. Additionally, Apelin36 significantly decreased the α-synuclein expression. Furthermore, the cytoprotective effect of Apelin-36 was weakened by pretreatment with Insulin-like Growth Factor-1 (IGF-1), an activator of PI3K/Akt, and MHY1485, an mTOR activator. SIGNIFICANCE: Our results demonstrated that Apelin-36 protects against MPP+-induced cytotoxicity through PI3K/Akt/mTOR autophagy pathway in PD model in vitro, which provides a new theoretical basis for the treatment of PD.

Simvastatin Protects Dopaminergic Neurons Against MPP+-Induced Oxidative Stress and Regulates the Endogenous Anti-Oxidant System Through ERK.

BACKGROUND/AIMS: Many clinical studies have demonstrated that statins, especially simvastatin, can decrease the incidence of Parkinson's disease (PD). However, the specific underlying mechanism remains unclear. This study aimed to investigate how simvastatin affects experimental parkinsonian models via the regulation of extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated activation of the anti-oxidant system. METHODS: l-Methyl-4-phenylpyridine ion (MPP+)-treated SH-SY5Y cells and substantia nigra neurons were used to investigate the neuroprotective effect of simvastatin. After incubation with MPP+ and/or simvastatin for 24 h, the MTT assay was used to assess cell viability. Reactive oxygen species (ROS) levels were measured using 2',7'-dichlorofluorescin diacetate, while cellular superoxide dismutase (SOD) levels were determined based on the blue formazan produced by the reduction of nitroblue tetrazolium. The level of cellular grade micro-reduced glutathione (GSH) was measured with 5,5'-dithiobis-(2-nitrobenzoic acid). Meanwhile, the malondialdehyde content released from SH-SY5Y cells and substantia nigra neuronal cells exposed to different culture media was calculated based on the condensation reaction involving thiobarbituric acid. The mRNA levels of genes encoding nuclear factor (erythroid-derived 2)-like 2 (Nrf2), heme oxygenase 1 (HO-1), and NAD(P)H dehydrogenase (quinone) 1 (NQO-1) were determined by a quantitative polymerase chain reaction assay, while the ERK, Nrf2, HO-1, NOX2, and NQO-1 protein levels were analyzed by western blot. Additionally, ERK small interfering RNA (siRNA) was used to investigate the mechanisms underlying MPP+-induced oxidative stress and the regulation of the endogenous anti-oxidant system. RESULTS: Simvastatin (1.5 µM) enhanced the viability of SH-SY5Y cells and primary neurons treated with MPP+, and significantly alleviated the oxidative stress induced by MPP+ in SH-SY5Y cells by regulating the production of SOD, analytical grade micro-reduced GSH, and ROS, which may be associated with the activation of the Nrf2 anti-oxidant system. An analysis involving ERK1/2 siRNA revealed that simvastatin can inhibit NOX2 expression via the activation of ERK1/2 in the MPP+-treated PD cell model. CONCLUSION: Our results provide strong evidence that ERK1/2-mediated modulation of the anti-oxidant system after simvastatin treatment may partially explain the anti-oxidant activity in experimental parkinsonian models. These findings contribute to a better understanding of the critical roles of simvastatin via the ERK1/2-mediated modulation of the anti-oxidant system, which may be relevant for treating PD.

Oxicam-derived non-steroidal anti-inflammatory drugs suppress 1-methyl-4-phenyl pyridinium-induced cell death via repression of endoplasmic reticulum stress response and mitochondrial dysfunction in SH-SY5Y cells.

We have previously reported that oxicam-derived non-steroidal anti-inflammatory drugs (oxicam-NSAIDs), including meloxicam, piroxicam and tenoxicam, elicit protective effects against 1-methyl-4-phenyl pyridinium (MPP+)-induced cell death in a fashion independent of cyclooxygenase (COX) inhibition. We have also demonstrated that oxicam-NSAIDs suppress the decrease in phosphorylation of Akt caused by MPP+. The molecular mechanism through which oxicam-NSAIDs provide cytoprotection remains unclear. In this study, we speculated a possibility that endoplasmic reticulum (ER) stress and/or mitochondrial dysfunction, which are both causative factors of Parkinson's disease (PD), may be involved in the neuroprotective mechanism of oxicam-NSAIDs. We demonstrated here that oxicam-NSAIDs suppressed the activation of caspase-3 and cell death caused by MPP+ or ER stress-inducer, tunicamycin, in SH-SY5Y cells. Furthermore, oxicam-NSAIDs suppressed the increases in the ER stress marker CHOP (apoptosis mediator) caused by MPP+ or tunicamycin, beside suppressing eukaryotic initiation factor 2α (eIF2α) phosphorylation and the increase in ATF4 caused by MPP+. Taken together, these results suggest that oxicam-NSAIDs suppress the eIF2α-ATF4-CHOP pathway, one of the three signaling pathways in the ER stress response. Oxicam-NSAIDs suppressed the decrease in mitochondrial membrane potential depolarization caused by MPP+, indicating they also rescue cells from mitochondrial dysfunction. Akt phosphorylation levels were suppressed after the incubation with MPP+, whereas phosphorylation of eIF2α was enhanced. These results suggest that oxicam-NSAIDs prevented eIF2α phosphorylation and mitochondrial dysfunction by maintaining Akt phosphorylation (reduced by MPP+), thereby preventing cell death.

