ABSTRACT
Oxidative stress and mitochondrial dysfunction are now widely accepted as the major factors involved in the pathogenesis of Parkinson's disease (PD). Rotenone, a commonly used environmental toxin also reproduces these principle pathological features of PD. Hence, it is used frequently to induce experimental PD in cells and animals. In this study, we evaluated the neuroprotective effects of metformin against rotenone-induced toxicity in SH-SY5Y cells. Metformin treatment clearly rescued these cells from rotenone-mediated cell death via the reduction of the cytosolic and mitochondrial levels of reactive oxygen species and restoration of mitochondrial function. Furthermore, metformin upregulated PGC-1α, the master regulator of mitochondrial biogenesis and key antioxidant molecules, including glutathione and superoxide dismutase. We demonstrated that the drug exerted its cytoprotective effects by activating nuclear factor erythroid 2-related factor 2 (Nrf2)/heme-oxygenase (HO)-1 pathway, which in turn, is dependent on AKT activation by metformin. Thus, our results implicate that metformin provides neuroprotection against rotenone by inhibiting oxidative stress in the cells by inducing antioxidant system via upregulation of transcription mediated by Nrf2, thereby restoring the rotenone-induced mitochondrial dysfunction and energy deficit in the cells.
Subject(s)
Hypoglycemic Agents/pharmacology , Metformin/pharmacology , Mitochondrial Diseases/prevention & control , NF-E2-Related Factor 2/genetics , Oncogene Protein v-akt/genetics , Oxidative Stress/drug effects , Rotenone/antagonists & inhibitors , Rotenone/toxicity , Signal Transduction/drug effects , Uncoupling Agents/toxicity , Cell Line , Cell Survival/drug effects , Humans , Reactive Oxygen Species/metabolismABSTRACT
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.
Subject(s)
Ghrelin/physiology , Mitochondria/drug effects , Mitophagy/physiology , Nerve Tissue Proteins/physiology , Rotenone/toxicity , Signal Transduction/drug effects , AMP-Activated Protein Kinases/antagonists & inhibitors , AMP-Activated Protein Kinases/physiology , Apoptosis/drug effects , Cell Line, Tumor , Drug Evaluation, Preclinical , Gene Expression Regulation/drug effects , Ghrelin/pharmacology , Humans , Membrane Potential, Mitochondrial/drug effects , Mitochondria/metabolism , Nerve Tissue Proteins/antagonists & inhibitors , Neuroblastoma , Oxidative Stress/drug effects , Parkinson Disease , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/physiology , Protein Kinases/metabolism , Protein Transport/drug effects , Reactive Oxygen Species , Rotenone/antagonists & inhibitors , Sirtuin 1/antagonists & inhibitors , Sirtuin 1/physiology , Ubiquitin-Protein Ligases/metabolism , alpha-Synuclein/biosynthesis , alpha-Synuclein/geneticsABSTRACT
Suntamide A (1), a new cyclic peptide, was isolated from Cicadidae Periostracum. The gross structure of 1 was elucidated by detailed analysis of HRMS and 1D/2D NMR spectra, and the absolute configuration was established by C3 Marfey's method. We extended our study to examine biological activity of 1, and found that 1 protected SH-SY5Y cells against rotenone-induced neurotoxicity. This effect of 1 seemed to be attributed to antioxidant induction and protection of mitochondria from rotenone-caused injury. Along with augmentation of the antioxidant system by 1, there was an evident activation of Nrf2, a transcription factor involved in the activation of the antioxidant system. These results indicate that 1 rescued the cells from rotenone-mediated neurotoxicity by enhancing antioxidant capacity via induction of Nrf2, suggesting that the compound could be used as a therapeutic intervention in neurodegenerative diseases such as Parkinson's disease.
Subject(s)
Antioxidants/pharmacology , Hemiptera/chemistry , NF-E2-Related Factor 2/metabolism , Neuroprotective Agents/pharmacology , Peptides, Cyclic/pharmacology , Animals , Antioxidants/chemistry , Antioxidants/isolation & purification , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Mitochondria/drug effects , Mitochondria/metabolism , Molecular Structure , Neuroprotective Agents/chemistry , Neuroprotective Agents/isolation & purification , Peptides, Cyclic/chemistry , Peptides, Cyclic/isolation & purification , Reactive Oxygen Species/antagonists & inhibitors , Reactive Oxygen Species/metabolism , Rotenone/antagonists & inhibitors , Rotenone/pharmacology , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
Olanzapine is an antipsychotic drug used to treat patients with schizophrenia due to its lower incidence of extrapyramidal symptoms. Previous studies have shown that olanzapine activates AMP-activated protein kinase (AMPK), and induce autophagy in SH-SY5Y cell line. In this study, we investigated whether olanzapine protected against rotenone-induced neurotoxicity in PC12 cells. We showed that treatment with olanzapine increased the phosphorylation of AMPK in both dose- and time-dependent manners in PC12 cells. In addition, olanzapine activated autophagy and increased autophagic vacuoles. Furthermore, olanzapine pretreatment could protect PC12 cells from rotenone-induced apoptosis. Besides, olanzapine pretreatment could suppress the rotenone-induced depolarization of mitochondrial potential and thus protect the cells. Moreover, pretreatment with specific AMPK inhibitor compound C or with autophagy inhibitor 3-methyladenine impaired the protective effect of olanzapine on rotenone-treated PC12 cells. In summary, our results show for the first time that olanzapine ameliorates rotenone-induced injury by activating autophagy through AMPK pathway.
