Ghrelin Antagonized 1-Methyl-4-Phenylpyridinium (MPP<Superscript>+</Superscript>)-Induced Apoptosis in MES23.5 Cells

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) acting to stimulate growth hormone release. In the previous study, we have observed the neuroprotective effects of ghrelin on dopaminergic neurons in vivo in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine -treated Parkinson’s disease mice. In order to illustrate the underlying mechanisms, in the present study, we conducted our experiment in vitro in 1-methyl-4-phenylpyridinium (MPP⁺)-treated MES23.5 cells that could express GHS-R1a. Ten- to 1,000-μmol/L MPP⁺ treatment caused decreased cell viability, with increased lactate dehydrogenase leakage. A 200-μmol/L MPP⁺ treatment was chosen to do the further experiments. MES23.5 cells treated with 200 μmol/L MPP⁺ showed decreased mitochondrial transmembrane potential, an elevated level of reactive oxidative species production and activation of caspase-3. Additionally, these cells also showed apoptotic morphological changes. Pretreatment with different doses of ghrelin (10⁻¹²–10⁻⁷ mol/L) could abolish the MPP⁺-induced apoptotic changes in a dose-dependent manner. These results suggested that ghrelin could antagonize MPP⁺-induced apoptosis in MES23.5 cells. The protective effects of ghrelin involved the restoration of mitochondria function. Juanjuan Dong and Ning Song contributed equally to this work.     

Ghrelin protects against rotenone-induced cytotoxicity: Involvement of mitophagy and the AMPK/SIRT1/PGC1α pathway

Parkinson's disease (PD) is the second most common neurodegenerative disorder, characterized by the loss of dopaminergic neurons in the substantia nigra and the deposition of Lewy bodies. Mitochondrial dysfunction, oxidative stress, and autophagy dysfunction are involved in the pathogenesis of PD. Ghrelin is a brain-gut peptide that has been reported that protected against 1-methyl-4-phenyl-1,2,3,6- tetrahydropyran (MPTP)/MPP⁺-induced toxic effects. In the present work, human neuroblastoma SH-SY5Y cells were exposed to rotenone as a PD model to explore the underlying mechanism of ghrelin. We found that ghrelin inhibited rotenone-induced cytotoxicity, mitochondrial dysfunction, and apoptosis by improving cell viability, increasing the ratio of red/green of JC-1, inhibiting the production of reactive oxidative species (ROS), and regulating Bcl-2, Bax, Cytochrome c, caspase-9, and caspase-3 expression. Besides, ghrelin promoted mitophagy accompanied by up-regulating microtubule-associated protein 1 Light Chain 3B-II/I(LC3B-II/I) and Beclin1 but decreasing the expression of p62. Moreover, ghrelin promoted PINK1/Parkin mitochondrial translocation. Additionally, we investigated that ghrelin activated the AMPK/SIRT1/PGC1α pathway and pharmacological inhibition of AMPK and SIRT1 abolished the cytoprotection of ghrelin, decreased the level of mitophagy, and PINK1/Parkin mitochondrial translocation. Taken together, our findings suggested that mitophagy and AMPK/SIRT1/PGC1α pathways were related to the cytoprotection of ghrelin. These findings provided novel insights into the underlying mechanisms of ghrelin, further mechanistic studies on preclinical and clinical levels are required to be conducted with ghrelin to avail and foresee it as a potential agent in the treatment and management of PD.     

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.     

1—甲基—4—苯基吡啶离子诱导MES23．5细胞线粒体功能异常的实验研究

目的：探讨1－甲基－4－苯基吡啶离子（MPP^ ）诱导多巴胺（DA）能神经元死亡的线粒体功能异常机制。方法：不同浓度的1－甲基－4－苯基－1,2,3,6－四氢吡啶（MPTP）、MPP 及还原型谷胱甘肽（GSH）与MES23．5细胞共同培养,采用MTT比色法及流式细胞术检测细胞线粒体膜电势（△Ψm）和氧自由基（ROS）的变.MES23\5在力在MPP^ 组显著减低,且与浓度呈正相关,GSH＋MPP^ 组轻度减低,而PTP组不同降低,此外,用MPP^ 处理后MES23．5细胞线粒体△Ψm明显下降和ROS生成显著增加,结论：线粒体△Ψm下降及ROS生成增多可能与了MPP^ 诱导黑质DNA能神经元死亡的机制。     