Polysulfide protects midbrain dopaminergic neurons from MPP+-induced degeneration via enhancement of glutathione biosynthesis.

Polysulfides are endogenous sulfur-containing molecular species that may regulate various cellular functions. Here we examined the effect of polysulfides exogenously applied to rat midbrain slice cultures, to address their potential neuroprotective actions. Na2S3 at concentrations of 10 µM or higher prevented 1-methyl-4-phenylpyridinium (MPP+)-induced loss of dopaminergic neurons. Na2S4 at 10 µM also protected dopaminergic neurons from MPP+ cytotoxicity, whereas Na2S and Na2S2 at the same concentration had no significant effect. We also found that Na2S3 (10 µM) prevented MPP+-induced increase in intracellular reactive oxygen species as detected by 2',7'-dichlorofluorescein fluorescence. In addition, the protective effect of Na2S3 was abolished by l-buthionine sulfoximine, an inhibitor of glutathione synthesis. In cellular models of neurons (SH-SY5Y cells) and glial cells (C6 cells), Na2S3 (30 and 100 µM) increased expression of mRNAs encoding the subunits of glutamate cysteine ligase, the rate-limiting enzyme for glutathione biosynthesis. Consistently, the cellular content of total glutathione was increased by Na2S3, and the effect was more prominent in SH-SY5Y cells than in C6 cells. These results suggest that polysulfides are efficient neuroprotectants superior to monosulfur species such as H2S and HS-, and that the neuroprotective effect of polysulfides is mediated by upregulation of glutathione biosynthesis.

Neuroprotective effects of Eucommia ulmoides Oliv. and its bioactive constituent work via ameliorating the ubiquitin-proteasome system.

BACKGROUND: Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by a loss of dopaminergic neurons in the substantia nigra, decreased striatal dopamine levels, and consequent extrapyramidal motor dysfunction. The purpose of this study was to investigate potential in vivo protective effects of Duzhong against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), as well as the bioactive constituents against 1-methyl-4-phenylpyridinium (MPP(+)) toxicity in vitro. METHODS: Male C57BL/6 mice were intraperitoneally administrated five consecutive injections of MPTP every 24 h at a dose of 30 mg/kg to induce an in vivo PD model. Pole and traction tests were performed in mice to evaluate motor deficits and bradykinesia after the final MPTP administration. The striatal levels of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanilic acid, were measured using a High-performance liquid chromatography-electrical conductivity detector. To further explore the bioactive constituents and protective mechanisms of Duzhong, seven compounds from Duzhong were tested on MPP(+)-treated SH-SY5Y cell lines in vitro. A proteasome enzymatic assay and Cell Counting Kit-8 were performed to examine proteasomal activity and cell viability of Duzhong-treated cells, respectively, after exposure to MPP(+) and proteasome inhibitor MG132. RESULTS: Duzhong antagonized the loss of striatal neurotransmitters and relieved the associated anomaly in ambulatory locomotor activity in PD mice after a 3-day pre-treatment of Duzhong crude extract. The five Duzhong compounds attenuated MPP(+)-induced dysfunction of protease activity and reduced MG132-induced cytotoxicity. CONCLUSION: Duzhong could serve as a potential candidate for PD treatment, and its mechanism involves the amelioration of the ubiquitin-proteasome system.

Neuroprotective effects of ginkgetin against neuroinjury in Parkinson's disease model induced by MPTP via chelating iron.