Subject(s)
Neuroprotective Agents/pharmacology , Olanzapine/pharmacology , Rotenone/antagonists & inhibitors , AMP-Activated Protein Kinases/antagonists & inhibitors , AMP-Activated Protein Kinases/metabolism , Animals , Autophagy/drug effects , Cell Survival/drug effects , PC12 Cells , Rats , Rotenone/toxicity , Tumor Cells, CulturedABSTRACT
Parkinson's disease (PD) is a neurodegenerative disease that is accompanied with the loss of dopaminergic neurons in the substantia nigra pars compacta which subsequently leads to a reduction in the dopamine level in the striatum. The flavonoids are gaining critical attention in the management of PD due to the toxic effects of the synthetic drugs. Naringin, a potent flavonoid, exerts neuroprotective activity against experimental animal models of PD. It also exhibits protective activity against rotenone-induced neurotoxicity in cell line studies. Therefore, the present study was designed to evaluate the therapeutic potential of naringin against rotenone-induced animal model of PD. The rotenone was injected through intracerebroventricular route into substantia nigra pars compacta (SNpc) to induce PD-like manifestations in the male rats. The behavioral deficits of the animals due to dopaminergic toxicity were evaluated in actophotometer, OFT, bar catalepsy, narrow beam walk, rota-rod, grip strength and foot print analysis. Naringin-attenuated rotenone-induced behavioral abnormalities in the experimental rats. Further, naringin reduced the rotenone-induced dopaminergic toxicity in striatum and SNpc the animals. At the sub-cellular level, naringin attenuated the rotenone-induced decrease in the mitochondrial function, integrity and bioenergetics in the SNpc of the animals. Furthermore, naringin reduced the rotenone-induced mitochondria-dependent apoptosis in the rat SNpc. However, Trigonelline significantly abolished the therapeutic effects of naringin on behavioral, biochemical and molecular observations in rotenone-induced PD-like animals. These observations indicate that naringin may exert neuroprotective activity against rotenone-induced toxicity in the animals possibly through Nrf2-mediated pathway. Thus, it can be presumed that naringin could be an alternative option in the management of PD.
Subject(s)
Flavanones/therapeutic use , Mitochondria/drug effects , Neuroprotection/drug effects , Neuroprotective Agents/therapeutic use , Parkinsonian Disorders/drug therapy , Rotenone/antagonists & inhibitors , Alkaloids/pharmacology , Animals , Dopamine/metabolism , Drug Evaluation, Preclinical , Electron Transport Chain Complex Proteins/metabolism , Exploratory Behavior/drug effects , Flavanones/pharmacology , Gait/drug effects , Hand Strength , Injections, Intraventricular , Locomotion/drug effects , Male , Mitochondria/metabolism , Neuroprotective Agents/pharmacology , Oxidative Stress/drug effects , Oxygen Consumption/drug effects , Parkinsonian Disorders/physiopathology , Parkinsonian Disorders/psychology , Rats , Rats, Wistar , Rotarod Performance Test , Rotenone/toxicity , Stereotaxic Techniques , Substantia Nigra/drug effectsABSTRACT
Oxidative stress and neuroinflammation play key roles in the initiation and progression of Parkinson's disease (PD), a neurodegenerative disorder, associated with the loss of nigrostriatal dopaminergic pathway. Thus, compounds that can mitigate oxidative stress and neuroinflammation are being investigated as promising agents for the treatment of PD. This study was designed to evaluate the effects of methyl jasmonate (MJ), a potent antioxidant and anti-inflammatory compound on parkinsonian-like symptoms and the underlying biochemical changes induced by rotenone (Rot) in mice. To this end, the effects of graded doses of MJ (25, 50 and100â¯mg/kg, i.p.) on motor dysfunctions, cognitive and depressive-like disorders induced by Rot (2.5â¯mg/kg, i.p.) were evaluated. The specific brain regions (striatum, prefrontal cortex and hippocampus) of the animals were processed for various biochemical studies. Rot-treated mice showed reduced motor activity, postural instability, cognitive and depressive-like disorders. Rot also increased brain levels of malondialdehyde (MDA), nitrite, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and acetyl-cholinesterase (AChE) activity. Moreover, Rot reduced the concentration of glutathione (GSH) and increased immnopositive cells of NF-κB and α-synuclein expressions in these brain regions. However, pretreatment with MJ, attenuated the parkinsonian-like symptoms and reduced the brain levels of MDA/nitrite, TNF-α and IL-6 induced by Rot. MJ also reduced AChE activity and down-regulate the expressions of NF-κB and α-synuclein in the brain of Rot-treated mice. These findings suggest that MJ has anti-parkinsonian-like activity, which may be related to the inhibition of oxidative stress, release of pro-inflammatory cytokines, and down regulation of NF-κB and α-synuclein expressions.