铜离子对MES23.5细胞的毒性作用机制与线粒体功能障碍有关

目的探讨铜离子对MES23．5多巴胺能细胞的毒性作用及可能机制。方法用MTF法检测铜离子对细胞存活率的影响;用半定量逆转录聚合酶链反应（RT-PCR）,Western blotting以及高效液相色谱电化学检测法（HPLC-ECD）检测细胞内酪氨酸羟化酶（tyrosine hydroxlase,TH）mRNA、蛋白的表达以及多巴胺含量的改变：用流式细胞仪检测线粒体跨膜电位的改变。结果100μmol／L和200／2mol／L铜离子对细胞存活率没有影响,400μmol／L和800μmol／L铜离子可以造成细胞存活率降低（P〈0．01）。200μmol／L铜离子孵育细胞24h,THmRNA、蛋白表达量以及多巴胺含量较正常对照组降低（P〈0．01,P〈0．01,P〈0．05）。200μmol／L铜离子孵育细胞24h,线粒体跨膜电位较正常对照组明显降低（P〈0．01）。结论铜离子对MES23．5多巴胺能细胞具有毒性作用,降低细胞功能的表达,其机制可能与线粒体功能障碍有关。     

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.     

Cyclosporin inhibition of apoptosis induced by mitochondrial complex I toxins

The cause of dopaminergic cell death in Parkinson's disease (PD) remains unknown, but may involve oxidative stress and mitochondrial complex I deficiency. Opening of the permeability transition pore and disruption of the mitochondrial transmembrane potential are known to be common events in the apoptotic pathway. Cyclosporin A and its non-immunosuppressant analogue, N-methyl-4-valine cyclosporin inhibit the opening of the mitochondrial megachannel. Complex I inhibitors, including MPP⁺, are known to induce both apoptosis in cell culture and parkinsonism in man and other primates. The present study using propidium iodide and FITC-TUNEL staining to identify apoptotic cells, demonstrates that rotenone, MPP⁺ and tetrahydroisoquinoline induce apoptosis in PC12 cells. Apoptosis induced by these agents was decreased by cyclosporin A and N-methyl-4-valine cyclosporin. Thus, apoptosis induced by inhibitors of mitochondrial complex I is probably mediated by permeability pore opening and collapse of the mitochondrial membrane potential. This observation may allow the development of novel neuroprotective strategies in disorders that may involve mitochondrial dysfunction and apoptotic cell death.     

D-beta-hydroxybutyrate protects dopaminergic SH-SY5Y cells in a rotenone model of Parkinson's disease

It has been postulated that the pathogenesis of Parkinson's disease (PD) is associated with mitochondrial dysfunction. Rotenone, an inhibitor of mitochondrial complex I, provides models of PD both in vivo and in vitro. We investigated the neuroprotective effect of D-beta-hydroxybutyrate (bHB), a ketone body, against rotenone toxicity by using SH-SY5Y dopaminergic neuroblastoma cells. SH-SY5Y cells, differentiated by all-trans-retinoic acid, were exposed to rotenone at concentrations ranging from 0 to 1,000 nM. We evaluated cellular oxidation reduction by the alamarBlue assay, viability by lactate dehydrogenase (LDH) assay, and survival/death ratio by live/dead assays. Exposure to rotenone for 48 hr oxidized cells and decreased their viability and survival rate in a concentration-dependent manner. Pretreatment of cells with 8 mM bHB provided significant protection to SH-SY5Y cells. Whereas rotenone caused the loss of mitochondrial membrane potential, released cytochrome c into the cytosol, and reduced cytochrome c content in mitochondria, addition of bHB blocked this toxic effect. bHB also attenuated the rotenone-induced activation of caspase-9 and caspase-3. Administration of 0-10 mM 3-nitropropionic acid, a complex II inhibitor, also decreased the reducing power of SH-SY5Y cells measured by alamarBlue assay. Pretreatment with 8 mM bHB attenuated the decrease of alamarBlue fluorescence. These data demonstrated that bHB had a neuroprotective effect that supported the mitochondrial respiration system by reversing the inhibition of complex I or II. Ketone bodies, the alternative energy source in the mammalian brain, appear to have therapeutic potential in PD.     

