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

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

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

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

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.     

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.     

β-Ecdysterone Protects SH-SY5Y Cells Against 6-Hydroxydopamine-Induced Apoptosis via Mitochondria-Dependent Mechanism: Involvement of p38<Superscript>MAPK</Superscript>–p53 Signaling Pathway

Parkinson’s disease (PD) is a neurological disorder pathologically characterized by loss of dopaminergic neurons in the substantia nigra. No curative therapy is available for PD. We recently found that phytoestrogen β-ecdysterone (β-Ecd) is able to reduce MPP⁺-induced apoptosis in PC12 cells. This study investigated the potential of β-Ecd to protect against SH-SY5Y cell apoptosis induced by the PD-related neurotoxin 6-hydroxydopamine (6-OHDA) and the underlying mechanism for this cytoprotection. In the present study, pretreatment with β-Ecd significantly reduced 6-OHDA-induced apoptosis of SH-SY5Y cells by a mitochondria-dependent pathway, as indicated by downregulation of Bax and PUMA (p53 upregulated modulator of apoptosis) expression, suppressing ΔΨm loss, inhibiting cytochrome c release, and attenuating caspase-9 activation. Furthermore, we showed that the inhibition of p38 mitogen-activated protein kinase (p38^MAPK)-dependent p53 promoter activity contributed to the protection of SH-SY5Y cells from apoptosis, which was validated by the use of SB203580 or p38β dominant negative (DN) mutants. Additionally, knock-down apoptosis signal-regulating kinase 1 (ASK1) by specific shRNA and blockade reactive oxygen species (ROS) by pharmacological inhibitor competently prevented β-Ecd-mediated inhibition of p38^MAPK and ASK1 phosphorylation, respectively. These data provide the first evidence that β-Ecd protects SH-SY5Y cells against 6-OHDA-induced apoptosis, possibly through mitochondria protection and p53 modulation via ROS-dependent ASK1–p38^MAPK pathways. The neuroprotective effects of β-Ecd make it a promising candidate as a therapeutic agent for PD.     

Characterization of the antiapoptotic effect of copper sulfate on striatal and midbrain damage induced by MPP+ in rats

1-Methyl-4-phenylpyridinium ion (MPP⁺)-induced neurotoxicity produces cellular damage resembling that encountered in Parkinson’s disease. The mechanisms of cellular death after MPP⁺ include the participation of oxidative stress in the loss of dopaminergic neurons. Among the mechanisms of defense against oxidative stress, several copper-dependent proteins have been implicated: Cu/Zn-SOD, ceruloplasmin, and metallothionein. Another important mechanism of damage, is MPP + interference with mitochondrial respiration. Both, oxidative stress and inhibition of mitochondrial respiration may trigger apoptosis in the neurons after MPP⁺. The aim of the present study was to characterize the time-course of apoptosis induced by MPP⁺ to determine if copper sulfate pretreatment is able to prevent the activation of caspases and decreased the neuronal apoptosis. MPP⁺ was microinjected into rat striatum using a stereotactic frame. The results showed increased activities of caspases 8, 9 and 3, between 72–120 hours after administration of MPP⁺, both in striatum and midbrain. After this study, we tested the effect of CuSO₄ on MPP⁺ neurotoxicity, showing a diminution of the apoptotic damage induced by MPP⁺, decreased levels of enzymatic activity of caspases: 8 (-34 and -25 %), 9 (-25 and -42 %) and 3 (-40 and -29 %) in striatum and midbrain, respectively. Finally, we performed an immunohistochemical analysis, evidencing a decreased number of apoptotic cells in the groups pretreated with copper sulfate pretreatment compared to the control group. With these findings, it is concluded that pretreatment with copper sulfate may be a good alternative to prevent MPP⁺-induced apoptosis.     

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.     

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.     

Proteomics analysis of MPP+-induced apoptosis in SH-SY5Y cells

Accumulating evidence suggests that oxidative stress plays a pivotal role in dopaminergic neurodegeneration. However, the kinds of proteins involved in the response to oxidative stress remain unclear. In the present study, SH-SY5Y cells were treated with neurotoxin 1-methyl-4-phenyl-pyridinium ion (MPP+) to induce apoptotic neuronal injury. 2D-DIGE followed by MALDI-TOF-MS was used to determine the changing protein levels. Proteomics analysis revealed that 22 proteins were differentially altered in MPP⁺-treated SH-SY5Y cells, of which 7 were up-regulated proteins and 15 were down-regulated proteins, respectively. Three protein spots were unambiguously identified as sorcin, annexin V, and ribosomal protein P0. The three proteins showed a significant increase in level, suggesting a role in MPP⁺-induced apoptosis. The functional roles of these three proteins collectively indicate that multiple mechanisms are pertinent in the underlying pathogenesis of Parkinson’s disease (PD), such as apoptosis, calcium homeostasis, and DNA insults.     

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.     

