Ghrelin prevents 1-methyl-4-phenylpyridinium ion-induced cytotoxicity through antioxidation and NF-κB modulation in MES23.5 cells

Ghrelin, a 28-amino acid peptide, is an endogenous ligand for the growth hormone secretagogue (GHS) receptor. Our previous data showed that ghrelin could inhibit apoptosis in Parkinson's disease (PD) models both in vitro and in vivo. There is now growing evidence that oxidative stress has a critical role in the etiology of PD. And ghrelin was reported to possess anti-inflammatory, antioxidant effects. Dose ghrelin protect dopaminergic neurons by its antioxidant effect? In the present study, 1-methyl-4-phenylpyridinium (MPP⁺) was used to evaluate the possible antioxidant effects of ghrelin on MPP⁺-induced neurotoxicity in MES23.5 cells and the underlying mechanisms. Our results showed that MPP⁺ significantly increased malonaldehyde (MDA) level and Bax/Bcl2 ratio, reduced the level of Cu-Zn superoxide dismutase (SOD) and catalase (CAT). Ghrelin protected MES23.5 cells against MPP⁺-induced neurotoxicity by reversing these changes. Furthermore, ghrelin pretreatment significantly inhibited MPP⁺-induced nuclear factor-kappaB translocation. These results suggest that the protective effects of ghrelin on MPP⁺-induced cytotoxicity may be ascribed to its antioxidative properties, and the modulation of nuclear factor-kappaB.     

Protection of MES23.5 dopaminergic cells by obestatin is mediated by proliferative rather than anti-apoptotic action

Obestatin is an endogenous peptide sharing a precursor with ghrelin. This study aims to investigate whether and how obestatin protects MES23.5 dopaminergic cells against 1-methyl-4-phenylpyridinium (MPP⁺)-induced neurotoxicity. MES23.5 cells were pretreated with obestatin (10⁻¹³–10⁻⁶ mol/L) for 20 min prior to incubation with 200 μmol/L MPP⁺ for 12 or 24 h, or treated with obestatin alone (10⁻¹³ to 10⁻⁶ mol/L) for 0, 6, 12, and 24 h. The methyl thiazolyl tetrazolium (MTT) assay was used to measure cell viability. Flow cytometry was used to measure the caspase-3 activity and the mitochondrial transmembrane potential. Proliferating cell nuclear antigen (PCNA) protein levels were determined by Western blotting. Obestatin (10⁻¹³ to 10⁻⁷ mol/L) pretreatment blocked or even reversed the MPP⁺-induced reduction of viability in MES23.5 cells, but had no effect on MPP⁺-induced mitochondrial transmembrane potential collapse and caspase-3 activation. When applied alone, obestatin increased viability. Elevated PCNA levels occurred with 10⁻⁷, 10⁻⁹, 10⁻¹¹ and 10⁻¹³ mol/L obestatin treatment for 12 h. The results suggest that the protective effects of obestatin against MPP⁺ in MES23.5 cells are due to its proliferation-promoting rather than anti-apoptotic effects.     

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.     

Isorhynchophylline Attenuates MPP<Superscript>+</Superscript>-Induced Apoptosis Through Endoplasmic Reticulum Stress- and Mitochondria-Dependent Pathways in PC12 Cells: Involvement of Antioxidant Activity

Endoplasmic reticulum stress (ERS) and mitochondrial dysfunctions are thought to be involved in the dopaminergic neuronal death in Parkinson’s disease (PD). In this study, we found that isorhynchophylline (IRN) significantly attenuated 1-methyl-4-phenylpyridinium (MPP⁺)-induced apoptotic cell death and oxidative stress in PC12 cells. IRN markedly reduced MPP⁺-induced-ERS responses, indicative of inositol-requiring enzyme 1 (IRE1) phosphorylation and caspase-12 activation. Furthermore, IRN inhibits MPP⁺-triggered apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal Kinase (JNK) signaling-mediated mitochondria-dependent apoptosis pathway. IRN-mediated attenuation of endoplasmic reticulum modulator caspase-12 activation was abolished by diphenyleneiodonium (DPI) or IRE-1α shRNA, but not by SP600125 or pifithrin-α in MPP⁺-treated PC12 cells. Inhibitions of MPP⁺-induced both cytochrome c release and caspase-9 activation by IRN were blocked by pre-treatment with DPI or pifithrin-α, but not by IRE-1α shRNA. IRN blocks the generation of reactive oxygen species upstream of both ASK1/JNK pathway and IRE1/caspase-12 pathway. Altogether, our in vitro findings suggest that IRN possesses potent neuroprotective activity and may be a potential candidate for the treatment of PD.     