Subject(s)
Acetates/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antioxidants/pharmacology , Cyclopentanes/pharmacology , Cytokines/metabolism , NF-kappa B/antagonists & inhibitors , Oxidative Stress/drug effects , Oxylipins/pharmacology , Parkinson Disease, Secondary/chemically induced , Parkinson Disease, Secondary/prevention & control , Rotenone/antagonists & inhibitors , Uncoupling Agents/toxicity , alpha-Synuclein/antagonists & inhibitors , Animals , Behavior, Animal/drug effects , Brain Chemistry/drug effects , Male , Mice , NF-kappa B/biosynthesis , Parkinson Disease, Secondary/psychology , Psychomotor Performance/drug effects , Rotenone/toxicity , alpha-Synuclein/biosynthesisABSTRACT
Sirtuin-3 (SIRT3) is a mitochondrial NADâ¯+â¯-dependent deacetylase that is essential in regulating mitochondrial proteins and maintaining cellular antioxidant properties. It has been reported that icariin (ICA) is neuroprotective over various neurotoxicant induced oxidative stress. This study aimed to determine whether ICA exerts neuroprotective effects on rotenone (ROT)-induced neurotoxicity through activation of SIRT3. Rats treated with ROT exhibited a marked loss of dopamine (DA) neurons and a decline in motor function, along with a decrease in protein expressions of SIRT3 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in the substantia nigra (SN). Administration of ICA significantly alleviated the loss of DA neurons, improved behavioral function, and concomitantly enhanced SIRT3 and PGC-1α expressions. The neuroprotective effect of ICA on ROT-induced cytotoxicity was further confirmed in the PC12 cell model, which showed significant improvement in the survival of ROT-treated cells with ICA pretreatment. The cytoprotective effect of ICA was abolished in ROT-treated cells by SIRT3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP), along with a resultant decrease in PGC-1α expression. In addition, knockdown of PGC-1α by siRNA suppressed ICA-mediated protective effects but did not affect SIRT3 expression, indicating the role of regulation of PGC-1α by SIRT3 in the protective action of ICA. Furthermore, we showed that ICA improved mitochondrial respiration, oxidative status, enhanced antioxidant enzyme SOD activity and GSH/GSSG ratio in cells treated with ROT. However, these protective effects of ICA on ROT-treated cells was markedly abolished by SIRT3 inhibitor 3-TYP. Our findings demonstrate that ICA exerts a neuroprotective role through upregulation of SIRT3.
Subject(s)
Flavonoids/pharmacology , Glucosides/pharmacology , Neuroprotective Agents/pharmacology , Neurotoxicity Syndromes/etiology , Rotenone/toxicity , Sirtuins/metabolism , Animals , Blotting, Western , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/pathology , Glutathione/metabolism , Glutathione Disulfide/metabolism , Male , Mitochondria/drug effects , Mitochondria/metabolism , Neurotoxicity Syndromes/pathology , Neurotoxicity Syndromes/prevention & control , Oxidative Stress/drug effects , Oxygen Consumption/drug effects , PC12 Cells/drug effects , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Rotarod Performance Test , Rotenone/antagonists & inhibitors , Superoxide Dismutase/metabolismABSTRACT
Herein we envisaged the possibility of exploiting alkyl nitrates as precursors of alcohol-bearing dual inhibitors targeting acetylcholinesterase (AChE) and monoamine oxidase B (MAO B), key enzymes in neurodegenerative syndromes such as Alzheimer's disease (AD), through biotransformation unmasking an alcoholic function upon nitric oxide (NO) release. The cooperation to neuroprotection of low fluxes of NO and target enzymes' inhibition by the alcohol metabolites might return a multitargeting effect. The in vitro screening towards ChEs and MAOs of a collection of 21 primary alcohols disclosed a subset of dual inhibitors, among which three diverse chemotypes were selected to study the corresponding nitrates. Nitrate 14 proved to be a brain permeant, potent AChE-MAO B inhibitor by itself. Moreover, it protected human SH-SY5Y lines against rotenone and hydrogen peroxide with a poor inherent cytotoxicity and showed a slow conversion profile to its alcohol metabolite 9d that still behaved as bimodal and neuroprotective molecule.