Potassium channels are involved in zinc-induced apoptosis in MES23.5 cells

There is evidence that zinc may be involved in the pathogenesis of Parkinson's disease by an apoptotic pathway. However, the mechanisms underlying zinc-induced apoptosis are unknown. Previous studies showed that 6-hydroxydopamine (6-OHDA)-enhanced potassium channels are involved in apoptosis of dopaminergic neurons. Our study was designed to test whether zinc-induced apoptosis was mediated by potassium channels. First we demonstrated cell apoptosis with zinc treatment by Hoechst staining assay. The results showed that 13.38% +/- 0.6% of MES23.5 cells were apoptotic after 24 hr of incubation with 60 microM zinc sulfate. Then we observed that the tyrosine hydroxylase (TH) protein expression and the dopamine content decreased, as detected by Western blots and high-performance liquid chromatography-electrochemical detection (HPLC-ECD). We further elucidated the mechanism of cell apoptosis by using whole-cell patch clamp recording. The data demonstrated that MES23.5 cells exhibited a tetraethylammonium (TEA)-sensitive outward K(+) current with delayed rectifier characteristics. Increases of K(+) current density were recorded following the treatment with 60 microM zinc for 4-8 hr. After incubation with 20 mM TEA, the zinc-induced enhancement of K(+) currents was fully blocked. Furthermore, incubation with TEA blocked zinc-mediated caspase-3 activation and cell apoptosis. These data suggest that zinc-induced apoptosis of MES23.5 dopaminergic cells may due to the enhancement of TEA-sensitive K(+) channel activity.     

CDK5在鱼藤酮诱导的多巴胺能神经元凋亡中的作用

目的探讨CDK5在鱼藤酮介导的SH-SY5Y细胞凋亡中的作用以及通过干预CDK5表达对细胞凋亡的影响。方法使用鱼藤酮处理多巴胺能神经元SH-SY5Y细胞,采用四甲基偶氮唑盐(MTT)法检测细胞活力,Annexin-V/PI染色,流式细胞仪检测不同实验组细胞凋亡率,采用Western blot法检测不同实验组CDK5的表达以及p25/p35蛋白的表达比。结果经鱼藤酮处理后细胞活力呈浓度依赖性下降,细胞凋亡率增高,且CDK5表达增高,p25/p35比值增高。CDK5抑制剂MDL28170或Olomoucine预处理细胞能够降低CDK5的表达水平,降低p25/p35蛋白的表达比,同时可以减轻鱼藤酮诱导的细胞损伤。结论 CDK5在鱼藤酮介导的SH-SY5Y细胞凋亡中可能发挥着重要作用,抑制CDK5的活性可能具有神经保护作用。     

利福平对鱼藤酮诱导大鼠的抗多巴胺神经元凋亡作用

目的探讨利福平抗鱼藤酮诱导帕金森病大鼠模型多巴胺神经元凋亡的作用。方法给SD大鼠背部皮下注射鱼藤酮1.5 mg/(kg.d)3周使其黑质多巴胺神经元发生凋亡,同时经灌胃给予利福平30 mg/(kg.d)干预,并通过对大鼠中脑切片进行TUNEL及Bax、Bcl-2和Caspase-3的免疫活性检测以明确利福平抗多巴胺神经元凋亡的作用。结果长期低剂量接触鱼藤酮可诱导SD大鼠中脑黑质部位出现凋亡细胞增加以及Bax、Bcl-2、Caspase-3的免疫活性的改变(P均<0.01),而应用利福平后可明显减轻这些变化(P均<0.01)。结论利福平具有抗鱼藤酮帕金森病大鼠模型的多巴胺神经元凋亡的作用,且此作用是通过上调Bcl-2和Bax的比值、抑制caspase通路而实现的。     