Ultrastructural alterations induced by 1-methyl-4-phenylpyridinium (MPP) in canine substantia nigra and rat mesencephalon in vitro

Explants of canine substantia nigra (SN) and rat mesencephalon (MES), grown in organotypic culture, were incubated with 1-methyl-4-phenylpyridinium (MPP⁺) and examined for ultrastructural changes. Prolonged exposure (3 days) to doses ranging from 0.1 nM to 10 μM MPP⁺ resulted in total destruction of all constituents (neuronal and glial) of canine SN cultures. No association was noted between MPP⁺-induced toxicity and age of canine SN cultures. The first ultrastructural change observed in canine SN cultures incubated with 0.1 nM MPP⁺ was at 3 h. Grossly swollen mitochondria were noted in large nerve cells. Swollen mitochondria were present in all cells of canine SN cultures by 8 h of incubation with MPP⁺. Only those rat MES cultures with relatively high preincubation levels of homovanillac acid, determined as an index of viable dopaminergic neurons, incubated with MPP⁺ (10 μM) for up to 8 days exhibited ultrastructural changes, namely, a swelling of mitochondria within the cytoplasm of large nerve cells. These findings suggest that continual exposure to MPP⁺in vitro results in a generalized, nonspecific toxicity in those species known to be susceptible to the parent compound 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine in vivo. However, the initial ultrastructural change, i.e., a swelling of mitochondria, may be the same in all species regardless of sensitivity suggesting that the ultimate mechanism underlying MPP⁺-toxicity relates to mitochondrial function.     

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     

Nitric oxide and MPP+-induced hydroxyl radical generation

Summary. Although neuroprotective effect of nitric oxide (NO) is discussed, NO has a role of pathogenesis of cellular injury. NO is synthesized from L-arginine by NO synthase (NOS). NO contributes to the extracellular potassium-ion concentration ([K⁺]_o)-induced hydroxyl radical (^•OH) generation. Cytotoxic free radicals such as peroxinitrite (ONOO⁻) and ^•OH may also be implicated in NO-mediated cell injury. NO activation was induced by K⁺ depolarization. NO may react with superoxide anion (O₂ ⁻) to form ONOO⁻ and its decomposition generates ^•OH. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) metabolite 1-methyl-4-phenylpyridinium ion (MPP⁺) involve toxicity induced by NO. Intraneuronal Ca²⁺ triggered by MPP⁺ may be detrimental to the functioning of dopaminergic nerve terminals in the striatum. Although the [K⁺]_o-induced depolarization enhances the formation of ^•OH product due to MPP⁺, the ^•OH generation via NOS activation may be unrelated the dopamine (DA)-induced ^•OH generation. Depolarization enhances the MPP⁺-induced ^•OH formation via NOS activation. NOS inhibition is associated with a protective effect due to suppression of depolarization-induced ^•OH generation. ONOO⁻ has been implicated as a causative factor under conditions in which DA neurons are damaged. These 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.     

Protective effect of ginsenoside Rg1 on MPP<Superscript>+</Superscript>-induced apoptosis in SHSY5Y cells

Summary. The neuroprotective mechanism of Rg1 was studied in this paper by means of its obvious anti-apoptotic effect on human SHSY5Y cells. SHSY5Y cells were treated with MPP⁺ (1-methyl-4-phenyl-pyridinium) for 72 hours to induce apoptosis. During the apoptosis, production of reactive oxygen species (ROS), activation of c-Jun N-terminal kinase (JNK) and activation of caspase-3 were observed. The results showed that the signal transduction pathway of MPP⁺-induced apoptosis might be ROS to JNK, then to caspase-3. MPP⁺-induced apoptosis in SHSY5Y cells was obviously inhibited in both NAC (N-acetylcysteine) pretreated groups and Rg1 pretreated groups. Meanwhile, compared to that of the controls, our results showed decreased level of ROS, less JNK activity and lower expression of cleaved caspase-3 in pretreated NAC groups and in Rg1 pretreated groups. The protection by Rg1 might be mediated by removing of ROS. The removal of ROS might inhibit the activity of JNK and the expression of cleaved caspase-3. These results suggest that ginsenoside Rg1 may take effect through its anti-apoptotic activity in neurodegenerative diseases.Received October 16, 2002; accepted March 5, 2003 Published online May 28, 2003     