Subject(s)
Acetylcholinesterase/metabolism , Cholinesterase Inhibitors/pharmacology , Monoamine Oxidase Inhibitors/pharmacology , Monoamine Oxidase/metabolism , Neuroprotective Agents/pharmacology , Nitrates/metabolism , Nitric Oxide/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cholinesterase Inhibitors/chemical synthesis , Cholinesterase Inhibitors/chemistry , Dose-Response Relationship, Drug , Humans , Hydrogen Peroxide/antagonists & inhibitors , Hydrogen Peroxide/pharmacology , Molecular Conformation , Monoamine Oxidase Inhibitors/chemical synthesis , Monoamine Oxidase Inhibitors/chemistry , Neuroprotective Agents/chemical synthesis , Neuroprotective Agents/chemistry , Rotenone/antagonists & inhibitors , Rotenone/pharmacology , Structure-Activity RelationshipABSTRACT
The study was conducted to evaluate the effect of minocycline against pesticide rotenone induced adverse effects in different rat brain regions. Assessment of oxidative stress, nitrite levels, degenerating neurons and level of cleaved caspase-3 was done in frontal cortex, mid brain, hippocampus and striatum regions of rat brain. In addition the expression profile of neuronal (MAP2), astrocytes (GFAP) and microglia (cd11b) markers was done after treatments. Rotenone induced DNA fragmentation was also assessed in all studied rat brain regions by utilizing comet assay. Rotenone administration caused significantly decreased level of glutathione along with increased level of nitrite and lipid peroxidation. Significant oxidative and nitrosative stress was also observed after rotenone administration which was considerably inhibited in minocycline treated rats in time dependent manner. Fluorojade staining and levels of cleaved caspase 3 showed the degeneration of neurons and apoptosis respectively in studied rat brain regions which were further inhibited with minocycline treatment. Rotenone administration caused significantly increased reactivity of astrocytes, microglia and altered neuronal morphology in rat brain regions which was also partially restored with minocycline treatment. In conclusion, present study showed that minocycline treatment attenuated the rotenone induced oxidative stress, nitrite level, degeneration of neurons, augmented glial reactivity and apoptosis.
Subject(s)
Apoptosis/drug effects , Microglia/drug effects , Minocycline/pharmacology , Nerve Degeneration/prevention & control , Nitrites/metabolism , Oxidative Stress/drug effects , Rotenone/antagonists & inhibitors , Rotenone/toxicity , Animals , Astrocytes/drug effects , Brain/metabolism , CD11b Antigen/metabolism , Caspase 3/metabolism , DNA Fragmentation/drug effects , Glutathione/metabolism , Lipid Peroxidation/drug effects , Male , Microglia/cytology , Microglia/metabolism , Microtubule-Associated Proteins/metabolism , Neuroprotective Agents/pharmacology , Neurotoxicity Syndromes/prevention & control , Rats , Stress, Physiological/drug effectsABSTRACT
Previous studies have shown that melatonin can protect cells against rotenone-induced cell death. Yet, the mechanism involved in this protection requires further research. In this study, we aimed to further investigate the effects of melatonin on inhibiting rotenone-induced SH-SY5Y cells and the underlying molecular mechanisms. Human neuroblastoma SH-SY5Y cells were treated with 0.3 or 1µM rotenone for 6 or 12h. Cell viability was measured with an MTS assay, the mitochondrial membrane potential was determined with a Rhodamine 123 staining assay, and the protein expression levels of the markers of autophagy, including cytochrome C release (Cyt C), light chain 3B (LC3 B) and Dynamin-Related Protein 1 (Drp1) were analyzed by western blotting. The co-localization of Drp1 and TOM20 proteins in the mitochondria of SH-SY5Y cells was measured by immunofluorescence coupled with confocal microscopy and the overexpression of the Drp1 gene was then conducted. The viability and expression levels of Cyt C and LC3 B in rotenone and melatonin + rotenone-treated Drp1-overexpressed SH-SY5Y cells were analyzed with MTS and western blotting, respectively. We found that rotenone effectively induced SH-SY5Y cell death by causing mitochondrial dysfunction and increasing Cyt C expression. Drp1 expression and its regulation of mitochondrial translocation mediated the rotenone-induced cell death and melatonin inhibited this process. Overexpression of Drp1 protein attenuated melatonin's inhibition of rotenone-induced SH-SY5Y cell death. In conclusion, melatonin effectively inhibits rotenone-induced neuronal cell death via the regulation of Drp1 expression.