Neuroprotective effect of 1-methyl-1,2,3,4-tetrahydroisoquinoline on cultured rat mesencephalic neurons in the presence or absence of various neurotoxins

1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) is an endogenous brain amine and its content in parkinsonian brain is decreased compared with that in control brain. There is some evidence that 1MeTIQ protects dopaminergic neurons against dysfunction such as that seen in Parkinson's disease. In this study, we examined the neuroprotective effect of 1MeTIQ against four dopaminergic neurotoxins, 1-methyl-4-phenylpyridinuim ion, 6-hydroxydopamine, rotenone, and l-benzyl-1,2,3,4-tetrahydroisoquinoline, in cultured rat mesencephalic neurons. 1MeTIQ exerted neuroprotective action against all these toxins. Furthermore, (R)-1MeTIQ was neuroprotective, while (S)-1MeTIQ had little effect, indicating that the effect is stereoselective. The protective action of 1MeTIQ was most effective in mesencephalic neurons, especially in tyrosine hydroxylase-positive neurons. 1MeTIQ showed no affinity for dopamine receptors and did not influence the inhibition of mitochondrial respiratory complex I by rotenone, 1-methyl-4-phenylpyridinuim ion, or 1-benzyl-1,2,3,4-tetrahydroisoquinoline. These results raise the possibility that 1MeTIQ indirectly acts as an anti-oxidant such as the induction of anti-oxidative enzymes, because all these four neurotoxins can burden oxidative stress in common. This is the first report to confirm a protective effect of 1MeTIQ at the cultured neuron level, and it may have potential as a lead compound for the development of new agents to treat Parkinson's disease.     

Rifampicin inhibits apoptosis in rotenone-induced differentiated PC12 cells by ameliorating mitochondrial oxidative stress

BACKGROUND: Previous studies have shown that rifampicin exhibits neuroprotective effects, but the precise mechanisms remain unclear. Rifampicin is thought to exert the neuroprotective effect as a hydroxyl free radical scavenger. OBJECTIVE: To investigate the protective effects of rifampicin pretreatment on rotenone-induced mitochondrial oxidative stress in differentiated PC12 cells. DESIGN, TIME AND SETTING: A repeated measure, cell-based study was performed at the Department of Neurology, Second Affiliated Hospital, Sun Yat-sen University, China between December 2007 and November 2008. MATERIALS: PC12 cells were a kind gift from the Physiology Laboratory of Zhongshan Medical School, Sun Yat-sen University, China. Rotenone and rifampicin were purchased from Sigma, USA. METHODS: PC12 cells were differentiated by culturing with 100 ng/mL 7S nerve growth factor for 9 days in Dulbecco's modified Eagle's medium/Nutrient Mix F12 (DMEM/F12) supplemented with 10% fetal bovine serum. The cells were assigned to six groups according to various treatment conditions: control, cultured with normal media; rifampicin group, treated with 300 μmol/L rotenone for 26 hours; rotenone group, treated with 2.5 μmol/L rotenone for 24 hours; rifampicin pretreatment groups, pretreated with 100,200, and 300 μmol/L rifampicin for 2 hours, respectively, followed by 2.5 μmol/L rotenone for 24 hours. MAIN OUTCOME MEASURES: Mitochondrial membrane potential was measured by fluorescence microscopy and flow cytometry, respectively, using rhodamine 123 staining. Intracellular reactive oxygen species formation was analyzed by flow cytometry using 2', 7'-dichlorofluorescin-diacetate staining, and intracellular reduced glutathione was measured with a microplate reader. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Cell apoptosis was detected by Hoechst 33342 staining and flow cytometry. RESULTS: Increased apoptosis in rotenone-induced, differentiated, PC12 cells was accompanied by the loss of mitochondrial transmembrane potential, the formation of reactive oxygen species, and reduced glutathione depletion (P < 0.01). Rotenone-induced mitochondrial dysfunction was blocked in a dose-dependent manner by rifampicin (P< 0.05 or P< 0.01). CONCLUSION: Pretreatment of differentiated PC12 cells with rifampicin blocked rotenone-induced apoptosis by ameliorating mitochondrial dysfunction and oxidative stress.     