Disruption of neuronal iron homeostasis and oxidative stress are closely related to the pathogenesis of Parkinson's disease (PD). Ginkgetin, a natural biflavonoid isolated from leaves of Ginkgo biloba L, has many known effects, including anti-inflammatory, anti-influenza virus, and anti-fungal activities, but its underlying mechanism of the neuroprotective effects in PD remains unclear. The present study utilized PD models induced by 1-methyl-4-phenylpyridinium (MPP(+)) and 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to explore the neuroprotective ability of ginkgetin in vivo and in vitro. Our results showed that ginkgetin could provide significant protection from MPP(+)-induced cell damage in vitro by decreasing the levels of intracellular reactive oxygen species and maintaining mitochondrial membrane potential. Meanwhile, ginkgetin dramatically inhibited cell apoptosis induced by MPP+ through the caspase-3 and Bcl2/Bax pathway. Moreover, ginkgetin significantly improved sensorimotor coordination in a mouse PD model induced by MPTP by dramatically inhibiting the decrease of tyrosine hydroxylase expression in the substantia nigra and superoxide dismutase activity in the striatum. Interestingly, ginkgetin could strongly chelate ferrous ion and thereby inhibit the increase of the intracellular labile iron pool through downregulating L-ferritin and upregulating transferrin receptor 1. These results indicate that the neuroprotective mechanism of ginkgetin against neurological injury induced by MPTP occurs via regulating iron homeostasis. Therefore, ginkgetin may provide neuroprotective therapy for PD and iron metabolism disorder related diseases.

Guanosine exerts neuroprotective effects by reversing mitochondrial dysfunction in a cellular model of Parkinson's disease.

The mitochondria are the most important cytoplasmic organelles in determining cell survival and death. Mitochondrial dysfunction leads to a wide range of disorders, including neurodegenerative diseases. The central events in the mitochondrial­dependent cell death pathway are the activation of the mitochodrial permeability transition pore (mPTP) and the disruption of mitochondrial membrane potential, which cause the release of apoptogenic molecules and finally lead to cell death. This is thought to be at least partly responsible for the loss of dopaminergic neurons in Parkinson's disease (PD); thus, the attenuation of mitochondrial dysfunction may contribute to alleviating the severity and progression of this disease. Guanosine is a pleiotropic molecule affecting multiple cellular processes, including cellular growth, differentiation and survival. Its protective effects on the central nervous system and and on several cell types by inhibiting apoptosis have been shown in a number of pathological conditions. This study aimed to analyze the ability of guanosine to protect neuronal PC12 cells from the toxicity induced by 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which mediates selective damage to dopaminergic neurons and causes irreversible Parkinson-like symptoms in humans and primates. Our results demonstrated that the apoptosis of PC12 cells induced by MPP+ was significantly prevented by pre-treatment for 3 h with guanosine. In addition, guanosine attenuated the MPP+-induced collapse of mitochondrial transmembrane potential and prevented the sebsequent activation of caspase-3, thereby protecting dopaminergic neurons against mitochondrial stress-induced damage.

Neuroprotective effects of mesenchymal stem cells through autophagy modulation in a parkinsonian model.

Autophagy is a major degradation pathway for abnormal aggregated proteins and organelles that cause various neurodegenerative diseases. Current evidence suggests a central role for autophagy in pathogenesis of Parkinson's disease, and that dysfunction in the autophagic system may lead to α-synuclein accumulation. In the present study, we investigated whether mesenchymal stem cells (MSCs) would enhance autophagy and thus exert a neuroprotective effect through the modulation of α-synuclein in parkinsonian models. In MPP(+)-treated neuronal cells, coculture with MSCs increased cellular viability, attenuated expression of α-synuclein, and enhanced the number of LC3-II-positive autophagosomes compared with cells treated with MPP(+) only. In an MPTP-treated animal model of Parkinson's disease, MSC administration significantly increased final maturation of late autophagic vacuoles, fusion with lysosomes. Moreover, MSC administration significantly reduced the level of α-synuclein in dopaminergic neurons, which was elevated in MPTP-treated mice. These results suggest that MSC treatment significantly enhances autophagolysosome formation and may modulate α-synuclein expression in parkinsonian models, which may lead to increased neuronal survival in the presence of neurotoxins.