Oxidative stress and 17-α- and 17-β-estradiol modulate neurofilaments differently

Oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD). Neuronal death in the substantia nigra of PD patients is partly caused by exacerbated oxidative damage. Our previous studies demonstrated that oxidative stress can alter the structure and stability of neuro-filament (NF) proteins and that 17-α- and 17-β-estradiol are potent neuroprotective agents. The aim of this study was to investigate the cytoskeletal target of neuroprotection by estrogens in neuronal PC12 cells. We induced oxidative stress by MPP⁺ administration for 24 h, and 17-α- and 17-β-estradiol were used as neuroprotective drugs. We measured gene expression and protein expression of each NF subunit, NFL, NFM, and NFH, by semiquantitative RT-PCR, Western blot, and immunofluorescence. Our results demonstrate that NFL mRNA and protein levels are not modulated by MPP⁺ or estradiol isomers, whereas NFM gene expression, as well as protein expression, are strongly influenced by MPP⁺, 17-α-, and 17-β-estradiol after a 24-h treatment. Finally, mRNA levels of the most phosphorylated subunits, NFH, are not changed by MPP⁺ or treatment with both estradiol isomers, whereas NFH protein expression is decreased by the same treatments. These results suggest that oxidative stress affects neuronal cytoskeleton, maybe though proteolysis and/or abnormal structural changes in NFs. Then, 17-α- and 17-β-estradiol might help the neuronal cell in recovering after oxidative stress by inducing protein expression of NFM and NFH subunits. These authors contributed equally to this paper.     

Cytotoxicity of chloral-derived β-carbolines is not specific towards neuronal nor dopaminergic cells

Summary. β-Carbolines structurally related to the selective dopaminergic neurotoxin 1-methyl-4- phenylpyridinium (MPP⁺) may contribute to dopaminergic neurodegeneration in Parkinson’s disease. The chloral-derived mammalian alkaloid derivative 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo) is formed endogenously by a Pictet-Spengler condensation from the biogenic amine tryptamine (Ta) and the hypnotic aldehyde chloral (Clo). Here we examine the dopaminergic toxicity of TaClo and related compounds by testing their differential cytotoxicities in dopaminergic SH-SY5Y and non-dopaminergic murine Neuro2A neuroblastoma cell lines as well as in heterologous expression systems of the dopamine transporter (DAT) using both HEK-293 and Neuro2A cells. All TaClo derivatives showed significant cytotoxicity in all cell lines after 72 hours with the following rank order of toxic potency: 1-Tribromomethyl-1,2,3,4-tetrahydro-β-carboline (TaBro) > TaClo > MPP⁺ > 1,2,3,4-tetrahydro-β-carboline (THβC) > 2[N]-methyl-TaClo > 2[N]-methyl-THβC. In contrast to MPP⁺, there was no selectivity towards dopaminergic cells or cells ectopically expressing the DAT in vitro. Our results suggest that TaClo and related analogs are strong cytotoxins without selectivity towards dopaminergic cells.     

Peripheral Administration of Tetanus Toxin Hc Fragment Prevents MPP<Superscript>+</Superscript> Toxicity In Vivo

Several studies have shown that intrastriatal application of 1-methyl-4-phenylpyridinium (MPP⁺) produces similar biochemical changes in rat to those seen in Parkinson’s disease (PD), such as dopaminergic terminal degeneration and consequent appearance of motor deficits, making the MPP⁺ lesion a widely used model of parkinsonism in rodents. Previous results from our group have shown a neuroprotective effect of the carboxyl-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) under different types of stress. In the present study, pretreatment with the intraperitoneal injection of Hc-TeTx in rats prevents the decrease of tyrosine hydroxylase immunoreactivity in the striatum due to injury with MPP⁺, when applied stereotaxically in the striatum. Similarly, striatal catecholamine contents are restored, as well as the levels of two other dopaminergic markers, the dopamine transporter (DAT) and the vesicular monoamine transporter-2 (VMAT-2). Additionally, uptake studies of [³H]-dopamine and [³H]-MPP⁺ reveal that DAT action is not affected by Hc-TeTx, discarding a protective effect due to a reduced entry of MPP⁺ into nerve terminals. Behavioral assessments show that Hc-TeTx pretreatment improves the motor skills (amphetamine-induced rotation, forelimb use, and adjusting steps) of MPP⁺-treated rats. Our results lead us to consider Hc-TeTx as a potential therapeutic tool in pathologies caused by impairment of dopaminergic innervation in the striatum, as is the case of PD.     