Subject(s)
Down-Regulation/drug effects , GTP Phosphohydrolases/metabolism , Melatonin/pharmacology , Microtubule-Associated Proteins/metabolism , Mitochondrial Proteins/metabolism , Rotenone/antagonists & inhibitors , Rotenone/pharmacology , Cell Death/drug effects , Cell Line, Tumor , Cytochromes c/metabolism , Cytoprotection/drug effects , Dose-Response Relationship, Drug , Dynamins , GTP Phosphohydrolases/genetics , Humans , Microtubule-Associated Proteins/genetics , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Neurons/cytology , Neurons/drug effects , Protein Transport/drug effectsABSTRACT
Currently, there is no viable treatment available for Parkinson's disease (PD) that stops or reverses disease progression. Interestingly, studies testing the glucagon-like-peptide-1 (GLP-1) mimetic Exendin-4 have shown neuroprotective/neurorestorative properties in pre-clinical tests and in a pilot clinical study of PD. Incretin analogues were originally developed to treat type 2 diabetes and several are currently on the market. In this study, we tested novel incretin analogues on the dopaminergic SH-SY5Y neuroblastoma cells against a toxic mitochondrial complex I inhibitor, Rotenone. Here, we investigate for the first time the effects of six different incretin receptor agonists - Liraglutide, D-Ser2-Oxyntomodulin, a GLP-1/GIP Dual receptor agonist, dAla(2)-GIP-GluPal, Val(8)GLP-1-GluPal and exendin-4. Post-treatment with doses of 1, 10 or 100 nM of incretin analogues for 12 h increased the survival of SH-SY5Y cells treated with 1 µM Rotenone for 12 h. Furthermore, we studied the post-treatment effect of 100 nM incretin analogues against 1 µM Rotenone stress on apoptosis, mitochondrial stress and autophagy markers. We found significant protective effects of the analogues against Rotenone stress on cell survival and on mitochondrial and autophagy-associated markers. The novel GLP-1/GIP Dual receptor agonist was superior and effective at a tenfold lower concentration compared to the other analogues. Using the Phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, we further show that the neuroprotective effects are partially PI3K-independent. Our data suggest that the neuroprotective properties exhibited by incretin analogues against Rotenone stress involve enhanced autophagy, increased Akt-mediated cell survival and amelioration of mitochondrial dysfunction. These mechanisms can explain the neuroprotective effects of incretin analogues reported in clinical trials. GLP-1, GIP and dual incretin receptor agonists showed protective effects in SH-SY5Y cells treated with the stressor Rotenone. The novel GLP-1/GIP dual receptor agonist was superior and effective at a tenfold lower concentration compared to the other analogues. The drugs protected the cells from rotenone-induced impairment in cell growth and Akt activation, mitochondrial damage, impairments of autophagy and apoptotic cell signalling. See paper for details.
Subject(s)
Autophagy/drug effects , Incretins/pharmacology , Mitochondria/drug effects , Neuroprotective Agents/pharmacology , Rotenone/antagonists & inhibitors , Rotenone/pharmacology , Stress, Physiological/drug effects , Uncoupling Agents/pharmacology , Apoptosis/drug effects , Cell Line , Cell Survival/drug effects , Chromones/pharmacology , Electron Transport Complex I/antagonists & inhibitors , Glucagon-Like Peptide 1/antagonists & inhibitors , Humans , Incretins/chemistry , Morpholines/pharmacology , Oncogene Protein v-akt/genetics , Oncogene Protein v-akt/metabolism , Oxidative Stress/drug effects , Phosphoinositide-3 Kinase InhibitorsABSTRACT
BACKGOUND: The purpose of this study was to assess the biological and clinical effects of n-acetyl-cysteine (NAC) in Parkinson's disease (PD). METHODS: The overarching goal of this pilot study was to generate additional data about potentially protective properties of NAC in PD, using an in vitro and in vivo approach. In preparation for the clinical study we performed a cell tissue culture study with human embryonic stem cell (hESC)-derived midbrain dopamine (mDA) neurons that were treated with rotenone as a model for PD. The primary outcome in the cell tissue cultures was the number of cells that survived the insult with the neurotoxin rotenone. In the clinical study, patients continued their standard of care and were randomized to receive either daily NAC or were a waitlist control. Patients were evaluated before and after 3 months of receiving the NAC with DaTscan to measure dopamine transporter (DAT) binding and the Unified Parkinson's Disease Rating Scale (UPDRS) to measure clinical symptoms. RESULTS: The cell line study showed that NAC exposure resulted in significantly more mDA neurons surviving after exposure to rotenone compared to no NAC, consistent with the protective effects of NAC previously observed. The clinical study showed significantly increased DAT binding in the caudate and putamen (mean increase ranging from 4.4% to 7.8%; p<0.05 for all values) in the PD group treated with NAC, and no measurable changes in the control group. UPDRS scores were also significantly improved in the NAC group (mean improvement of 12.9%, p = 0.01). CONCLUSIONS: The results of this preliminary study demonstrate for the first time a potential direct effect of NAC on the dopamine system in PD patients, and this observation may be associated with positive clinical effects. A large-scale clinical trial to test the therapeutic efficacy of NAC in this population and to better elucidate the mechanism of action is warranted. TRIAL REGISTRATION: ClinicalTrials.gov NCT02445651.