Neuropep-1 ameliorates learning and memory deficits in an Alzheimer's disease mouse model, increases brain-derived neurotrophic factor expression in the brain, and causes reduction of amyloid beta plaques.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by amyloid beta (Aß) deposits, hyperphosphorylated tau deposition, and cognitive dysfunction. Abnormalities in the expression of brain-derived neurotrophic factor (BDNF), which plays an important role in learning and memory formation, have been reported in the brains of AD patients. A BDNF modulating peptide (Neuropep-1) was previously identified by positional-scanning synthetic peptide combinatorial library. Here we examine the neuroprotective effects of Neuropep-1 on several in vitro neurotoxic insults, and triple-transgenic AD mouse model (3xTg-AD). Neuropep-1 protects cultured neurons against oligomeric Aß1-42, 1-methyl-4-phenylpyridinium, and glutamate-induced neuronal cell death. Neuropep-1 injection also significantly rescues the spatial learning and memory deficits of 3xTg-AD mice compared with vehicle-treated control group. Neuropep-1 treatment markedly increases hippocampal and cortical BDNF levels. Furthermore, we found that Neuropep-1-injected 3xTg-AD mice exhibit dramatically reduced Aß plaque deposition and Aß levels without affecting tau pathology. Neuropep-1 treatment does not alter the expression or activity of full-length amyloid precursor protein, α-, ß-, or γ-secretase, but levels of insulin degrading enzyme, an Aß degrading enzyme, were increased. These findings suggest Neuropep-1 may be a therapeutic candidate for the treatment of AD.

Therapeutic effects of multifunctional tetramethylpyrazine nitrone on models of Parkinson's disease in vitro and in vivo.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Although the etiology of PD is not completely understood, it is well-documented that oxidative stress and Ca(2+)-mediated cellular damage play important roles in the progression of PD. 2-[[(1,1-Dimethylethyl)oxidoimino]-methyl]-3,5,6-trimethylpyrazine (TBN), a novel nitrone derivative of tetramethylpyrazine, has shown significant therapeutic effects in stroke models due to its multiple functions, including calcium overload blockade and free radical-scavenging. In this study, we investigated the neuroprotective and neurorescue effects of TBN on various in vitro and in vivo models of PD and explored its possible mechanisms of action. The results show that TBN exerted significant neuroprotection on 1-methyl-4-phenylpyridinium (MPP(+))-induced damage in SH-SY5Y cells and primary dopaminergic neurons, as well as on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neuron loss in zebrafish (TBN and MPTP were added simultaneously into the fish embryo medium and the treatment period was 48 h). In the MPTP-induced mouse and 6-hydroxydopamine (6-OHDA)-induced rat PD models, TBN administrated orally twice daily for 14 d (3 d post-MPTP lesion in mice and 7 d post-6-OHDA lesion in rats) exhibited remarkable neurorescue effects to increase the number of dopaminergic neurons. In addition, TBN improved apomorphine-induced rotational behavior in the 6-OHDA-lesioned PD rats. TBN suppressed the MPP(+)-induced intracellular reactive oxygen species (ROS) in SH-SY5Y cells, increased the superoxide dismutase (SOD) activity and glutathione (GSH) concentration in the substantial nigra of MPTP-treated mice. These data indicate that TBN protects and rescues dopaminergic neurons from MPP(+) and MPTP/6-OHDA-induced damage by reducing ROS and increasing cellular antioxidative defense capability.

[Protective effects of crude polysaccharides from Chroogomphus rutilus on SH-SY5Y cells impaired by MPP(+)].

The aim of the present study was to investigate the protective effects of crude polysaccharides from Chroogomphus rutilus on dopaminergic neurons impaired by MPP(+). SH-SY5Y cells were pretreated with crude polysaccharides (200, 400 and 800 µg/mL), and then MPP(+) was added to cell medium. After 48 h of incubation, MTT method was used to detect the survival rate of SH-SY5Y cells damaged by MPP(+). Annexin V-FITC staining and flow cytometry were used to detect apoptotic rate. The results showed that pretreating SH-SY5Y cells with crude polysaccharides (400 and 800 µg/mL) increased the survival rates, and reduced the apoptotic rates of SH-SY5Y cells. To rule out the possibility that crude polysaccharides may decrease actual concentration of MPP(+) by direct binding, we washed off crude polysaccharides before MPP(+) addition. Under this experimental condition, MTT results showed the survival rates of the SH-SY5Y cells were still significantly increased by 800 µg/mL crude polysaccharides pretreatment. These results suggest a protective effect of polysaccharides on the SH-SY5Y cells. Most of this protection is contributed by direct action of polysaccharides on the cells, not by binding with MPP(+). It is indicated that the crude polysaccharides from Chroogomphus rutilus can be developed as a potential drug for Parkinson's disease prevention and treatment in the future.