Evaluation of acute antiapoptotic effects of Li<Superscript>+</Superscript> in neuronal cell cultures

Summary Li⁺ exerts protective effect against several neurotoxins in neuronal cell preparations. Here we examined the antiapoptotic effects of GSK3β in cerebellar granule neurons (CGNs) in the presence of several neurotoxins. Acute treatment with Li⁺ protected neurons against nocodazole and serum/potassium (S/K) deprivation, but were ineffective against kainic acid and MPP⁺. Li⁺ 5 mM also decreased caspase-3 activation induced by nocodazole and S/K deprivation as measured by Ac-DEVD-p-nitroaniline and the breakdown of α-spectrin. All the neurotoxins used in the present study activated GSK3β, evaluated with a specific antibody phospho-GSK-3β (Ser9) by Western-blot and immunocytochemistry and were always inhibited by Li⁺ 5 mM. Our results implicate Li⁺ in the regulation of apoptosis mediated by caspase activation (Type I). Furthermore inhibition of GSK3β by acute treatment with Li⁺ 5 mM is not an indicator of neuroprotection. The acute antiapoptotic function of Li⁺ is discussed in terms of its inhibition of Type I pathway, the intrinsic (mitochondrial) apoptotic pathway in cerebellar granule cells.     

Neuronal nitric oxide synthase inhibition reduces MPP+-evoked hydroxyl radical formation but not dopamine efflux in rat striatum

Summary. Nigral cell degeneration induced by 1-methyl-4-phenyl-1,2,3,6-tertrahydropyridine (MPTP) or its metabolite 1-methy1-4-phenyl pyridinium (MMP⁺) may involve toxicity induced by nitric oxide. In the present study a microdialysis procedure incorporating salicylate hydroxylation was used to measure striatal hydroxyl radical production through the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA). MPP⁺ (5–20 mM for 20 min) increased 2,3-DHBA formation in the rat striatum in a concentration-dependent manner with a concomitant increase in dopamine release and decrease in 3,4-dihydroxyphenyl acetic acid (DOPAC) formation. Inhibition of NO synthesis following N^G-nitro-L-arginine methyl ester (L-NAME; 1 mM) and 7-nitroindazole monosodium salt (7-NINA; 1 mM), but not N^G-nitro-D-arginine methyl ester (D-NAME; 1 mM) attenuated the MPP⁺-induced increase in hydroxyl radical formation. However, neither L-NAME nor 7-NINA had any effect on the MPP⁺-induced increase in dopamine efflux measured in vivo by microdialysis or in vitro using superfused striatal slices, although nomifensine (10 μM) abolished the MPP⁺-evoked dopamine efflux in vitro. These data suggest that NO formation is necessary for the production of hydroxyl radical following MPP⁺ treatment, but is not involved in the MPP⁺-evoked dopamine release. Received September 11, 1998; accepted November 10, 1998     

1-Methyl-4-phenyl-pyridinium ion (MPP) causes DNA fragmentation and increases the Bcl-2 expression in human neuroblastoma, SH-SY5Y cells, through different mechanisms