Subject(s)
Acetylcysteine/therapeutic use , Antioxidants/therapeutic use , Neuroprotective Agents/therapeutic use , Neurotoxins/antagonists & inhibitors , Parkinson Disease/drug therapy , Rotenone/antagonists & inhibitors , Aged , Caudate Nucleus/diagnostic imaging , Caudate Nucleus/drug effects , Caudate Nucleus/metabolism , Caudate Nucleus/pathology , Cell Differentiation , Cell Survival/drug effects , Dopamine/metabolism , Dopamine Plasma Membrane Transport Proteins/metabolism , Dopaminergic Neurons/cytology , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/metabolism , Female , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/metabolism , Humans , Male , Mesencephalon/diagnostic imaging , Mesencephalon/drug effects , Mesencephalon/metabolism , Mesencephalon/pathology , Middle Aged , Neurotoxins/toxicity , Parkinson Disease/diagnostic imaging , Parkinson Disease/metabolism , Parkinson Disease/pathology , Pilot Projects , Putamen/diagnostic imaging , Putamen/drug effects , Putamen/metabolism , Putamen/pathology , Rotenone/toxicity , Single Photon Emission Computed Tomography Computed Tomography , Tissue Culture TechniquesABSTRACT
The present study investigated the neuroprotective effects of Forsythia suspense extract in a rotenone-induced neurotoxic model. FS8, one of the herbal extracts, markedly protected PC12 cells against rotenone toxicity and was selected for the in vivo study. Gavage administration of FS8 (50 and 200mg/kg, but not 10mg/kg) for 25 days significantly improved the behavior function, decreased the loss of dopaminergic neurons in substantia nigra (SN), and maintained the level of dopamine in striatum after unilateral infusion of rotenone in SN. Wherein, the protective effects of FS8 at the dose of 200mg/kg were better than selegiline. Further study indicated the excellent antioxidant activity of FS8 on the 5th and 21st days after intranigral injection of rotenone. Moreover, FS8 could inhibit microglia activity and accumulation in SN, and obviously decreased the expression of pro-inflammatory molecules (IL-6, TNF-α, iNOS and COX-2), which indicated the anti-inflammatory effects of FS8. In the PI3K/Akt/NF-κB and MAPK pathways, FS8 significantly down-regulated the protein expression of p-PI3K, p-Akt, p-IκB, p-P65, cleaved Caspase 8, p-p38 and p-JNK but not p-mTOR, cleaved Caspase 3 and p-ERK. Therefore, FS8 protected dopamine neurons against rotenone toxicity via antioxidant and anti-inflammatory effects, which suggested the promising application of FS8 in the prevention and treatment of Parkinson disease.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Antioxidants/pharmacology , Forsythia/chemistry , Neuroprotective Agents/pharmacology , Plant Extracts/pharmacology , Rotenone/toxicity , Animals , Anti-Inflammatory Agents/chemistry , Antioxidants/chemistry , Apomorphine/pharmacology , Corpus Striatum/metabolism , Dopamine/metabolism , Dose-Response Relationship, Drug , Fruit/chemistry , Inflammation Mediators/metabolism , MAP Kinase Signaling System/drug effects , Male , Motor Activity/drug effects , Neuroprotective Agents/chemistry , PC12 Cells , Plant Extracts/adverse effects , Plant Extracts/analysis , Plant Extracts/chemistry , Rats , Rotarod Performance Test , Rotenone/antagonists & inhibitorsABSTRACT
In the present study, primary mesencephalic cell cultures prepared from embryonic mouse mesencephala were used to investigate the neuroprotective effect of cabergoline, an ergoline D2 receptor agonist, against the pesticide and neurotoxin rotenone relevant to Parkinson disease (PD). Treatment of cultures with cabergoline alone significantly increased the number of tyrosine hydroxylase immunoreactive (THir) neurons and reduced the release of lactate dehydrogenase (LDH) into the culture medium compared to untreated controls. Against rotenone toxicity, cabergoline significantly rescued degenerating THir neurons, reduced the release of LDH into the culture medium and improved the morphology of surviving THir neurons. The neuroprotective effects afforded by cabergoline were independent of dopaminergic stimulation as blocking of dopamine receptors by the dopamine receptor antagonist sulpiride did not prevent them. Furthermore, rotenone-induced formation of reactive oxygen species (ROS) was significantly reduced by cabergoline. Although cabergoline increased the glutathione (GSH) content in the culture, the protective effect for dopaminergic neurons seemed not to be predominantly mediated by increasing GSH, as depletion of GSH by L-buthionine-(S,R)-sulfoximine (BSO), a GSH biosynthesis inhibitor, did not prevent cabergoline-mediated neuroprotection of THir neurons in rotenone-treated cultures. Moreover, cabergoline significantly increased the ATP/protein ratio in primary mesencephalic cell cultures when added alone or prior to rotenone treatment. These results indicate a neuroprotective effect of cabergoline for dopaminergic neurons against rotenone toxicity. This effect was independent of dopamine receptor stimulation and was at least partially mediated by reducing ROS production and increasing the ATP/protein ratio.