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

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

Involvement of K(ATP)/PI (3)K/AKT/Bcl-2 pathway in hydrogen sulfide-induced neuroprotection against the toxicity of 1-methy-4-phenylpyridinium ion

We previously reported that hydrogen sulfide (H₂S) produces protection in PC12 cells during 1-methy-4-phenylpyridinium ion (MPP⁺) challenge. The present study aims to clarify the mechanisms underlying the neuroprotective effects of H₂S. We showed that both glybenclamide, an ATP-sensitive potassium (K_ATP) channel blocker, and LY294002, a specific PI₃K–AKT pathway inhibitor, reversed the neuroprotective effect of NaHS (a H₂S donor) against MPP⁺-induced cytotoxicity to PC12 cells and that NaHS up-regulated the activity of AKT in PC12 cells, which was abolished by blockade of K_ATP channels with glybenclamide. In addition, NaHS up-regulated the expression of Bcl-2 and blocked MPP⁺-induced down-regulation of Bcl-2, and this augmentation of Bcl-2 expression was prevented by both glybenclamide and LY294002. These data provided the evidence that the neuroprotective action of H₂S against MPP⁺ toxicity to PC12 cells is via the K_ATP/PI₃K/AKT/Bcl-2 pathway. We also demonstrated that NaHS attenuated the inhibitory effect of MPP⁺ ERK1/2 activation in PC12 cells, whereas U0126, a specific MEK inhibitor, did not reverse the neuroprotective effect of NaHS, which indicated that attenuating MPP⁺-triggered down-regulation of ERK1/2 activation is involved in the protection of H₂S against MPP⁺ neurotoxicity, but ERK1/2 is not an essential effector mediating the neuroprotective effect of H₂S. In conclusion, the present observations identify a crucial role of the K_ATP/PI₃K/AKT/Bcl-2 pathway in H₂S-exerted neuroprotection against the toxicity of MPP⁺. Findings from the present study will help shed light on the mechanisms of H₂S-elicited neuroprotective effects on MPP⁺ toxicity.     

Δ(9) -tetrahydrocannabinol (Δ(9) -THC) exerts a direct neuroprotective effect in a human cell culture model of Parkinson's disease

C. B. Carroll, M.‐L. Zeissler, C. O. Hanemann and J. P. Zajicek (2012) Neuropathology and Applied Neurobiology 38, 535–547 Δ⁹‐tetrahydrocannabinol (Δ⁹‐THC) exerts a direct neuroprotective effect in a human cell culture model of Parkinson's disease Aims: Δ⁹‐tetrahydrocannabinol (Δ⁹‐THC) is neuroprotective in models of Parkinson's disease (PD). Although CB1 receptors are increased within the basal ganglia of PD patients and animal models, current evidence suggests a role for CB1 receptor‐independent mechanisms. Here, we utilized a human neuronal cell culture PD model to further investigate the protective properties of Δ⁹‐THC. Methods: Differentiated SH‐SY5Y neuroblastoma cells were exposed to PD‐relevant toxins: 1‐methyl‐4‐phenylpyridinium (MPP⁺), lactacystin and paraquat. Changes in CB1 receptor level were determined by quantitative polymerase chain reaction and Western blotting. Cannabinoids and modulatory compounds were co‐administered with toxins for 48 h and the effects on cell death, viability, apoptosis and oxidative stress assessed. Results: We found CB1 receptor up‐regulation in response to MPP⁺, lactacystin and paraquat and a protective effect of Δ⁹‐THC against all three toxins. This neuroprotective effect was not reproduced by the CB1 receptor agonist WIN55,212‐2 or blocked by the CB1 antagonist AM251. Furthermore, the antioxidants α‐tocopherol and butylhydroxytoluene as well as the antioxidant cannabinoids, nabilone and cannabidiol were unable to elicit the same neuroprotection as Δ⁹‐THC. However, the peroxisome proliferator‐activated receptor‐gamma (PPARγ) antagonist T0070907 dose‐dependently blocked the neuroprotective, antioxidant and anti‐apoptotic effects of Δ⁹‐THC, while the PPARγ agonist pioglitazone resulted in protection from MPP⁺‐induced neurotoxicity. Furthermore, Δ⁹‐THC increased PPARγ expression in MPP⁺‐treated SH‐SY5Y cells, another indicator of PPARγ activation. Conclusions: We have demonstrated up‐regulation of the CB1 receptor in direct response to neuronal injury in a human PD cell culture model, and a direct neuronal protective effect of Δ⁹‐THC that may be mediated through PPARγ activation.     