Neuroprotective Effect of CNB-001, a Novel Pyrazole Derivative of Curcumin on Biochemical and Apoptotic Markers Against Rotenone-Induced SK-N-SH Cellular Model of Parkinson’s Disease

Oxidative stress and mitochondrial dysfunction are underpinned for initiating a cascade of toxic events leading to dopaminergic neuronal death in Parkinson’s disease (PD) and identified as vital target for therapeutic intervention. Curcumin, a potent antioxidant has been reported to display diverse neuroprotective properties against various neurodegenerative diseases including PD. In this present study, we investigated the protective effect of CNB-001, a pyrazole derivative of curcumin on rotenone-induced toxicity and its possible mechanisms in neuroblastoma SK-N-SH cells. Rotenone insult significantly reduced cell viability (MTT assay) and resulted in 78 % apoptosis (dual staining) by altering Bcl-2, Bax, caspase-3, and cytochrome C expression. Moreover, rotenone enhanced ROS production and disrupts mitochondrial membrane potential. These resultant phenotypes were distinctly alleviated by CNB-001. Pretreatment with CNB-001(2 μM) 2 h before rotenone exposure (100 nM) increased cell viability, decreased ROS formation, maintained normal physiological mitochondrial membrane potential, and reduced apoptosis. Furthermore, CNB-001 inhibited downstream apoptotic cascade by increasing the expression of vital antiapoptotic protein Bcl-2 and decreased the expression of Bax, caspase-3, and cytochrome C. Collectively, the results suggest that CNB-001 protects neuronal cell against toxicity through antioxidant and antiapoptotic properties through its action on mitochondria. Therefore, it may be concluded that CNB-001 can be further developed as a promising drug for treatment of PD.     

An inhibitor of mitochondrial complex I, rotenone, inactivates proteasome by oxidative modification and induces aggregation of oxidized proteins in SH-SY5Y cells

In Parkinson's disease, characteristic pathological features are the cell death of nigrostriatal dopamine neurons and the formation of Lewy bodies composed of oxidized proteins. Mitochondrial dysfunction and aggregation of abnormal proteins have been proposed to cause the pathological changes. However, the relation between these two factors remains to be clarified. In this study, the effects of mitochondrial dysfunction on the oxidative modification and accumulation of proteins were analyzed using an inhibitor of mitochondrial complex I, rotenone, and antibodies against acrolein- and dityrosine-modified proteins. Under conditions inducing mainly apoptosis in neuroblastoma SH-SY5Y cells, rotenone markedly increased oxidized proteins, especially those modified with acrolein, even though the increase in intracellular reactive oxygen and nitrogen species was only transient and was not so marked. In addition, the activity of the proteasome system degrading oxidized proteins was reduced profoundly after treatment with rotenone. The 20S beta subunit of proteasome was modified with acrolein, to which other acrolein-modified proteins were found to bind, as shown by coprecipitation with the antibody against 20S beta subunit. These results suggest that mitochondrial dysfunction, especially decreased activity of complex I, may reduce proteasome activity through oxidative modification of proteasome itself and aggregation with other oxidized proteins. This mechanism might account for the accumulation of modified protein and, at least partially, for cell death of the dopamine neurons in Parkinson's disease.     