Subject(s)
Dopamine Agonists/pharmacology , Dopaminergic Neurons/metabolism , Ergolines/pharmacology , Mesencephalon/metabolism , Neuroprotective Agents/pharmacology , Rotenone/toxicity , Animals , Cabergoline , Cell Death/drug effects , Cell Death/physiology , Cells, Cultured , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/pathology , Female , Male , Mesencephalon/drug effects , Mesencephalon/pathology , Mice , Mice, Inbred C57BL , Reactive Oxygen Species/metabolism , Rotenone/antagonists & inhibitorsABSTRACT
There are convincing evidences that oxidative stress has an important role in both the initiation and progression of Parkinson's disease. N-acetylcysteine (NAC) is shown to have antioxidant properties via fortifying glutathione which is one of the main endogenous antioxidant systems. Therefore our study was aimed to evaluate the effect of NAC in management of Parkinson's disease. To this aim, male Wistar rats (10-12 months) received rotenone 2.5mg/kg/48 h intraperitoneally (ip) to induce a Parkinson's disease model. Pretreatment with NAC (25 and 50mg/kg/48 h ip) was administered 1h before the rotenone injection. Three behavioral tests (rotarod, rearing and bar tests) were performed for motor function assessment. Dopamine levels of dopaminergic areas in rat brain including substantia nigra (SN) and striatum (ST) were assessed using high performance liquid chromatography analysis to measure the loss of dopamine. Western blot analysis was also done for parkin and Drp1 (dynamin related protein-1) proteins quantification in SN and ST. Our results indicated that NAC significantly ameliorated the rotenone-induced motor dysfunction and dopamine loss. Furthermore, NAC was able to prevent the rotenone-induced changes in parkin and Drp1 levels in the both studied areas. In conclusion we found that NAC delayed the Parkinson's disease induction by rotenone and this effect might be related to its proved antioxidant effect.
Subject(s)
Acetylcysteine/pharmacology , Dynamins/metabolism , Parkinsonian Disorders/chemically induced , Parkinsonian Disorders/prevention & control , Rotenone/antagonists & inhibitors , Ubiquitin-Protein Ligases/metabolism , Animals , Corpus Striatum/drug effects , Corpus Striatum/metabolism , Disease Models, Animal , Dopamine/metabolism , Dopaminergic Neurons/metabolism , Drug Interactions , Male , Oxidative Stress/drug effects , Parkinsonian Disorders/metabolism , Protein Binding , Random Allocation , Rats , Rats, Wistar , Rotarod Performance Test , Rotenone/pharmacology , Substantia Nigra/drug effects , Substantia Nigra/metabolismABSTRACT
Environmental toxin exposure is associated with the development of Parkinson's disease (PD), and environmental factors can influence the onset of the majority of sporadic PD cases via genetically mediated pathways. Rotenone, a widespread pesticide, induces Parkinsonism and the formation of Lewy bodies in animals; however, the molecular mechanism that underlies α-synuclein aggregation remains unclear. Here, we assessed the aggregation of α-synuclein in PC12 cells with or without cross-linking following rotenone exposure via a variety of methods, including western blotting, immunofluorescence and electron microscopy. We demonstrated that rotenone increased the intracellular calcium levels and induced the aggregation and phosphorylation of α-synuclein in a calcium-dependent manner. Aggregated α-synuclein is typically degraded by autophagy, and rotenone impaired this process. The attenuation of autophagy and α-synuclein alterations were reversed by scavenging calcium. Calcium regulates the activity of AKT-glycogen synthase kinase 3 (GSK3)ß. We demonstrated that rotenone attenuated the phosphorylation of AKT and GSK3ß, and the elimination of calcium reversed these phenomena. As a GSK3ß inhibitor, lithium promoted autophagy and decreased the aggregation and phosphorylation of α-synuclein. GSK3ß activation through overexpression depressed autophagy and increased the total protein level and phosphorylation of α-synuclein. These results suggest that rotenone-induced α-synuclein aggregation is mediated by the calcium/GSK3ß signaling pathway.
Subject(s)
Calcium/metabolism , Glycogen Synthase Kinase 3/drug effects , Insecticides/toxicity , Rotenone/toxicity , Uncoupling Agents/toxicity , alpha-Synuclein/drug effects , Animals , Autophagy/drug effects , Dose-Response Relationship, Drug , Glycogen Synthase Kinase 3 beta , Insecticides/antagonists & inhibitors , Male , Mice , Mice, Inbred C57BL , Microinjections , PC12 Cells , Rats , Rotenone/antagonists & inhibitors , Signal Transduction/drug effects , Substantia NigraABSTRACT
Betaine protects cells from environmental stress and serves as a methyl donor in several biochemical pathways. It reduces cardiovascular disease risk and protects liver cells from alcoholic liver damage and nonalcoholic steatohepatitis. Its pretreatment can rescue cells exposed to toxins such as rotenone, chloroform, and LiCl. Furthermore, it has been suggested that betaine can suppress cancer cell growth in vivo and in vitro. Mitochondrial electron transport chain (ETC) complexes generate the mitochondrial membrane potential, which is essential to produce cellular energy, ATP. Reduced mitochondrial respiration and energy status have been found in many human pathological conditions including aging, cancer, and neurodegenerative disease. In this study we investigated whether betaine directly targets mitochondria. We show that betaine treatment leads to an upregulation of mitochondrial respiration and cytochrome c oxidase activity in H2.35 cells, the proposed rate limiting enzyme of ETC in vivo. Following treatment, the mitochondrial membrane potential was increased and cellular energy levels were elevated. We propose that the anti-proliferative effects of betaine on cancer cells might be due to enhanced mitochondrial function contributing to a reversal of the Warburg effect.