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     

Autophagy Activation Alleviates Amyloid-β-Induced Oxidative Stress,Apoptosis and Neurotoxicity in Human Neuroblastoma SH-SY5Y Cells

Autophagy is an evolutionary conserved catabolic process that ensures continuous removal of damaged cell organelles and long-lived protein aggregates to maintain cellular homeostasis. Although autophagy has been implicated in amyloid-β (Aβ) production and deposition, its role in pathogenesis of Alzheimer’s disease remains elusive. Thus, the present study was undertaken to assess the cytoprotective and neuroprotective potential of autophagy on Aβ-induced oxidative stress, apoptosis and neurotoxicity in human neuroblastoma SH-SY5Y cells. The treatment of Aβ1-42 impaired the cell growth and redox balance, and induced apoptosis and neurotoxicity in SH-SY5Y cells. Next, the treatment of rapamycin (RAP) significantly elevated the expression of autophagy markers such as microtubule-associated protein-1 light chain-3 (LC3), sequestosome-1/p62, Beclin-1, and unc-51-like kinase-1 (ULK1) in SH-SY5Y cells. RAP-induced activation of autophagy notably alleviated the Aβ1-42-induced impairment of redox balance by decreasing the levels of pro-oxidants such as reactive oxygen species, lipid peroxidation and Ca²⁺ influx, and concurrently increasing the levels of antioxidant enzymes such as superoxide dismutase and catalase. The RAP-induced autophagy also ameliorated Aβ1-42-induced loss of mitochondrial membrane potential and apoptosis. Additionally, the activated autophagy provided significant neuroprotection against Aβ1-42-induced neurotoxicity by elevating the expression of neuronal markers such as synapsin-I, PSD95, NCAM, and CREB. However, 3-methyladenine treatment significantly exacerbated the neurotoxic effects of Aβ1-42. Taken together, our study demonstrated that the activation of autophagy provided possible neuroprotection against Aβ-induced cytotoxicity, oxidative stress, apoptosis, and neurotoxicity in SH-SY5Y neuronal cells.     

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

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

Silibinin prevents dopaminergic neuronal loss in a mouse model of Parkinson's disease via mitochondrial stabilization

Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the selective loss of dopaminergic neurons in the nigrostriatal pathway. The lipophile 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) can cross the blood–brain barrier and is subsequently metabolized into toxic1‐methyl‐4‐phenylpyridine (MPP⁺), which causes mitochondrial dysfunction and the selective cell death of dopaminergic neurons. The present article reports the neuroprotective effects of silibinin in a murine MPTP model of PD. The flavonoid silibinin is the major active constituent of silymarin, an extract of milk thistle seeds, and is known to have hepatoprotective, anticancer, antioxidative, and neuroprotective effects. In the present study, silibinin effectively attenuated motor deficit and dopaminergic neuronal loss caused by MPTP. Furthermore, in vitro study confirmed that silibinin protects primary cultured neurons against MPP⁺‐induced cell death and mitochondrial membrane disruption. The findings of the present study indicate that silibinin has neuroprotective effects in MPTP‐induced models of PD rather than antioxidative or anti‐inflammatory effects and that the neuroprotection afforded might be mediated by the stabilization of mitochondrial membrane potential. Furthermore, these findings suggest that silibinin protects mitochondria in MPTP‐induced PD models and that it offers a starting point for the development of treatments that ameliorate the symptoms of PD. © 2015 Wiley Periodicals, Inc.     

Transforming growth factor-β1 acts via TβR-I on microglia to protect against MPP+-induced dopaminergic neuronal loss

Neuroinflammation is associated with pathogenesis of Parkinson’s disease (PD), a neurodegenerative disorder characterized by a progressive loss of dopaminergic (DAergic) neurons within the substantia nigra. Transforming growth factor (TGF)-β1 exerts anti-inflammatory and neuroprotective properties. However, it is unclear if microglia are required for TGF-β1 neuroprotection in PD. Here we used both shRNA and pharmacologic inhibition to determine the role of microglial TGF-β receptor (TβR)-I and its downstream signaling pathways in 1-methyl-4-phenylpyridinium (MPP⁺)-induced DAergic neuronal toxicity. As expected, MPP⁺ reduced the number of tyrosine hydroxylase (TH)-immunoreactive cells in ventral mesencephalic cell cultures. We found that MPP⁺ activated microglia as determined by an upregulation in expression of CD11b and inducible nitric oxide synthase (iNOS), an increase in expression and secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-1β, and a decrease in expression and secretion of the neurotrophic factor, insulin-like growth factor (IGF)-1. Pretreatment with TGF-β1 significantly inhibited all these changes caused by MPP⁺. Expression of microglial TβR-I was upregulated by TGF-β1. Silencing of the TβR-I gene in microglia abolished both the neuroprotective and anti-inflammatory properties of TGF-β1. TGF-β1 increased microglial p38 MAPK and Akt phosphorylation, both of which were blocked by the p38 inhibitor SB203580 and the PI3K inhibitor LY294002, respectively. Pretreatment of microglia with either SB203580 or LY294002 impaired the ability of TGF-β1 to inhibit MPP⁺-induced DAergic neuronal loss and microglial activation. These findings establish that TGF-β1 activates TβR-I and its downstream p38 MAPK and PI3K–Akt signaling pathways in microglia to protect against DAergic neuronal loss that characterizes in PD.