Curcumin protects nigral dopaminergic neurons by iron-chelation in the 6-hydroxydopamine rat model of Parkinson’s disease

Objective Curcumin is a plant polyphenolic compound and a major component of spice turmeric (Curcuma longa). It has been reported to possess free radical-scavenging, iron-chelating, and anti-inflammatory properties in dif- ferent tissues. Our previous study showed that curcumin protects MES23.5 dopaminergic cells from 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in vitro. The present study aimed to explore this neuroprotective effect in the 6-OHDA-lesioned rat model of Parkinson’s disease in vivo. Methods Rats were given intragastric curcumin for 24 days. 6-OHDA lesioning was conducted on day 4 of curcumin treatment. Dopamine content was assessed by high-performance liquid chromatography with electrochemical detection, tyrosine hydroxylase (TH)-containing neurons by immunohistochemistry, and iron-containing cells by Perls’ iron staining. Results The dopamine content in the striatum and the number of TH-immunoreactive neurons decreased after 6-OHDA treatment. Curcumin pretreatment reversed these changes. Further studies demonstrated that 6-OHDA treatment increased the number of iron-staining cells, which was dramatically decreased by curcumin pretreatment. Conclusion The protective effects of curcumin against 6-OHDA may be attributable to the ironchelating activity of curcumin to suppress the iron-induced degeneration of nigral dopaminergic neurons.     

Dietary restriction and 2-deoxyglucose administration improve behavioral outcome and reduce degeneration of dopaminergic neurons in models of Parkinson's disease.

Parkinson's disease (PD) is an age-related disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra (SN) and corresponding motor deficits. Oxidative stress and mitochondrial dysfunction are implicated in the neurodegenerative process in PD. Although dietary restriction (DR) extends lifespan and reduces levels of cellular oxidative stress in several different organ systems, the impact of DR on age-related neurodegenerative disorders is unknown. We report that DR in adult mice results in resistance of dopaminergic neurons in the SN to the toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP-induced loss of dopaminergic neurons and deficits in motor function were ameliorated in DR rats. To mimic the beneficial effect of DR on dopaminergic neurons, we administered 2-deoxy-D-glucose (2-DG; a nonmetabolizable analogue of glucose) to mice fed ad libitum. Mice receiving 2-DG exhibited reduced damage to dopaminergic neurons in the SN and improved behavioral outcome following MPTP treatment. The 2-DG treatment suppressed oxidative stress, preserved mitochondrial function, and attenuated cell death in cultured dopaminergic cells exposed to the complex I inhibitor rotenone or Fe2+. 2-DG and DR induced expression of the stress proteins heat-shock protein 70 and glucose-regulated protein 78 in dopaminergic cells, suggesting involvement of these cytoprotective proteins in the neuroprotective actions of 2-DG and DR. The striking beneficial effects of DR and 2-DG in models of PD, when considered in light of recent epidemiological data, suggest that DR may prove beneficial in reducing the incidence of PD in humans.     

Prohibitin reduces mitochondrial free radical production and protects brain cells from different injury modalities

Prohibitin is an essential mitochondrial protein that has been implicated in a wide variety of functions in many cell types, but its role in neurons remains unclear. In a proteomic screen of rat brains in which ischemic tolerance was induced by electrical stimulation of the cerebellar fastigial nucleus, we found that prohibitin is upregulated in mitochondria. This observation prompted us to investigate the role of prohibitin in neuronal death and survival. We found that prohibitin is upregulated also in the ischemic tolerance induced by transient ischemia in vivo, or oxygen-glucose deprivation in neuronal cultures. Cell fractionation and electron-microscopic immunolabeling studies demonstrated that prohibitin is localized to neuronal mitochondria. Upregulation of prohibitin in neuronal cultures or hippocampal slices was markedly neuroprotective, whereas prohibitin gene silencing increased neuronal vulnerability, an effect associated with loss of mitochondrial membrane potential and increased mitochondrial production of reactive oxygen species. Prohibitin upregulation was associated with reduced production of reactive oxygen species in mitochondria exposed to the complex I inhibitor rotenone. In addition, prohibitin protected complex I activity from the inhibitory effects of rotenone. These observations, collectively, establish prohibitin as an endogenous neuroprotective protein involved in ischemic tolerance. Prohibitin exerts beneficial effects on neurons by reducing mitochondrial free radical production. The data with complex I activity suggest that prohibitin may stabilize the function of complex I. The protective effect of prohibitin has potential translational relevance in diseases of the nervous system associated with mitochondrial dysfunction and oxidative stress.