Subject(s)
Antineoplastic Agents/pharmacology , Betaine/pharmacology , Cytoprotection , Mitochondria/drug effects , Adenosine Triphosphate/metabolism , Animals , Cell Line , Cell Respiration/drug effects , Electron Transport Complex IV/metabolism , Energy Metabolism/drug effects , Membrane Potential, Mitochondrial/drug effects , Mice , Mitochondria/metabolism , Rotenone/antagonists & inhibitorsABSTRACT
The α7 acetylcholine nicotine receptor is a ligand-gated ion channel that is involved in cognition disorders, schizophrenia, pain and inflammation among other diseases. Therefore, the development of new agents that target this receptor has great significance. Positive allosteric modulators might be advantageous, since they facilitate receptor responses without directly interacting with the agonist binding site. Here we report the search for and further design of new positive allosteric modulators having the relatively simple chalcone structure. From the natural product isoliquiritigenin as starting point, chalcones substituted with hydroxyl groups at defined locations were identified as optimal and specific promoters of α7 nicotinic function. The most potent compound (2,4,2',5'-tetrahydroxychalcone, 111) was further characterized showing its potential as neuroprotective, analgesic and cognitive enhancer, opening the way for future developments around the chalcone structure.
Subject(s)
Analgesics/pharmacology , Chalcones/pharmacology , alpha7 Nicotinic Acetylcholine Receptor/antagonists & inhibitors , Allosteric Regulation/drug effects , Analgesics/chemical synthesis , Analgesics/chemistry , Animals , Behavior, Animal/drug effects , Cell Death/drug effects , Cell Survival/drug effects , Chalcones/chemical synthesis , Chalcones/chemistry , Dose-Response Relationship, Drug , Humans , Male , Maze Learning/drug effects , Mice , Molecular Structure , Oligomycins/antagonists & inhibitors , Oligomycins/pharmacology , Pain/drug therapy , Rats , Rats, Wistar , Rotenone/antagonists & inhibitors , Rotenone/pharmacology , Structure-Activity Relationship , alpha7 Nicotinic Acetylcholine Receptor/metabolismABSTRACT
We have investigated whether polyamidoamine (PAMAM), phosphorus (pd) and viologen-phosphorus (vpd) dendrimers can prevent damage to embryonic mouse hippocampal cells (mHippoE-18) caused by rotenone, which is used as a pesticide, insecticide, and as a nonselective piscicide, that works by interfering with the electron transport chain in mitochondria. Several basic aspects, such as cell viability, production of reactive oxygen species and changes in mitochondrial transmembrane potential, were analyzed. mHippoE-18 cells were treated with these structurally different dendrimers at 0.1µM. A 1h incubation with dendrimers was followed by the addition of rotenone at 1µM, and a further 24h incubation. PAMAM, phosphorus and viologen-phosphorus dendrimers all increased cell viability (reduced cell death-data need to be compared with untreated controls). A lower level of reactive oxygen species and a favorable effect on mitochondrial system were found with PAMAM and viologen-phosphorus dendrimers. These results indicate reduced toxicity in the presence of dendrimers.
Subject(s)
Dendrimers/pharmacology , Phosphorus/pharmacology , Polyamines/pharmacology , Rotenone/antagonists & inhibitors , Rotenone/pharmacology , Viologens/pharmacology , Animals , Cell Survival/drug effects , Cells, Cultured , Dendrimers/chemistry , Dose-Response Relationship, Drug , Membrane Potential, Mitochondrial/drug effects , Mice , Molecular Structure , Phosphorus/chemistry , Polyamines/chemistry , Reactive Oxygen Species/metabolism , Rotenone/chemistry , Structure-Activity RelationshipABSTRACT
Dendropanax morbifera Leveille (Araliaceae) is well known in Korean traditional medicine for a variety of diseases. Rotenone is a commonly used neurotoxin to produce in vivo and in vitro Parkinson's disease models. This study was designed to elucidate the processes underlying neuroprotection of rutin, a bioflavonoid isolated from D. morbifera Leveille in cellular models of rotenone-induced toxicity. We found that rutin significantly decreased rotenone-induced generation of reactive oxygen species levels in SH-SY5Y cells. Rutin protected the increased level of intracellular Ca(2+) and depleted level of mitochondrial membrane potential (ΔΨm) induced by rotenone. Furthermore, it prevented the decreased ratio of Bax/Bcl-2 caused by rotenone treatment. Additionally, rutin protected SH-SY5Y cells from rotenone-induced caspase-9 and caspase-3 activation and apoptotic cell death. We also observed that rutin repressed rotenone-induced c-Jun N-terminal kinase and p38 mitogen-activated protein kinase phosphorylation. These results suggest that rutin may have therapeutic potential for the treatment of neurodegenerative diseases associated with oxidative stress.