Apoptosis has been shown to be induced by some pathological stimuli. MPP⁺ is a neurotoxin and an inducer of parkinsonism. When SH-SY5Y cells, human neuroblastoma cell line, were treated with MPP⁺, cell death estimated by lactate dehydrogenase (LDH) leakage assay occurred. The cell death was associated with the DNA fragmentation into nucleosomal fragments at 180 bp, suggesting that MPP⁺-induced cell death of SH-SY5Y cells occurs through apoptosis. Although SH-SY5Y cells natively express Bcl-2 protein, which inhibits apoptosis, the level of Bcl-2 protein in SH-SY5Y cells increased with increases in the treatment periods of MPP⁺. MPP⁺ inhibits the mitochondrial respiratory chain. The other inhibitors of the mitochondrial respiratory chain, antimycin A and oligomycin, also caused cell death associated with DNA fragmentation, but did not increase the Bcl-2 protein level, suggesting that an MPP⁺-induced apoptosis may be due to the inhibition of the mitochondrial respiratory chain but the MPP⁺-induced increase in the Bcl-2 protein level is not due to it. A protein kinase inhibitor, staurosporine, inhibited the MPP⁺-induced increase in the Bcl-2 protein level, but not the MPP⁺-induced cell death. These results also suggest that the mechanism by which MPP+ increases the Bcl-2 protein level is different from that of MPP⁺-induced cell death.     

Death by a dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP) and protection by EGF in GH3 cells

In this study we investigated the uptake and effect of a dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP⁺) on a clonal strain, GH3 cells, established from rat anterior pituitary. Although the level was very low compared with that in PC12 cells, a clonal rat pheochromocytoma cell line, there was a detectable amount of tyrosine hydroxylase protein in GH3 cells. The levels of monoamines including dopamine in GH3 cells were also very low compared with those in PC12 cells.

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was incorporated to GH3 cells in a concentration-dependent manner and the uptake was inhibited by nomifensine, an inhibitor of dopamine transporter. Addition of 200 μM MPP⁺ stimulated the leakage of lactate dehydrogenase (LDH) after a lag of 24 h. Pretreatment with 50 ng/ml of epidermal growth factor (EGF), but not nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF), protected against MPP⁺-induced cell death. These findings show that: (1) MPP⁺ uptake to GH3 cells was via an effective dopamine transport system and causes delayed cell death, and (2) EGF protects against MPP⁺-induced cell death. A possible role for GH3 cells as dopaminergic neurons is discussed.     

Attenuation of 1-methyl-4-phenylpyridinium (MPP+) neurotoxicity by deprenyl in organotypic canine substantia nigra cultures

Summary Systemic administration of MPTP to experimental animals induces neurodegeneration of dopaminergic neurons in the central nervous system. MPTP crosses the blood-brain barrier where it is taken up by astrocytes and converted to MPP⁺ by monamine oxidase-B (MAO-B). Subsequently, MPP⁺ is selectively taken up by dopaminergic neurons upon which it exerts intracellular neurotoxic effects. Systemic administration of the selective MAO-B inhibitor deprenyl prevents the conversion of MPTP to MPP⁺ and by this mechanism is able to protect against MPTP neurotoxicity. Deprenyl has also been reported to exert neuroprotective effects that are independent of its MAO-B inhibitory properties, but since MPP⁺ itself does not cross the blood-brain barrier it is difficult to directly study the MAO-B independentin vivo effects of MPP⁺ itself. One approach is to use organotypic tissue cultures of the canine substantia nigra (CSN) which permit administration of precise concentrations of pharmacological agents directly to mature, well-developed and metabolically active dopaminergic neurons. These neurons as well as other components of the cultures exhibit morphological and biochemical characteristics identical to theirin vivo counterparts. This study was undertaken to evaluate the neuroprotective effects of deprenyl in MPP⁺-treated cultures by measuring changes in the levels of HVA as an indicator of dopamine release and metabolism by dopaminergic neurons and to correlate this indication of dopaminergic function with morphological evidence of survival or loss of dopaminergic neurons in mature CSN cultures. Mature CSN cultures, at 44 days in vitro (DIV), were exposed to either MPP⁺ alone, deprenyl alone or simultaneously to both deprenyl and MPP⁺ or to MPP⁺ following 4 day pretreatment with deprenyl. Exposure to MPP⁺ alone caused significant reduction in HVA levels, evidence of widespread injury and ultimate disappearance of large neurons in the cultures. These effects were attenuated by simultaneous exposure to MPP⁺ and deprenyl and the destructive effects of MPP⁺ appeared to be prevented by pretreatment with deprenyl. Thus the neuroprotective effects of deprenyl on MPP⁺-induced reduction of HVA levels in living cultures appears similar to the effects of deprenyl on dopamine levels and tyrosine hydroxylase activity reported by others in cultures previously exposed to deprenyl and MPP⁺. These studies also confirm that the neuroprotective effects of deprenyl against MPP⁺ in dopaminergic neurons are, at least in part, independent of deprenyl's inhibition of MAO-B.     

GPR30 Activation Contributes to the Puerarin-Mediated Neuroprotection in MPP<Superscript>+</Superscript>-Induced SH-SY5Y Cell Death

The neuroprotective action of puerarin in Parkinson’s disease (PD) models has been well investigated. However, the mechanisms involved in protection have not been completely understood. G protein-coupled receptor 30 (GPR30) is a G protein-coupled estrogen receptor and considered a potential target in the neuroprotection against PD. In this study, we investigated whether puerarin prevented against 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell death via GPR30. Our results showed that the GPR30 agonist, G1, exhibited puerarin-mediated neuroprotection against MPP⁺-induced cell death of SH-SY5Y cells. This protective action was reversed by the GPR30 antagonist. Moreover, a time- and concentration-dependent effect of puerarin on GPR30 expression was verified at the protein level but not at the mRNA level. Further, we showed that an mTor-dependent new GPR30 synthesis contributed to the protection conferred by puerarin. Finally, glial cell line-derived neurotrophic factor (GDNF) levels were enhanced by puerarin and G1 in both control and MPP⁺-lesioned cells via GPR30. Taken together, our data strongly suggest that puerarin prevents MPP⁺-induced cell death via facilitating GPR30 expression and GDNF release.     

Protective effect of BAG5 on MPP+-induced apoptosis in PC12 cells

Abstract

Objectives: Parkinson’s disease (PD) is the most common neurodegenerative disease in humans, and an abundance of evidence has implicated apoptosis signaling pathways in the neurodegeneration of PD. The purpose of this study was to assess the role of B-cell lymphoma 2 (Bcl-2)-associated athanogene 5 (BAG5) protein, which was previously confirmed to play an important role in the pathogenesis of PD, in the regulation of apoptosis induced by 1-methyl-4-phenyl-pyridinium (MPP⁺) in PC12 cells.

Methods: PC12 cells were treated with MPP⁺ for 48 hours to induce apoptosis, and activation of Bcl-2, Bcl-xl, and caspase 3 was measured by western blot.

Results: The upregulation of BAG5 in PC12 cells inhibited apoptosis and increased the expression of anti-apoptotic proteins, including Bcl-2 and Bcl-xl, after MPP⁺ treatment. In addition, downregulation of BAG5 in PC12 cells enhanced apoptosis and decreased the expression of these proteins after MPP⁺ treatment.

Discussion: The data suggest that BAG5 inhibits MPP⁺-induced apoptosis through both endogenous and mitochondria-mediated pathways of apoptosis. Through this mechanism, the upregulation of BAG5 levels may occur through its anti-apoptotic activity in PD.     

The atypical antipsychotic,olanzapine, potentiates ghrelin-induced receptor signaling: An in vitro study with cells expressing cloned human growth hormone secretagogue receptor

The growth hormone secretagogue receptor (GHS-R) belongs to Gαq-coupled G protein-coupled receptor (GPCR) that mediates growth hormone release, food intake, appetite, glucose metabolism and body composition. Ghrelin has been identified as an endogenous ligand for GHS-R, and it is the only orexigenic peptide found in the peripheral organs. Olanzapine, an atypical antipsychotic agent that binds to and inhibits the activation of GPCR for several neurotransmitters, has metabolic side effects such as excessive appetite and weight gain. Recently, studies have revealed that the orexigenic mechanism of olanzapine is mediated via GHS-R signaling, although the precise mechanisms have not been clarified.In this study, we investigated the effect of olanzapine on ghrelin-mediated GHS-R signaling by using an electrical impedance-based receptor biosensor assay system (CellKey™). Olanzapine at concentrations of 10^− 7 and 10^− 6 mol/L enhanced ghrelin-induced (10^− 10–10^− 8 mol/L) GHS-R activation. A Ca^2 + imaging assay revealed that olanzapine (10^− 7 and 10^− 6 mol/L) enhanced ghrelin (10^− 7 M)-induced GHS-R activity. In contrast, haloperidol (an antipsychotic agent) failed to enhance this ghrelin-mediated GHS-R activation, as demonstrated by both the CellKey™ and Ca^2 + imaging assays. Together, these results suggest that olanzapine, but not haloperidol, promotes appetite by enhancing ghrelin-mediated GHS-R signaling.